Substrate Specificity Research Papers - Academia.edu (original) (raw)

Phenylpropenes such as chavicol, t-anol, eugenol, and isoeugenol are produced by plants as defense compounds against animals and microorganisms and as floral attractants of pollinators. Moreover, humans have used phenylpropenes since... more

Phenylpropenes such as chavicol, t-anol, eugenol, and isoeugenol are produced by plants as defense compounds against animals and microorganisms and as floral attractants of pollinators. Moreover, humans have used phenylpropenes since antiquity for food preservation and flavoring and as medicinal agents. Previous research suggested that the phenylpropenes are synthesized in plants from substituted phenylpropenols, although the identity of the enzymes and the nature of the reaction mechanism involved in this transformation have remained obscure. We show here that glandular trichomes of sweet basil (Ocimum basilicum), which synthesize and accumulate phenylpropenes, possess an enzyme that can use coniferyl acetate and NADPH to form eugenol. Petunia (Petunia hybrida cv. Mitchell) flowers, which emit large amounts of isoeugenol, possess an enzyme homologous to the basil eugenol-forming enzyme that also uses coniferyl acetate and NADPH as substrates but catalyzes the formation of isoeugenol. The basil and petunia phenylpropene-forming enzymes belong to a structural family of NADPH-dependent reductases that also includes pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase.

The precursor protein honey bee prepromelittin has been expressed as a fusion protein in Escherichia coli joined to the C-terminus of a truncated form of the bacteriophage gene 10 protein via an engineered recognition sequence for Factor... more

The precursor protein honey bee prepromelittin has been expressed as a fusion protein in Escherichia coli joined to the C-terminus of a truncated form of the bacteriophage gene 10 protein via an engineered recognition sequence for Factor Xa. Factor Xa was found to cleave poorly at the engineered site, giving a low yield of the required prepromelittin. In contrast, cleavage on the C-terminal side of the sequence VLGR at residue 67 in the gene 10 sequence proceeded in high yield. Factor Xa may be inhibited by adjacent hydrophobic sequences on the C-terminal side of a potential cleavage site.

N-acetyl-β-D-hexosaminidase was purified from wheat bran and characterized. The purified enzyme showed two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular mass of 75 and 78 kDa. The... more

N-acetyl-β-D-hexosaminidase was purified from wheat bran and characterized. The purified enzyme showed two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular mass of 75 and 78 kDa. The enzyme exhibited optimum pH and temperature at 5.0 and 50 o C, respectively. The enzyme was active on the substrates of p-nitrophenyl-Nacetyl-β-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-Nacetyl-β-D-galactosaminide (pNP-GalNAc), whereas inactive on pNP-β-D-glucopyranoside, pNP-β-D-galactopyranoside, swollen chitin, and colloidal chitin, suggesting high substrate specificity. The enzyme activity for pNP-GlcNAc was stable at pH 3-6 and under 50 o C. The K m , V max and K cat for pNP-GlcNAc were 0.014 mM, 0.05 µmol/min, and 3.01×10 6 min −1 , respectively. The enzyme could be completely inhibited at 1-10 mM HgCl 2 and AgNO 3, suggesting that the intact thiol group is essential for activity. β-N-Acetylhexosaminidase from wheat bran could inhibit the conidial germination and digest the hyphae of Fusarium solani.

Although the precise mechanisms explaining loss of, and failure to regain, function after spinal cord injury are unknown, there is increasing interest in the role of "secondary cell death." One prevalent theme in cell loss in other... more

Although the precise mechanisms explaining loss of, and failure to regain, function after spinal cord injury are unknown, there is increasing interest in the role of "secondary cell death." One prevalent theme in cell loss in other regions of the CNS involves apoptosis executed by the intracellular caspase proteases. A recent study demonstrated that spinal cord injury rapidly increased the activation of caspase-3. Our previous studies demonstrated peak apoptosis in three of four cellular compartments 3 days after controlled contusion in the rat. We have extended these analyses to include enzyme and substrate studies of caspase subfamilies both in rostral and in caudal adjacent segments compared to the lesion site. Although presumed activation of programmed proenzyme is considered the mechanism for enhanced caspases, our novel analyses were designed to detect upregulation of gene expression. We surveyed traumatically injured spinal cord for caspase family messages with a modified differential mRNA display approach and found that the caspase-3 (CASP3) message was present and upregulated severalfold after injury. Our results clearly demonstrate that cell death in the spinal cord occurs after posttranslational activation of caspases that follow, at least for caspase-3, initial upregulation of CASP3 mRNA levels.

Lignin is a major component of plant secondary cell walls. Here we describe caffeoyl shikimate esterase (CSE) as an enzyme central to the lignin biosynthetic pathway. Arabidopsis thaliana cse mutants deposit less lignin than do wild-type... more

Lignin is a major component of plant secondary cell walls. Here we describe caffeoyl shikimate esterase (CSE) as an enzyme central to the lignin biosynthetic pathway. Arabidopsis thaliana cse mutants deposit less lignin than do wild-type plants, and the remaining lignin is enriched in p-hydroxyphenyl units. Phenolic metabolite profiling identified accumulation of the lignin pathway intermediate caffeoyl shikimate in cse mutants as compared to caffeoyl shikimate levels in the wild type, suggesting caffeoyl shikimate as a substrate for CSE. Accordingly, recombinant CSE hydrolyzed caffeoyl shikimate into caffeate. Associated with the changes in lignin, the conversion of cellulose to glucose in cse mutants increased up to fourfold as compared to that in the wild type upon saccharification without pretreatment. Collectively, these data necessitate the revision of currently accepted models of the lignin biosynthetic pathway.

PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in Escherichia coli, the hydrolysis... more

PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in Escherichia coli, the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and two pepN mutants, 9218 and DH5aDpepN. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in both pepN mutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in both pepN mutants. This decreased ability to cleave these substrates is pepN-specific as there is no reduction in the ability to hydrolyse exopeptidase substrates in E. coli mutants lacking other peptidases, pepA, pepB or pepE. PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in E. coli. Further in vitro studies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference of E. coli PepN to be Arg>Ala>Lys>Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress in E. coli, demonstrating a physiological role for this aminoendopeptidase under some stress conditions.

Carnosinases are Xaa-His dipeptidases that play diverse functions throughout all kingdoms of life. Human isoforms of carnosinase (CN1 and CN2) under appropriate conditions catalyze the hydrolysis of the dipeptides carnosine... more

Carnosinases are Xaa-His dipeptidases that play diverse functions throughout all kingdoms of life. Human isoforms of carnosinase (CN1 and CN2) under appropriate conditions catalyze the hydrolysis of the dipeptides carnosine (β-alanyl-L-histidine) and homocarnosine (γ-aminobutyryl-L-histidine). Alterations of serum carnosinase (CN1) activity has been associated with several pathological conditions, such as neurological disorders, chronic diseases and cancer. For this reason the use of carnosinase levels as a biomarker in cerebrospinal fluid (CSF) has been questioned. The hydrolysis of imidazolerelated dipeptides in prokaryotes and eukaryotes is also catalyzed by aminoacyl-histidine dipeptidases like PepD (EC 3.4.13.3), PepV (EC 3.4.13.19) and anserinase (EC 3.4.13.5). The review deals with the structure and function of this class of enzymes in physiological and pathological conditions. The main substrates of these enzymes, i.e., carnosine, homocarnosine and anserine (β-alanyl-3-methyl-L-histidine) will also be described.

Supplemental Figure 1. Ribose-614 cleavage is unaffected by the addition of 10% complementary strand. Addition of equimolar amounts of the complement greatly lowers cleavage rate and plateau. (A) Cleavage profile for ribose-614 (100... more

Supplemental Figure 1. Ribose-614 cleavage is unaffected by the addition of 10% complementary strand. Addition of equimolar amounts of the complement greatly lowers cleavage rate and plateau. (A) Cleavage profile for ribose-614 (100 nM)(triangles), or ribose-614 (115 nM) plus complementary strand (11.5 nM)(squares). (B) Cleavage profile for ribose-614 (222 nM)(triangles), or ribose-614 (200 nM) plus equimolar complementary strand (circles). Unless otherwise stated, all kinetics were performed under standard reaction conditions, namely 1M NaCl, 1 mM MgCl 2 , 50 mM HEPES, 25°C, and errors in percent cleaved are < ±2 percentage points. Supplemental Figure 2. Cleavage products do not affect ribose-614 cleavage, and only minimally alter ribose-library cleavage. Ribose-614 or ribose-library were incubated either alone (400 nM), or with the addition of the 28-nt product of 614 cleavage (400 nM), the 78-nt product of 614 cleavage (400 nM), or both (400 nM each). Data are fit to a single exponential curve. Errors in percent cleaved are < ±2 percentage points. Supplemental Figure 3. Gel-purification of ribose-614 at cleavage plateau results in additional cleaveage, indicating a fraction of 614 is folded into an inactive conformation. Ribose-614 (222 nM) was allowed to self-cleave for 140 hours, nearing cleavage plateau

The two opponents, toxin (CcdB, LetB or LetD, protein G, LynB) and antidote (CcdA, LetA, protein H, LynA), in the plasmid addiction system ccd of the F plasmid were studied by different biophysical methods. The thermodynamic stability was... more

The two opponents, toxin (CcdB, LetB or LetD, protein G, LynB) and antidote (CcdA, LetA, protein H, LynA), in the plasmid addiction system ccd of the F plasmid were studied by different biophysical methods. The thermodynamic stability was measured at different temperatures combining denaturant and thermally induced unfolding. It was found that both proteins denature in a two-state equilibrium (native dimer versus unfolded monomer) and that CcdA has a signi®cantly lower thermodynamic stability. Using a numerical model, which was developed earlier by us, and on the basis of the determined thermodynamic parameters the concentration dependence of the denaturation transition temperature was obtained for both proteins. This concentration dependence may be of physiological signi®cance, as the concentration of both ccd addiction proteins cannot exceed a certain limit because their expression is controlled by autoregulation. The in¯uence of DNA on the thermal stability of the two proteins was probed. It was found that cognate DNA increases the melting temperature of CcdA. In the presence of non-speci®c DNA the thermal stability was not changed. The melting temperature of CcdB was not in¯uenced by the applied double-stranded oligonucleotides, neither cognate nor unspeci®c.

Myrosinases (␤-thioglucoside glucohydrolase, TGG; EC 3.2.1.147) catalyze the hydrolysis of glucosinolates, a structurally distinct group of nitrogen-and sulfur-containing secondary metabolites, to give a chemically unstable intermediate,... more

Myrosinases (␤-thioglucoside glucohydrolase, TGG; EC 3.2.1.147) catalyze the hydrolysis of glucosinolates, a structurally distinct group of nitrogen-and sulfur-containing secondary metabolites, to give a chemically unstable intermediate, glucose and sulfate. This catalysis initiates a chemical defense in crucifer plants as a response to the tissue-damaging activities of herbivores and pathogens. To characterize the individual and collective biochemical properties of the myrosinase enzymes found in the aerial tissues of Arabidopsis thaliana, we purified TGG1 and TGG2, which share 73% amino acid identity, individually from T-DNA insertion lines of Arabidopsis using lectin affinity and anion exchange chromatography. Electrophoresis under denaturing conditions and the mobility of nondenatured TGG1 and TGG2 protein on gel filtration chromatography indicated that the native proteins exist as dimers of 150 and 126 kDa, respectively. Despite their relatively similar kinetic parameters, both enzymes had distinct physicochemical properties such as extractability in low ionic strength buffer and electrophoretic mobility following deglycosylation treatment. Deglycosylation under nondenaturing conditions had limited effects on TGG1 and no effect on TGG2 activity. Both enzymes functioned across a broad range of temperatures (up to 60 • C) and pH values (5-10). These results demonstrate that myrosinases have the ability to function in environments like the digestive tract of insect herbivores that are significantly different from the environment in a damaged plant.

The crystal structures of the 4-methoxybenzoate bound forms of cytochrome P450 enzymes CYP199A2 and CYP199A4 from the Rhodopseudomonas palustris strains CGA009 and HaA2 have been solved. The structures of these two enzymes, which share... more

The crystal structures of the 4-methoxybenzoate bound forms of cytochrome P450 enzymes CYP199A2 and CYP199A4 from the Rhodopseudomonas palustris strains CGA009 and HaA2 have been solved. The structures of these two enzymes, which share 86% sequence identity, are very similar though some differences are found on the proximal surface. In these structures the enzymes have a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. In both structures the substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of these enzymes for oxidative demethylation of the substrate. Mutagenesis studies on CYP199A4 highlight the involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates by these enzymes. † Electronic supplementary information (ESI) available: Comparison of active site and substrate access channel residues of CYP199A2 and CYP199A4, structural comparisons of the active site, substrate access channel and anion binding sites of CYP199A4 to those of CYP199A2, substrate binding and kinetic analysis of CYP199A4 variants, sequence alignment of CYP199 family members and SDS-PAGE analysis of CYP199A4.

portant tool for studying phagocyte function. The ini-The enzymatic determination of hydrogen peroxide tial product of NADPH oxidase-mediated oxygen can be accomplished with high sensitivity and specificreduction is primarily superoxide... more

portant tool for studying phagocyte function. The ini-The enzymatic determination of hydrogen peroxide tial product of NADPH oxidase-mediated oxygen can be accomplished with high sensitivity and specificreduction is primarily superoxide anion. Subsequently, ity using N-acetyl-3,7-dihydroxyphenoxazine (Amplex superoxide is converted to H 2 O 2 either spontaneously Red), a highly sensitive and chemically stable fluoroor by superoxide dismutase. Because of its stability and genic probe for the enzymatic determination of H 2 O 2 . membrane permeability, H 2 O 2 is usually chosen as an Enzyme-catalyzed oxidation of Amplex Red, which is a analyte among other oxidants to quantitate the extracolorless and nonfluorescent derivative of dihydrorescellular release of reactive oxygen reduction products orufin, produces highly fluorescent resorufin, which in phagocytes. H 2 O 2 is also a coproduct of a variety of has an excitation maximum at 563 nm and emission oxidases, and its detection is the basis for measuring maximum at 587 nm. The reaction stoichiometry of the activity of these oxidases (1, 2). Both chromogenic Amplex Red and H 2 O 2 was determined to be 1:1. This and fluorogenic substrates are currently used to meaprobe allows detection of 5 pmol H 2 O 2 in a 96-well fluosure the activity of NADPH oxidase, HRP, 1 and other rescence microplate assay. When applied to the meaoxidases. While assays using chromogenic substrates surement of NADPH oxidase activation, the Amplex require only a regular spectrophotometer, they are usu-Red assay can detect H 2 O 2 release from as few as 2000 ally less sensitive than assays that use fluorogenic phorbol myristate acetate-stimulated neutrophils with substrates. Absorption-and fluorescence-detecting a sensitivity 5-to 20-fold greater than that attained in microplate readers, which are particularly useful inthe scopoletin assay under the same experimental construments for enzyme assays, are now available in ditions. Furthermore, the oxidase-catalyzed assay usmany research and clinical laboratories.

Abstract: Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A... more

Abstract: Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase reaction do not follow the classical Michaelis-Menten model. With only a few exceptions, bacterial lipases are able to completely hydrolyze a triacylglycerol substrate although a certain preference for primary ester bonds has been observed. Numerous lipase assay methods are available using coloured or fluorescent substrates which allow spectroscopic and fluorimetric detection of lipase activitiy. Another important assay is based on titration of fatty acids released from the substrate. Newly developed methods allow to exactly determine lipase activity via controlled surface pressure or by means of a computer-controlled oil drop tensiometer. The synthesis and secretion of lipases by bacteria is influenced by a variety of environmental factors like ions, carbon sources, or presence of non-metabolizable polysaccharides. The secretion pathway is known for Pseudomonas lipases with P. aeruginosa lipase using a two-step mechanism and P. fluorescens lipase using a one-step mechanism. Additionally, some Pseudomonas lipases need specific chaperone-like proteins assisting their correct folding in the periplasm. These lipase-specific foldases (Lif-proteins) which show a high degree of amino acid sequence homology among different Pseudomonas species are coded for by genes located immediately downstream the lipase structural genes. A comparison of different bacterial lipases on the basis of primary structure revealed only very limited sequence homology. However, determination of the three-dimensional structure of the P. glumae lipase indicated that at least some of the bacterial lipases will presumably reveal a conserved folding pattern called the α/β-hydrolase fold, which has been described for other microbial and human lipases. The catalytic site of lipases is buried inside the protein and contains a serine-protease-like catalytic triad consisting of the amino acids serine, histidine, and aspartate (or glutamate). The Ser-residue is located in a strictly conserved β-ε-Ser-α motif. The active site is covered by a lid-like a-helical structure which moves away upon contact of the lipase with its substrate, thereby exposing hydrophobic residues at the protein's surface mediating the contact between protein and substrate. This movable lid-like α-helix explains at a molecular level the lipase-specific phenomenon of interfacial activation. At least some of the pathogenic bacterial species produce a lipase which has been studied with respect to its role as a virulence factor. Lipases of Propionibacterium acnes and Staphylococcus epidermidis may be involved in colonization and persistence of these bacteria on the human skin. Lipases of S. aureus and P. aeruginosa are produced during the bacterial infection process and, at least in vitro, considerably impair the function of different cell types involved in the human immune response like macrophages or platelets. The present state of knowledge suggests to classify the lipases as important bacterial virulence factors which exert their harmful effects in combination with other bacterial enzymes, in particular the phospholipases C. Most of the steadily increasing interest in bacterial lipases is based on their biotechnological applications which are partly based on their potential to catalyze not only hydrolysis but also synthesis of a variety of industrially valuable products. Optically active compounds, various esters and lactones are among the substances synthesized using bacterial lipases. Recently, an important application emerged with the addition of bacterial lipases to household detergents in order to reduce or even replace synthetic detergent chemicals which pose considerable environmental problems. As a main conclusion, [ipases represent an extremely versatile group of bacterial extracellular enzymes that are capable of performing a variety of important reactions, thereby presenting a fascinating field tot future research.

Soluble enzymes from sage (Salvia oflicinalis) and tansy (Tanacetum vulgare), which catalyze the cyclization of geranyl pyrophosphate and the presumptive intermediate linalyl pyrophosphate to the (+) and (-) enantiomers, respectively, of... more

Soluble enzymes from sage (Salvia oflicinalis) and tansy (Tanacetum vulgare), which catalyze the cyclization of geranyl pyrophosphate and the presumptive intermediate linalyl pyrophosphate to the (+) and (-) enantiomers, respectively, of 2-bornyl pyrophosphate, were employed to evaluate mechanistic alternatives for the pyrophosphate migration in monoterpene cyclization reactions. Separate incubation of [ 1-3H2,(u-32P]-and [ 1 -3H2,@-32P]geranyl and (f)-linalyl pyrophosphates with partially purified preparations of each enantiomer-generating cyclase gave [3H,32P] bornyl pyrophosphates, which were selectively hydrolyzed

Fruit flies in the family Tephritidae are rated among the world's most destructive agricultural pests. The Mediterranean fruit fly Ceratitis capitata is emerging as a model organism to study the fertilization in Insects. Three integral... more

Fruit flies in the family Tephritidae are rated among the world's most destructive agricultural pests. The Mediterranean fruit fly Ceratitis capitata is emerging as a model organism to study the fertilization in Insects. Three integral proteins with glycosidase activity are present in the plasma membrane of spermatozoa. The glycosidases have been purified and characterized. We have demonstrated the presence of three enzymes, a b-N-acetylhexosaminidase, an a-mannosidase and an a-L-fucosidase. The molecular mass of the native enzymes estimated by gel filtration was 160 kDa for b-N-acetylhexosaminidase, 310 kDa for a-mannosidase and 140 kDa for a-L-fucosidase. SDS-PAGE showed that b-N-acetylhexosaminidase is a dimer of a single protein of 73 kDa, a-mannosidase consists of six subunits with different molecular weights and a-L-fucosidase is a dimer made up by two different monomers. Characterization of the purified enzymes included glycosylation pattern, pI, optimal pH, substrate preference, kinetic properties and thermal stability. Soluble forms similar to the sperm associated glycosidases are present. Polyclonal antibodies raised against synthetic peptides designed from the predicted products of the Drosophila melanogaster genes encoding b-N-acetylhexosaminidase and a-L-fucosidase were used.

Sulfation of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and structurally related hydroxamic acids by rat and human sulfotransferases was studied. There was a clear sex and age difference in the sulfation of N-OH-AAF and the... more

Sulfation of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and structurally related hydroxamic acids by rat and human sulfotransferases was studied. There was a clear sex and age difference in the sulfation of N-OH-AAF and the other hydroxamic acids by rat liver cytosols: adult male rats had the highest sulfation activity. Experiments with purified aryl sulfotransferase IV (AST IV) indicated that the high expression of this enzyme in male rat liver may be responsible for these differences. No such sex or age difference was found for the sulfation of aromatic hydroxylamines. In cytosols of adult human livers, sulfation activity towards aromatic hydroxamic acids and hydroxylamines was clearly present, but activities were much lower than in rat liver cytosols. Sulfation activity towards these compounds was also found in fetal and neonatal liver and adrenals. These compounds probably are sulfated by several different sulfotransferases in humans

Cardiovascular disease is a major cause of death and thus a great deal of effort has been made in salvaging the diseased myocardium. Although various factors have been identified as possible causes of different cardiac diseases such as... more

Cardiovascular disease is a major cause of death and thus a great deal of effort has been made in salvaging the diseased myocardium. Although various factors have been identified as possible causes of different cardiac diseases such as heart failure and ischemic heart disease, there is a real need to elucidate their role for the better understanding of the cardiac disease pathology and formulation of strategies for developing newer therapeutic interventions. In view of the intimate involvement of different types of proteases in maintaining cellular structure, the role of proteases in various cardiac diseases has become the focus of recent research. Proteases are present in the cytosol as well as are localized in a number of subcellular organelles in the cell. These are known to use extracellular matrix, cytoskeletal, sarcolemmal, sarcoplasmic reticular, mitochondrial and myofibrillar proteins as substrates. Work from different laboratories using a wide variety of techniques has shown that the activation of proteases causes alterations of a number of specific proteins leading to subcellular remodeling and cardiac dysfunction. Inhibition of protease action by different drugs and agents, therefore, has a clinical relevance and is expected to form a part of new treatment paradigm for improving heart function. This review examines the biochemistry and localization of some of the proteases in the cardiac tissue in addition to identification of the sites of action of some protease inhibitors. (Mol Cell Biochem 263: 241–256, 2004)

Functional identity and significant similarities in cofactors and sequence exist between the L and M reaction center proteins of the photosynthetic bacteria and the D l and D2 photosystem-I1 reaction center proteins of cyanobacteria,... more

Functional identity and significant similarities in cofactors and sequence exist between the L and M reaction center proteins of the photosynthetic bacteria and the D l and D2 photosystem-I1 reaction center proteins of cyanobacteria, algae, and plants. A model of the quinone (QB) binding site of the D1 protein is presented based upon the resolved structure of the QB binding pocket of the L subunit, and introducing novel quantitative notions of complementarity and contact surface between atoms. This model, built without using traditional methods of molecular mechanics and restricted to residues in direct contact with QB, accounts for the experimentally derived functional state of mutants of the D 1 protein in the region of QB. It predicts the binding of both the classical and phenol-type PSI1 herbicides and rationalizes the relative levels of tolerance of mutant phenotypes. o 1995 Wiey-Liss, Inc.

Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular R-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively.... more

Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular R-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the R-galactosidases were identical. Intracellular R-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular R-galactosidase presented higher k cat than the intracellular enzyme (7.16 vs 3.29 s-1 , respectively) for the p-nitrophenyl-R-D-galactopyranoside substrate. The K m for hydrolysis of pNPRGal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was acompetitively inhibited by galactose (K i) 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55°C reduced stachyose and raffinose amounts by 100 and 73%, respectively.

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type HSV blocks the execution of the cell death program triggered by viral gene products, by the effectors of the immune... more

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type HSV blocks the execution of the cell death program triggered by viral gene products, by the effectors of the immune system such as the Fas and tumor necrosis factor pathways, or by nonspecific stress agents such as either osmotic shock induced by sorbitol or thermal shock. A report from this laboratory showed that caspase inhibitors do not block DNA fragmentation induced by infection with the HSV-1 d120 mutant. To identify the events in programmed cell death induced and blocked by HSV-1, we examined cells infected with wild-type virus or the d120 mutant or cells infected and exposed to sorbitol. We report that: (i) the HSV-1 d120 mutant induced apoptosis by a caspase-3-independent pathway inasmuch as caspase 3 was not activated and DNA fragmentation was not blocked by caspase inhibitors even though the virus caused cytochrome c release and depolarization of the inner mitochondrial membrane. (ii) Cells infected with wild-type HSV-1 exhibited none of the manifestations associated with programmed cell death assayed in these studies. (iii) Uninfected cells exposed to osmotic shock succumbed to caspase-dependent apoptosis inasmuch as cytochrome c was released, the inner mitochondrial potential was lost, caspase-3 was activated, and chromosomal DNA was fragmented. (iv) Although caspase-3 was activated in cells infected with wild-type HSV-1 and exposed to sorbitol, cytochrome c outflow, depolarization of the inner mitochondrial membrane, and DNA fragmentation were blocked. We conclude that although d120 induces apoptosis by a caspase-3-independent pathway, the wild-type virus blocks apoptosis induced by this pathway and also blocks the caspase-dependent pathway induced by osmotic shock. The block in the caspase-dependent pathway may occur downstream of caspase-3 activation.

Staphylococcus epidermidis, a Gram-positive, coagulase-negative bacterium is a predominant inhabitant of human skin and mucous membranes. Recently, however, it has become one of the most important agents of hospital-acquired bacteriemia,... more

Staphylococcus epidermidis, a Gram-positive, coagulase-negative bacterium is a predominant inhabitant of human skin and mucous membranes. Recently, however, it has become one of the most important agents of hospital-acquired bacteriemia, as it has been found to be responsible for surgical wound infections developed in individuals with indwelling catheters or prosthetic devices, as well as in immunosupressed or neutropenic patients. Despite their medical significance, little is known about proteolytic enzymes of S. epidermidis and their possible contribution to the bacterium's pathogenicity; however, it is likely that they function as virulence factors in a manner similar to that proposed for the proteases of Staphylococcus aureus. Here we describe the purification of a cell wall-associated cysteine protease from S. epidermidis, its biochemical properties and specificity. A homology search using N-terminal sequence data revealed similarity to staphopain A (ScpA) and staphopain B (SspB), cysteine proteases from S. aureus. Moreover, the gene encoding S. epidermidis cysteine protease (Ecp) and a downstream gene coding for a putative inhibitor of the protease form an operon structure which resembles that of staphopain A in S. aureus. The active cysteine protease was detected on the bacterial cell surface as well as in the culture media and is apparently produced in a growth phase-dependent manner, with initial expression occurring in the mid-logarithmic phase. This enzyme, with elastinolytic properties, as well as the ability to cleave a 1 PI, fibrinogen and fibronectin, may possibly contribute to the invasiveness and pathogenic potential of S. epidermidis.

Transaldolase (Tal) is involved in the central carbon metabolism, i.e. the non-oxidative pentose phosphate pathway, and is therefore a ubiquitous enzyme. However, Tals show a low degree in sequence identity and vary in length within the... more

Transaldolase (Tal) is involved in the central carbon metabolism, i.e. the non-oxidative pentose phosphate pathway, and is therefore a ubiquitous enzyme. However, Tals show a low degree in sequence identity and vary in length within the enzyme family which previously led to the definition of five subfamilies. We wondered how this variation is conserved in structure and function. To answer this question we characterised and compared the Tals from Bacillus subtilis, Corynebacterium glutamicum and Escherichia coli, each belonging to a different subfamily, with respect to their biochemical properties and structures. The overall structure of the Tal domain, a (b ⁄ a) 8 -barrel fold, is well conserved between the different subfamilies but the enzymes show different degrees of oligomerisation (monomer, dimer and decamer). The substrate specificity of the three enzymes investigated is quite similar which is reflected in the conservation of the active site, the phosphate binding site as well as the position of a catalytically important water molecule. All decameric enzymes characterised so far appear to be heat stable no matter whether they originate from a mesophilic or thermophilic organism. Hence, the thermostability might be due to the structural properties, i.e. tight packing, of these enzymes.

GDP-4-keto-6-deoxy-D-mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP-L-fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte... more

GDP-4-keto-6-deoxy-D-mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP-L-fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte Lewis system and cell-surface antigens in pathogenic bacteria, depend on the availability of GDP-L-fucose for their expression. Therefore, the enzyme is a potential target for therapy in pathological states depending on selectin-mediated cell-to-cell interactions. Previous crystallographic investigations have shown that GDP-4-keto-6-deoxy-Dmannose epimerase/reductase belongs to the short-chain dehydrogenase/reductase protein homology family. The enzyme active-site region is at the interface of an N-terminal NADPH-binding domain and a C-terminal domain, held to bind the substrate. The design, expression and functional characterization of seven site-speci®c mutant forms of GDP-4-keto-6-deoxy-D-mannose epimerase/reductase are reported here. In parallel, the crystal structures of the native holoenzyme and of three mutants (Ser107Ala, Tyr136Glu and Lys140Arg) have been investigated and re®ned at 1.45-1.60 A Ê resolution, based on synchrotron data (R-factors range between 12.6 % and 13.9 %). The re®ned protein models show that besides the active-site residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic activity and may affect the coenzyme binding mode, side-chains capable of proton exchange, located around the expected substrate (GDP-4-keto-6-deoxy-D-mannose) binding pocket, are selectively required during the epimerization and reduction steps. Among these, Cys109 and His179 may play a primary role in proton exchange between the enzyme and the epimerization catalytic intermediates. Finally, the additional role of mutated active-site residues involved in substrate recognition and in enzyme stability has been analyzed.

Aims: The present investigation deals with the development of thermotolerant mutant strain of yeast for studying enhanced productivity of ethanol from molasses in a fully controlled bioreactor. Methods and Results: The parental culture of... more

Aims: The present investigation deals with the development of thermotolerant mutant strain of yeast for studying enhanced productivity of ethanol from molasses in a fully controlled bioreactor. Methods and Results: The parental culture of Saccharomyces cerevisiae ATCC 26602 was mutated using UV treatment. A single thermotolerant mutant was isolated after extensive screening and optimization, and grown on molasses medium in liquid cultures. The mutant was 1AE45-fold improved than its wild parent with respect to ethanol productivity (7AE2 g l )1 h )1 ), product yield (0AE44 g ethanol g )1 substrate utilized) and specific ethanol yield (19AE0 g ethanol g )1 cells). The improved ethanol productivity was directly correlated with titres of intracellular and extracellular invertase activities. The mutant supported higher volumetric and product yield of ethanol, significantly (P £ 0AE05) higher than the parental and other strains. The mutated cultures produced 1AE8-and 2AE6-fold more extracellular and intracellular invertase productivity, respectively, than that produced by its wild parent at 40°C. Thermodynamic studies revealed that the cell system exerted protection against thermal inactivation during formation of products. Conclusions: A mutant derivative of Sacchromyces cerevisiae with improved productivity of ethanol and invertases has been obtained, which showed concomitant improvement in thermostability of endogenous metabolism for formation of both ethanol and invertases. Significance and Impact of the Study: The results of the present study are of commercial value as the mutant can be used for ethanol production in parts of Pakistan where the temperature may go up to 40°C in April. Ethanol product yield coefficient and volumetric productivity, revealed the hyper-productivity of ethanol from molasses at 40°C, which is not appropriate for wild organism.

The importance of carbohydrates in a variety of biological functions is the reason that interest has recently increased in these compounds as possible components of therapeutic agents. Thus, the need for a technique allowing the easy... more

The importance of carbohydrates in a variety of biological functions is the reason that interest has recently increased in these compounds as possible components of therapeutic agents. Thus, the need for a technique allowing the easy synthesis of carbohydrates and glucoconjugates is an emerging challenge for chemists and biologists involved in this field. At present, enzymatic synthesis has resulted in the most promising approach for the production of complex oligosaccharides. In this respect, the enzymological characteristics of the catalysts, in term of regioselectivity, substrate specificity, and operational stability, are of fundamental importance to improve the yields of the process and to widen the repertoire of the available products. Here, two methods of oligosaccharide synthesis performed by a glycosynthase and by an α-xylosidase from the hyperthermophilic archaeon Sulfolobus solfataricus are briefly reviewed. The approaches used and the biodiversity of the catalysts together are key features for their possible utilization in the synthesis of oligosaccharides.

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. Oxidants produce modifications to proteins leading to loss of function (or gain of undesirable function) and very often to an enhanced... more

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. Oxidants produce modifications to proteins leading to loss of function (or gain of undesirable function) and very often to an enhanced degradation of the oxidized proteins. For several years it has been known that the proteasome is involved in the degradation of oxidized proteins. This review summarizes our knowledge about the recognition of oxidized protein substrates by the proteasome in in vitro systems and its applicability to living cells. The majority of studies in the field agree that the degradation of mildly oxidized proteins is an important function of the proteasomal system. The major recognition motif of the substrates seems to be hydrophobic surface patches that are recognized by the 20S ÔcoreÕ proteasome. Such hydrophobic surface patches are formed by partial unfolding and exposure of hydrophobic amino acid residues during oxidation. Oxidized proteins appear to be relatively poor substrates for ubiquitination, and the ubiquitination system does not seem to be involved in the recognition or targeting of oxidized proteins. Heavily oxidized proteins appear to first aggregate (new hydrophobic and ionic bonds) and then to form covalent cross-links that make them highly resistant to proteolysis. The inability to degrade extensively oxidized proteins may contribute to the accumulation of protein aggregates during diseases and the aging process.

We found a tumor me&stasis-associa ted heparan sulfate (HS)degrading endoglycosidase in melanoma cells that is a unique endo-&ghzuronidase (hepamnam ) capable of speciftcally cleaving HS at intrachain sites (M. Nakajima, T. Irimura, N.... more

We found a tumor me&stasis-associa ted heparan sulfate (HS)degrading endoglycosidase in melanoma cells that is a unique endo-&ghzuronidase (hepamnam ) capable of speciftcally cleaving HS at intrachain sites (M. Nakajima, T. Irimura, N. DiFerrame, and G. L. Nicolson, 1984, J. Biof. Chem. 259.2283-2290. To perform rapid and microscale quantitative assays of heparanase we developed a solid-phase HS substrate by crosslinking radiolabeled HS onto agarose gel beads using one covalent linkage. The HS from bovine lung was partially Ndesulfated and labeled with ["C]acetic anhydride. Free HS amino groups were completely acetylated, and reducing terminal saccharides were reductively aminated. The HS derivatives with amino groups at their reducing termini were coupled to amino-reactive agarose beads Incubation of the solid-phase HS substrates with B 16 melanoma cell extracts in the presence of ~saccharic acid 1 +lactone (a potent exo-j3glucuronidase inhibitor) resulted in the time-and dosedependent release of [%]HS fragments.

Substrate reduction therapy (SRT) is considered to be a potential therapeutic option for juvenile GM2 gangliosidosis (jGM2g). We evaluated the efficacy of SRT in jGM2g, assessing neurological, neuropsychological and brain magnetic... more

Substrate reduction therapy (SRT) is considered to be a potential therapeutic option for juvenile GM2 gangliosidosis (jGM2g). We evaluated the efficacy of SRT in jGM2g, assessing neurological, neuropsychological and brain magnetic resonance imaging (MRI) outcomes over a 24-month period of treatment. In an open-label and single-center study, five jGM2g patients (mean age 14.6+/-4.5 years) received oral miglustat at doses of 100-200mg t.i.d. adjusted to body surface area. Patients underwent general and neurological examinations, neuropsychological, electrophysiological, and brain MRI studies. All patients showed neurological deterioration over the period of the study, with particularly notable worsening of gait, speech and coordination. One patient experienced acute psychosis, and another showed worsening of pre-existing epilepsy. Some neuropsychological tests showed no evidence of deterioration in the three patients with high enough cognitive functioning for reliable assessment. Profound cognitive impairment in two children precluded neuropsychological evaluation. In four patients, evaluation of brain MRI showed no changes in white matter signal abnormalities and cerebellar atrophy noted at baseline, while one patient showed progression of cerebellar and supratentorial brain atrophy. Transmission electron microscopy analysis of peripheral mononuclear cells showed reduction of intracytoplasmatic inclusions with treatment. SRT with miglustat of patients with jGM2g failed to ameliorate progressive neurological deterioration, but apparently no worsening of some areas of cognitive function tested and brain MRI lesions was noted over 24 months of treatment. The results must be interpreted with care owing to the small sample of patients and the lack of a control-arm.

Thyroid hormones (THs) are transported across cell membranes by different transmembrane transporter proteins. In previous studies, we showed marked 3,3′-diiodothyronine (3,3′-T2) but moderate T3 uptake by the L-type amino acid transporter... more

Thyroid hormones (THs) are transported across cell membranes by different transmembrane transporter proteins. In previous studies, we showed marked 3,3′-diiodothyronine (3,3′-T2) but moderate T3 uptake by the L-type amino acid transporter 2 (Lat2). We have now studied the structure-function relationships of this transporter and TH-like molecules. Our Lat2 homology model is based on 2 crystal structures of the homologous 12-transmembrane helix transporters arginine/agmatine antiporter and amino acid/polyamine/organocation transporter. Model-driven mutagenesis of residues lining an extracellular recognition site and a TH-traversing channel identified 9 sensitive residues. Using Xenopus laevis oocytes as expression system, we found that side chain shortening (N51S, N133S, N248S, and Y130A) expanded the channel and increased 3,3′-T2 transport. Side chain enlargements (T140F, Y130R, and I137M) decreased 3,3′-T2 uptake, indicating channel obstructions. The opposite results with mutations ...

Invertase and urease are enzyme entities highly associated with the cells of the astaxanthin-producer yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) during any stage of its cell growth cycle. In this study cellobiose was a more... more

Invertase and urease are enzyme entities highly associated with the cells of the astaxanthin-producer yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) during any stage of its cell growth cycle. In this study cellobiose was a more efficient carbon source ...

Reversible protein phosphorylation is a prevalent signaling mechanism which modulates cellular metabolism in response to changing environmental conditions. In this study, we focus on previously uncharacterized Mycobacterium tuberculosis... more

Reversible protein phosphorylation is a prevalent signaling mechanism which modulates cellular metabolism in response to changing environmental conditions. In this study, we focus on previously uncharacterized Mycobacterium tuberculosis Ser/ Thr protein kinase (STPK) PknJ, a putative transmembrane protein. PknJ is shown to possess autophosphorylation activity and is also found to be capable of carrying out phosphorylation on the artificial substrate myelin basic protein (MyBP). Previous studies have shown that the autophosphorylation activity of M. tuberculosis STPKs is dependent on the conserved residues in the activation loop. However, our results show that apart from the conventional conserved residues, additional residues in the activation loop may also play a crucial role in kinase activation. Further characterization of PknJ reveals that the kinase utilizes unusual ions (Ni 2+ , Co 2+ ) as cofactors, thus hinting at a novel mechanism for PknJ activation. Additionally, as shown for other STPKs, we observe that PknJ possesses the capability to dimerize. In order to elucidate the signal transduction cascade emanating from PknJ, the M. tuberculosis membrane-associated protein fraction is treated with the active kinase and glycolytic enzyme Pyruvate kinase A (mtPykA) is identified as one of the potential substrates of PknJ. The phospholabel is found to be localized on serine and threonine residue(s), with Ser 37 identified as one of the sites of phosphorylation. Since Pyk is known to catalyze the last step of glycolysis, our study shows that the fundamental pathways such as glycolysis can also be governed by STPK-mediated signaling.

Abbreviations: FUS/TLS: fusion involved in t(12:16) in malignant liposarcoma / translocation in liposarcoma; ; LC-MS/MS: liquid chromatography tandem mass spectrometry; OTUB1: otubain 1; OTUB2: otubain 2; Rack 1: receptor for activated... more

Abbreviations: FUS/TLS: fusion involved in t(12:16) in malignant liposarcoma / translocation in liposarcoma; ; LC-MS/MS: liquid chromatography tandem mass spectrometry; OTUB1: otubain 1; OTUB2: otubain 2; Rack 1: receptor for activated kinase 1; Ub: ubiquitin Word counts: Total: 8121; Abstract: 191 1 Abstract

Previously, the cDNA and the respective gene for a presumed tauropine dehydrogenase (TaDH) from Suberites domuncula (GenBank accession nos. AM712888, AM712889) had been annotated. The conclusion that the sequences encode a TaDH had been... more

Previously, the cDNA and the respective gene for a presumed tauropine dehydrogenase (TaDH) from Suberites domuncula (GenBank accession nos. AM712888, AM712889) had been annotated. The conclusion that the sequences encode a TaDH had been inferred from the 68% identity with the TaDH protein from the marine demosponge Halichondria japonica. However, subsequent enzymatic assays shown here indicate that the presumed S. domuncula opine dehydrogenase is in fact a strombine dehydrogenase (StDH). The enzyme StDH is highly specific for glycine and is inhibited by an excess of the substrate pyruvate. Besides kinetic data, we report in this study also on the predicted tertiary and quaternary structure of the sponge StDH. It is concluded that the dimer (75 kDa) has a novel structure, distinguishing it from other known marine invertebrate OpDHs that exist as monomers.

Lubrication of mammalian joints is mediated by lubricin, a product of megakaryocyte stimulating factor gene (MSF; GenBank accession #U70136) expression. Lubricin (M(r) approximately 240 kDa) is a mucinous glycoprotein which is 50% (w/w)... more

Lubrication of mammalian joints is mediated by lubricin, a product of megakaryocyte stimulating factor gene (MSF; GenBank accession #U70136) expression. Lubricin (M(r) approximately 240 kDa) is a mucinous glycoprotein which is 50% (w/w) post-translationally modified with beta(1-3)Gal-GalNAc incompletely capped with NeuAc, and lubricates apposed cartilaginous surfaces in the boundary mode through an unknown mechanism. Both bovine and human lubricin were purified from synovial fluid and digested with recombinant glycosidases. Released oligosaccharides were identified and quantified by fluorophore assisted carbohydrate electrophoresis (FACE). Corresponding digests of human lubricin were also assayed in a friction apparatus oscillating latex rubber against polished glass at a pressure of 0.35 x 10(6) N/m(2) and the coefficient of friction (mu) was measured. Digestion with alpha2,3-neuraminidase decreased lubricating ability by 19.3%. Partial removal of beta(1-3)Gal-GalNAc moieties by en...

Background and purpose: d-Aminolevulinic acid (ALA) is used in cancer patients for photodynamic diagnosis or therapy. Oral administration of ALA has been used in patients with prostate and bladder cancer. The present aim was to... more

Background and purpose: d-Aminolevulinic acid (ALA) is used in cancer patients for photodynamic diagnosis or therapy. Oral administration of ALA has been used in patients with prostate and bladder cancer. The present aim was to investigate the mechanism of intestinal absorption of ALA and its transport via the amino acid transporter SLC36A1. Experimental approach: In vitro investigations of ALA affinity for and uptake via SLC36A1 and SLC15A1 were performed in Caco-2 cell monolayers. Interaction of ALA with SLC15A1 was investigated in MDCK/SLC15A1 cells, whereas interactions with SLC36A1 were investigated in COS-7 cells transiently expressing SLC36A1. Key results: ALA inhibited SLC36A1-mediated L-[ 3 H]Pro and SLC15A1-mediated [ 14 C]Gly-Sar uptake in Caco-2 cell monolayers with IC50 values of 11.3 and 2.1 mM respectively. In SLC36A1-expressing COS-7 cells, the uptake of [ 14 C]ALA was saturable with a Km value of 6.8 Ϯ 3.0 mM and a Vmax of 96 Ϯ 13 pmol·cm -2 ·min -1 . Uptake of [ 14 C]ALA was pH and concentration dependent, and could be inhibited by glycine, proline and GABA. In a membrane potential assay, translocation of ALA via SLC36A1 was concentration dependent, with a Km value of 3.8 Ϯ 1.0 mM. ALA is thus a substrate for

A number of C4--C9 aliphatic ketones are acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitors, with Ki values in the 0.7-5 mM range. Comparison to analogous substrates would suggest that these ketones are transition state... more

A number of C4--C9 aliphatic ketones are acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitors, with Ki values in the 0.7-5 mM range. Comparison to analogous substrates would suggest that these ketones are transition state analogs; e.g. 2-pentanone binds to the enzyme approx. 550 times more tightly than ethylacetate. However, a number of other criteria contradict this conclusions: (1) the binding is insensitive to ketone structure: isomeric ketones, cycloalkanones, and sterically hindered ketones have similar inhibitory potencies.

Trichomonas vaginalis, Ecto-ATPase, Ecto-phosphatase, Galactose This work describes the ability of living Trichomonas vaginalis to hydrolyze extracellular ATP (164.0 ð 13.9 nmol Pi / h ¥ 10 7 cells). This ecto-enzyme was stimulated by... more

Trichomonas vaginalis, Ecto-ATPase, Ecto-phosphatase, Galactose This work describes the ability of living Trichomonas vaginalis to hydrolyze extracellular ATP (164.0 ð 13.9 nmol Pi / h ¥ 10 7 cells). This ecto-enzyme was stimulated by ZnCl 2 , CaCl 2 and MgCl 2 , was insensitive to several ATPase and phosphatase inhibitors and was able to hydrolyze several nucleotides besides ATP. The activity was linear with cell density and with time for at least 60 min. The optimum pH for the T. vaginalis ecto-ATPase lies in the alkaline range. d-galactose, known to be involved in adhesion of T. vaginalis to host cells, stimulated this enzyme by more than 90%. A comparison between two strains of T. vaginalis showed that the ecto-ATPase activity of a fresh isolate was twice as much as that of a strain axenically maintained in culture, through daily passages, for several years. The results suggest a possible role for this ecto-ATPase in adhesion of T. vaginalis to host cells and in its pathogenicity.

A continuous spectrophotometric assay has been developed for detecting beta-glucuronidase activity. In the assay, Para-nitrophenyl beta-D-glucuronide is cleaved to yield a chromophoric product. With the commercial E. coli enzyme, it is... more

A continuous spectrophotometric assay has been developed for detecting beta-glucuronidase activity. In the assay, Para-nitrophenyl beta-D-glucuronide is cleaved to yield a chromophoric product. With the commercial E. coli enzyme, it is demonstrated that the reactions can be continuously monitored by the increase of absorbance at 405 nm. The method is highly sensitive and able to detect less than 1.4 x 10(-4) U/mL of the enzyme activity in solution. Such a new assay offers significant advantages over the existing discontinuous methods and should be useful for both routine enzyme assay and accurate kinetic studies.

In this paper we describe the cloning and optimization of a nitrilase for a regio-and stereo-specific synthesis of (3S)-3-cyano-5-methyl hexanoic acid (2) from isobutylsuccinonitrile (IBSN, 1). Ten representative plant and bacterial... more

In this paper we describe the cloning and optimization of a nitrilase for a regio-and stereo-specific synthesis of (3S)-3-cyano-5-methyl hexanoic acid (2) from isobutylsuccinonitrile (IBSN, 1). Ten representative plant and bacterial nitrilases have been cloned and their substrate specificity was studied using a fluorescent assay. The desired nitrilase AtNit1 from Arabidopsis thaliana was identified with high enantioselectivity (E > 150). This enzyme was then purified and characterized to be an oligomer of 12 subunits by size exclusion chromatography. AtNit1 was subsequently optimized to increase expression and engineered to improve activity. Preliminary screening of a small percentage (1%) of the mutant library shows that the mutant C236S has a nearly 3-fold increase in reactivity in the hydrolysis of IBSN. (J. Tao). NH 3 byproduct. The desired nitrilase AtNit1 from Arabidopsis thaliana was then optimized to increase expression, purified for physical characteirzation and engineered to improve activity.

Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl group(s) from polyphenolic acetates (PAs) to functional proteins, such as Glutathione S-transferase (GST), NADPH Cytochrome c reductase and Nitric Oxide Synthase (NOS)... more

Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl group(s) from polyphenolic acetates (PAs) to functional proteins, such as Glutathione S-transferase (GST), NADPH Cytochrome c reductase and Nitric Oxide Synthase (NOS) resulting in the modulation of biological activities. A comparison of the specificities of the acetoxy derivatives of coumarins, biscoumarins, chromones, flavones, isoflavones and xanthones has been carried out earlier by us with an aim to study the effect of nature and position of the acetoxy groups on the benzenoid ring and the position of the carbonyl group with respect to oxygen/ nitrogen heteroatom for the catalytic activity of CRTAase. In this communication for the first time, we have studied the influence of differently substituted benzofurans on the CRTAase activity to study the effect of the replacement of pyran ring of coumarin with furan ring, presence of carbonyl at C-3, substitution of C-3 carbonyl group with acetoxy group and presence of various substituents (OAc/OH/Cl) on the benzenoid ring. It was observed that acetoxy derivatives of benzofurans lead to inhibition of ADP induced platelet aggregation by the activation of platelet Nitric Oxide Synthase catalyzed by CRTAase. Accordingly, the formation of NO in platelets by 3-oxo-2,3-dihydrobenzofuran-6,7-diyl diacetate (3a) was found to be comparable with that of model polyphenolic acetate (PA), 7,8-diacetoxy-4-methylcoumarin (DAMC).