Mohinder Sardana - Academia.edu (original) (raw)
Papers by Mohinder Sardana
Journal of Biological Chemistry, May 1, 1990
In vitro hydroxylation of aspartic acid has recently been demonstrated in a synthetic peptide bas... more In vitro hydroxylation of aspartic acid has recently been demonstrated in a synthetic peptide based on the structure of the first epidermal growth factor domain in human factor IX (Gronke, R. S., VanDusen, W. J., Garsky, V. M., Jacobs, J. W., Sardana, M. K., Stern, A. M., and Friedman, P. A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3609-3613). The putative enzyme responsible for the posttranslational modification, aspartyl beta-hydroxylase, has been shown to be a member of a class of 2-ketoglutarate-dependent dioxygenases, which include prolyl-4- and lysyl-hydroxylases. In the present study, we describe the solubilization with nonionic detergent of the enzyme from bovine liver microsomes and its purification using DEAE-cellulose followed by heparin-Sepharose. No additional detergent was required during purification. The partially purified enzyme preparation was found to contain no prolyl-4- or lysyl-hydroxylase activity. Using a synthetic peptide based on the structure of the epidermal growth factor-like region in human factor X as substrate, the apparent Km values for iron and alpha-ketoglutarate were 3 and 5 microM, respectively. The enzyme hydroxylated the factor X peptide with the same stereospecificity (erythro beta-hydroxyaspartic acid) and occurred only at the aspartate corresponding to the position seen in vivo. Furthermore, the extent to which either peptide (factor IX or X) was hydroxylated reflected the extent of hydroxylation observed for both human plasma factors IX and X.
Biochemical Pharmacology, Feb 1, 1986
ABSTRACT
Proceedings of the National Academy of Sciences of the United States of America, Apr 1, 1987
Tin(IV)-protoporphyrin (Sn-protoporphyrin) potently inhibits heme degradation to bile pigments in... more Tin(IV)-protoporphyrin (Sn-protoporphyrin) potently inhibits heme degradation to bile pigments in vitro and in vivo, a property that confers upon this synthetic compound the ability to suppress a variety of experimentally
![Research paper thumbnail of [1] Purification of viral polymerases: General considerations](https://attachments.academia-assets.com/112018799/thumbnails/1.jpg)
](Methods in Enzymology, 1996
Biochemical journal. Cellular aspects, May 15, 1977
By using 3-amino-1,2,4-triazole, an inhibitor of haem synthesis, and 2-allyl-2-isopropylacetamide... more By using 3-amino-1,2,4-triazole, an inhibitor of haem synthesis, and 2-allyl-2-isopropylacetamide, a drug that degrades the haem moiety of cytochrome P-450, the involvement of haem in cytochrome P-450 synthesis and assembly was investigated. Phenobarbital was used to stimulate apo-(cytochrome P-450) synthesis. Degradation of preformed cytochrome P-450 haem does not result in a concomitant release of the apoprotein from the endoplasmic reticulum. The availability of haem for cytochrome P-450 synthesis in the normal animal is not rate-limiting. Prolonged inhibition of haem synthesis in vivo decreases the rate of apo-(cytochrome P-450) synthesis, although this effect is not discernible under conditions of short-term inhibition of haem synthesis. Under the former conditions exogenous haemin is able to counteract the decrease in the rate of apoprotein synthesis. In animals receiving successive injections of phenobarbital plus 3-amino-1 ,2,4-triazole, compared with those receiving phenobarbital only, the holo-(cytochrome P-450) content measured spectrally shows a greater decrease than could be accounted for by the decrease in the content of the total apoprotein. In addition to less haem being available under these conditions, the free apoprotein appears to have undergone some modification, such that its haem-binding capacity is considerably decreased. This particular effect could be due to a direct interaction of 3-amino-1,2,4-triazole or its meta
Biochemical journal. Cellular aspects, Aug 15, 1976
A single administration of 2-allyl-2-isopropylacetamide, a porphyrinogenic drug, enhanced the 32P... more A single administration of 2-allyl-2-isopropylacetamide, a porphyrinogenic drug, enhanced the 32P-labelling of nucleoplasmic as well as cytoplasmic poly(A)-containing RNA in rat liver. The synthesis of total microsomal RNA is only marginally increased under these conditions. The drug enhances the labelling of a variety of cytoplasmic poly(A)-containing RNA species, and this effect is counteracted by the simultaneous administration of haemin. 2-Allyl-2-isopropylacetamide also enhanced the release of RNA from the nucleus to the cytoplasm. 2-Allyl-2-isopropylacetamide is a powerful inducer of 5-aminolaevulinate synthetase, the first and ratelimiting enzyme of the haem-biosynthetic pathway (Granick, 1966). Haem has been implicated as a regulator of this enzyme induction (Granick, 1966; De Matteis 1971; Padmanaban et at., 1973). On the basis of susceptibility of the induction process to inhibitors of nucleic acid synthesis, it has been suggested that RNA synthesis mediates the effects observed (Granick, 1966; Sassa & Granick, 1970). It has been shown that 2-allyl-2-isopropylacetamide increases nucleoplasmic RNA synthesis in rat liver (Sardana et al., 1975). Nawata & Kato (1973) have reported that 3,5-diethoxycarbonyl-1,4-dihydrocollidine, another potent inducer of 5-aminolaevulinate synthetase, increases total nuclear RNA synthesis in rat liver. Incefy etal. (1974) have reported the increase in nucleoplasmic RNA polymerase activity in chickembryo liver cell cultures exposed to 2-allyl-2isopropylacetamide. The present studies reveal that 2-allyl-2-isopropylacetamide enhances the labelling of cytoplasmic poly(A)-containing RNA associated with microsonlal/polyribosomal fractions by activating the transcription process as well as the transport of the RNA from the nucleus to the cytoplasm. On the basis of the fact that most eukaryotic mRNA has poly(A) at the 3'-end (Brawerman, 1974), it is concluded that 2-allyl-2-isopropylacetamide increases the amounts of possibly several mRNA species and that this early effect of 2-allyl-2-isopropylacetamide is counteracted by haemin. Materials and Methods Male abino rats (1 10-120g) of the Indian Institute of Science strain were used in these experiments. 32P (carrier-free) was purchased from the Bhabha Atomic Research Centre, Trombay, India. 2-Allyl-Vol. 158 2-isopropylacetamide was generously supplied by Hoffmann
Journal of Biological Chemistry, Apr 1, 1992
Human papillomaviruses (HPVs) are the etiologic agents responsible for genital warts and are cont... more Human papillomaviruses (HPVs) are the etiologic agents responsible for genital warts and are contributing factors in the pathogenesis of human cervical cancer. The HPV E7 gene is transcriptionally active in these diseases and has been shown to transform mammalian cells in vitro. We have expressed and purified the HPV-16 E7 gene product in Escherichia coli. The isolated E7 protein contains zinc in a 1:l molar ratio. X-ray absorption fine structure studies demonstrated that the zinc is coordinated by 4 sulfur ligands. We sequentially derivatized the E7 cysteines to differentiate between solvent-exposed, metal-bound, and disulfide-associated cysteines. Our results demonstrate that CysZ4 and Cyses are accessible to solvent, while cysteines in the two conserved Cys-X-X-Cys motifs are likely involved in binding zinc. We observed no evidence for the existence of disulfide bonds in recombinant E7 protein under the conditions tested. Nearly half of the known human papilloma viruses (HPVs)' can infect the genital mucosa and produce benign epithelial lesions. Epidemiological and molecular genetic studies have demonstrated that only a few of the HPV isolates that infect the genital mucosa are associated with most (75-100%) cases of cervical cancer (1). In these cases two viral genes, E6 and E7, are transcriptionally active (2). The most common HPV strain associated with cervical neoplasia is HPV-16. The HPV-16 E6 and E7 genes together have been shown to be necessary and sufficient for transformation of primary human keratinocytes (3). The HPV-16 E7 gene is able to transform established rodent fibroblasts (4), and, in conjunction with ras, primary rat epithelial cells (5). The HPV-16 E7 gene encodes a protein of 98 amino acids (6) which shares a region of homology (amino acids 2-35) with the adenovirus E1A protein and simian virus-40 large T antigen. E7, like ElA, can transactivate transcription from ~~ ~ * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Biochimica Et Biophysica Acta - General Subjects, Sep 4, 1981
1. Biochim Biophys Acta. 1981 Sep 4;676(3):289-99. Porphyrogenic effects and induction of heme ox... more 1. Biochim Biophys Acta. 1981 Sep 4;676(3):289-99. Porphyrogenic effects and induction of heme oxygenase in vivo by delta-aminolevulinic acid. Anderson KE, Drummond GS, Freddara U, Sardana MK, Sassa S. PMID: 6895184 [PubMed - indexed for MEDLINE]. ...
Journal of Biological Chemistry, Jul 1, 1994
Journal of Biological Chemistry, Feb 1, 1998
Hepatology, Dec 1, 1991
delta-Aminolevulinic acid-4-[14C] and [3H]-bilirubin were administered intravenously to five pati... more delta-Aminolevulinic acid-4-[14C] and [3H]-bilirubin were administered intravenously to five patients with Gilbert's syndrome and four healthy control subjects on two occasions: before and on days 10 through 14 of a course of phenobarbital (2.5 mg/kg/day). The resulting curves of [3H]-bilirubin and [14C]-bilirubin in plasma were analyzed by computer to determine a number of parameters of physiological interest. As expected, phenobarbital produced a highly significant fall in the plasma concentration of unconjugated bilirubin as a result of a significant increase in hepatic bilirubin clearance in all subjects; plasma bilirubin turnover was unaltered. Surprisingly, the drug produced no change in the incorporation of [14C]-delta-aminolevulinic acid into [14C]-early labeled bilirubin. To explain this unexpected finding, the effects of phenobarbital (75 mg/kg/day for 6 days) on incorporation of [14C]-delta-aminolevulinic acid and 2-[14C]-glycine into [14C]-early labeled bilirubin and on the activity of the enzyme delta-aminolevulinic acid synthase were studied in nonfasted, adult, male Sprague-Dawley rats. At the dose and duration of treatment used, phenobarbital administration increased total hepatic delta-aminolevulinic acid synthase activity and produced a significant increase of 70% in the incorporation of [14C]-glycine into early labeled bilirubin. By contrast, no increase in the incorporation of [14C]-delta-aminolevulinic acid into early labeled bilirubin was observed. These data suggest that delta-aminolevulinic acid is an inappropriate precursor for studies of the rate of heme biosynthesis, presumably because it bypasses delta-aminolevulinic acid synthase, the physiological rate-limiting enzyme in the heme biosynthetic pathway.
Journal of Biological Chemistry, Jul 1, 1991
Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of blood coagulation factor ... more Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D.E., Arcuri, K.E., and Vlasuk, G.P. (1990) Science 248, 593-596). The 60-amino acid sequence of TAP shows limited homology to Kunitz-type inhibitors, including cysteines at positions 5, 15, 33, 39, 55, and 59. For detailed biochemical and pharmacological studies, a recombinant version of TAP (rTAP) has been produced in yeast. To determine the arrangement of the disulfide bonds, rTAP was cleaved with trypsin and chymotrypsin and the purified peptides sequenced using a gas-phase sequenator. The positions of the disulfide bonds were assigned by identifying the cycle(s) at which di-phenylthiohydan-toin-cystine was released. The specific disulfide bridges, Cys-5 to Cys-59, Cys-15 to Cys-39, and Cys-33 to Cys-55, are analogous to those in the prototype Kunitz-type inhibitor, bovine pancreatic trypsin inhibitor (BPTI). While treatment of BPTI with dithiothreitol rapidly and specifically reduced one disulfide bond, the reduction of disulfide bonds in rTAP proceeded at a slower rate and appeared to be nonspecific, reaching a maximum of two disulfides reduced. Reduced rTAP derivatized with either iodoacetic acid or iodoacetamide lost 59% of its inhibitory activity. In contrast, BPTI alkylated with iodoacetic acid inhibited trypsin half as well as the iodoacetamide derivative. Although the arrangement of disulfides in the two inhibitors is the same, their susceptibility to reduction is markedly different.
Journal of Medicinal Chemistry, Oct 31, 2001
Proceedings of the National Academy of Sciences of the United States of America, May 9, 2000
γ-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amy... more γ-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two Aβ peptide isoforms, Aβ40 and Aβ42. Here we report the detergent solubilization and partial characterization of γ-secretase. The activity of solubilized γ-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the β-secretase cleavage site. Cleavage of C100Flag by γ-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Aβ40 or Aβ42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Aβ40 and Aβ42 termini. Recovery of catalytically competent, soluble γ-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized γ-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Aβ40 and Aβ42 in mammalian cells. Upon gel exclusion chromatography, solubilized γ-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 × 106. Anti-PS1 antibody immunoprecipitates γ-secretase activity from the solubilized γ-secretase preparation. These data suggest that γ-secretase activity is catalyzed by a PS1-containing macromolecular complex.
Biochemical Journal, Apr 1, 1975
The porphyrogenic drug allylisopropylacetamide, a potent inducer of 5-aminolaevulinate synthetase... more The porphyrogenic drug allylisopropylacetamide, a potent inducer of 5-aminolaevulinate synthetase, specifically increases nucleoplasmic RNA synthesis in rat liver. The drugmediated increase in nucleoplasmic RNA synthesis is blocked by cycloheximide and haemin, which also inhibit the enzyme induction.
Archives of Biochemistry and Biophysics, Mar 1, 1993
S. Karger AG eBooks, Apr 16, 2015
Applied and Environmental Microbiology, 2000
A tripeptidase (PepT) from a thermophilic dairy starter strain of Lactobacillus helveticus was pu... more A tripeptidase (PepT) from a thermophilic dairy starter strain of Lactobacillus helveticus was purified by four chromatographic steps. PepT appeared to be a trimeric metallopeptidase with a molecular mass of 150 kDa. PepT exhibited maximum activity against hydrophobic tripeptides, with the highest activity for Met-Gly-Gly ( K m , 2.6 mM; V max , 80.2 μmol · min −1 · μg −1 ). Some of the hydrophobic dipeptides were slowly hydrolyzed, distinguishing the Lactobacillus PepT from its counterpart in mesophilic Lactococcus lactis . No activity against tetrapeptides or amino acid p -nitroanilide derivatives was observed. The pepT gene and its flanking regions were isolated by PCR and sequenced by cyclic sequencing. The sequence analyses revealed open reading frames (ORFs) 816 bp (ORF1) and 1,239 bp (ORF2) long. ORF2 encoded a 47-kDa PepT protein which exhibited 53% identity with the PepT from L. lactis . The mRNA analyses indicated that pepT conforms a novel operon structure with an ORF1 lo...
Journal of Biological Chemistry, May 1, 1990
In vitro hydroxylation of aspartic acid has recently been demonstrated in a synthetic peptide bas... more In vitro hydroxylation of aspartic acid has recently been demonstrated in a synthetic peptide based on the structure of the first epidermal growth factor domain in human factor IX (Gronke, R. S., VanDusen, W. J., Garsky, V. M., Jacobs, J. W., Sardana, M. K., Stern, A. M., and Friedman, P. A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3609-3613). The putative enzyme responsible for the posttranslational modification, aspartyl beta-hydroxylase, has been shown to be a member of a class of 2-ketoglutarate-dependent dioxygenases, which include prolyl-4- and lysyl-hydroxylases. In the present study, we describe the solubilization with nonionic detergent of the enzyme from bovine liver microsomes and its purification using DEAE-cellulose followed by heparin-Sepharose. No additional detergent was required during purification. The partially purified enzyme preparation was found to contain no prolyl-4- or lysyl-hydroxylase activity. Using a synthetic peptide based on the structure of the epidermal growth factor-like region in human factor X as substrate, the apparent Km values for iron and alpha-ketoglutarate were 3 and 5 microM, respectively. The enzyme hydroxylated the factor X peptide with the same stereospecificity (erythro beta-hydroxyaspartic acid) and occurred only at the aspartate corresponding to the position seen in vivo. Furthermore, the extent to which either peptide (factor IX or X) was hydroxylated reflected the extent of hydroxylation observed for both human plasma factors IX and X.
Biochemical Pharmacology, Feb 1, 1986
ABSTRACT
Proceedings of the National Academy of Sciences of the United States of America, Apr 1, 1987
Tin(IV)-protoporphyrin (Sn-protoporphyrin) potently inhibits heme degradation to bile pigments in... more Tin(IV)-protoporphyrin (Sn-protoporphyrin) potently inhibits heme degradation to bile pigments in vitro and in vivo, a property that confers upon this synthetic compound the ability to suppress a variety of experimentally
Methods in Enzymology, 1996
Biochemical journal. Cellular aspects, May 15, 1977
By using 3-amino-1,2,4-triazole, an inhibitor of haem synthesis, and 2-allyl-2-isopropylacetamide... more By using 3-amino-1,2,4-triazole, an inhibitor of haem synthesis, and 2-allyl-2-isopropylacetamide, a drug that degrades the haem moiety of cytochrome P-450, the involvement of haem in cytochrome P-450 synthesis and assembly was investigated. Phenobarbital was used to stimulate apo-(cytochrome P-450) synthesis. Degradation of preformed cytochrome P-450 haem does not result in a concomitant release of the apoprotein from the endoplasmic reticulum. The availability of haem for cytochrome P-450 synthesis in the normal animal is not rate-limiting. Prolonged inhibition of haem synthesis in vivo decreases the rate of apo-(cytochrome P-450) synthesis, although this effect is not discernible under conditions of short-term inhibition of haem synthesis. Under the former conditions exogenous haemin is able to counteract the decrease in the rate of apoprotein synthesis. In animals receiving successive injections of phenobarbital plus 3-amino-1 ,2,4-triazole, compared with those receiving phenobarbital only, the holo-(cytochrome P-450) content measured spectrally shows a greater decrease than could be accounted for by the decrease in the content of the total apoprotein. In addition to less haem being available under these conditions, the free apoprotein appears to have undergone some modification, such that its haem-binding capacity is considerably decreased. This particular effect could be due to a direct interaction of 3-amino-1,2,4-triazole or its meta
Biochemical journal. Cellular aspects, Aug 15, 1976
A single administration of 2-allyl-2-isopropylacetamide, a porphyrinogenic drug, enhanced the 32P... more A single administration of 2-allyl-2-isopropylacetamide, a porphyrinogenic drug, enhanced the 32P-labelling of nucleoplasmic as well as cytoplasmic poly(A)-containing RNA in rat liver. The synthesis of total microsomal RNA is only marginally increased under these conditions. The drug enhances the labelling of a variety of cytoplasmic poly(A)-containing RNA species, and this effect is counteracted by the simultaneous administration of haemin. 2-Allyl-2-isopropylacetamide also enhanced the release of RNA from the nucleus to the cytoplasm. 2-Allyl-2-isopropylacetamide is a powerful inducer of 5-aminolaevulinate synthetase, the first and ratelimiting enzyme of the haem-biosynthetic pathway (Granick, 1966). Haem has been implicated as a regulator of this enzyme induction (Granick, 1966; De Matteis 1971; Padmanaban et at., 1973). On the basis of susceptibility of the induction process to inhibitors of nucleic acid synthesis, it has been suggested that RNA synthesis mediates the effects observed (Granick, 1966; Sassa & Granick, 1970). It has been shown that 2-allyl-2-isopropylacetamide increases nucleoplasmic RNA synthesis in rat liver (Sardana et al., 1975). Nawata & Kato (1973) have reported that 3,5-diethoxycarbonyl-1,4-dihydrocollidine, another potent inducer of 5-aminolaevulinate synthetase, increases total nuclear RNA synthesis in rat liver. Incefy etal. (1974) have reported the increase in nucleoplasmic RNA polymerase activity in chickembryo liver cell cultures exposed to 2-allyl-2isopropylacetamide. The present studies reveal that 2-allyl-2-isopropylacetamide enhances the labelling of cytoplasmic poly(A)-containing RNA associated with microsonlal/polyribosomal fractions by activating the transcription process as well as the transport of the RNA from the nucleus to the cytoplasm. On the basis of the fact that most eukaryotic mRNA has poly(A) at the 3'-end (Brawerman, 1974), it is concluded that 2-allyl-2-isopropylacetamide increases the amounts of possibly several mRNA species and that this early effect of 2-allyl-2-isopropylacetamide is counteracted by haemin. Materials and Methods Male abino rats (1 10-120g) of the Indian Institute of Science strain were used in these experiments. 32P (carrier-free) was purchased from the Bhabha Atomic Research Centre, Trombay, India. 2-Allyl-Vol. 158 2-isopropylacetamide was generously supplied by Hoffmann
Journal of Biological Chemistry, Apr 1, 1992
Human papillomaviruses (HPVs) are the etiologic agents responsible for genital warts and are cont... more Human papillomaviruses (HPVs) are the etiologic agents responsible for genital warts and are contributing factors in the pathogenesis of human cervical cancer. The HPV E7 gene is transcriptionally active in these diseases and has been shown to transform mammalian cells in vitro. We have expressed and purified the HPV-16 E7 gene product in Escherichia coli. The isolated E7 protein contains zinc in a 1:l molar ratio. X-ray absorption fine structure studies demonstrated that the zinc is coordinated by 4 sulfur ligands. We sequentially derivatized the E7 cysteines to differentiate between solvent-exposed, metal-bound, and disulfide-associated cysteines. Our results demonstrate that CysZ4 and Cyses are accessible to solvent, while cysteines in the two conserved Cys-X-X-Cys motifs are likely involved in binding zinc. We observed no evidence for the existence of disulfide bonds in recombinant E7 protein under the conditions tested. Nearly half of the known human papilloma viruses (HPVs)' can infect the genital mucosa and produce benign epithelial lesions. Epidemiological and molecular genetic studies have demonstrated that only a few of the HPV isolates that infect the genital mucosa are associated with most (75-100%) cases of cervical cancer (1). In these cases two viral genes, E6 and E7, are transcriptionally active (2). The most common HPV strain associated with cervical neoplasia is HPV-16. The HPV-16 E6 and E7 genes together have been shown to be necessary and sufficient for transformation of primary human keratinocytes (3). The HPV-16 E7 gene is able to transform established rodent fibroblasts (4), and, in conjunction with ras, primary rat epithelial cells (5). The HPV-16 E7 gene encodes a protein of 98 amino acids (6) which shares a region of homology (amino acids 2-35) with the adenovirus E1A protein and simian virus-40 large T antigen. E7, like ElA, can transactivate transcription from ~~ ~ * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Biochimica Et Biophysica Acta - General Subjects, Sep 4, 1981
1. Biochim Biophys Acta. 1981 Sep 4;676(3):289-99. Porphyrogenic effects and induction of heme ox... more 1. Biochim Biophys Acta. 1981 Sep 4;676(3):289-99. Porphyrogenic effects and induction of heme oxygenase in vivo by delta-aminolevulinic acid. Anderson KE, Drummond GS, Freddara U, Sardana MK, Sassa S. PMID: 6895184 [PubMed - indexed for MEDLINE]. ...
Journal of Biological Chemistry, Jul 1, 1994
Journal of Biological Chemistry, Feb 1, 1998
Hepatology, Dec 1, 1991
delta-Aminolevulinic acid-4-[14C] and [3H]-bilirubin were administered intravenously to five pati... more delta-Aminolevulinic acid-4-[14C] and [3H]-bilirubin were administered intravenously to five patients with Gilbert's syndrome and four healthy control subjects on two occasions: before and on days 10 through 14 of a course of phenobarbital (2.5 mg/kg/day). The resulting curves of [3H]-bilirubin and [14C]-bilirubin in plasma were analyzed by computer to determine a number of parameters of physiological interest. As expected, phenobarbital produced a highly significant fall in the plasma concentration of unconjugated bilirubin as a result of a significant increase in hepatic bilirubin clearance in all subjects; plasma bilirubin turnover was unaltered. Surprisingly, the drug produced no change in the incorporation of [14C]-delta-aminolevulinic acid into [14C]-early labeled bilirubin. To explain this unexpected finding, the effects of phenobarbital (75 mg/kg/day for 6 days) on incorporation of [14C]-delta-aminolevulinic acid and 2-[14C]-glycine into [14C]-early labeled bilirubin and on the activity of the enzyme delta-aminolevulinic acid synthase were studied in nonfasted, adult, male Sprague-Dawley rats. At the dose and duration of treatment used, phenobarbital administration increased total hepatic delta-aminolevulinic acid synthase activity and produced a significant increase of 70% in the incorporation of [14C]-glycine into early labeled bilirubin. By contrast, no increase in the incorporation of [14C]-delta-aminolevulinic acid into early labeled bilirubin was observed. These data suggest that delta-aminolevulinic acid is an inappropriate precursor for studies of the rate of heme biosynthesis, presumably because it bypasses delta-aminolevulinic acid synthase, the physiological rate-limiting enzyme in the heme biosynthetic pathway.
Journal of Biological Chemistry, Jul 1, 1991
Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of blood coagulation factor ... more Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D.E., Arcuri, K.E., and Vlasuk, G.P. (1990) Science 248, 593-596). The 60-amino acid sequence of TAP shows limited homology to Kunitz-type inhibitors, including cysteines at positions 5, 15, 33, 39, 55, and 59. For detailed biochemical and pharmacological studies, a recombinant version of TAP (rTAP) has been produced in yeast. To determine the arrangement of the disulfide bonds, rTAP was cleaved with trypsin and chymotrypsin and the purified peptides sequenced using a gas-phase sequenator. The positions of the disulfide bonds were assigned by identifying the cycle(s) at which di-phenylthiohydan-toin-cystine was released. The specific disulfide bridges, Cys-5 to Cys-59, Cys-15 to Cys-39, and Cys-33 to Cys-55, are analogous to those in the prototype Kunitz-type inhibitor, bovine pancreatic trypsin inhibitor (BPTI). While treatment of BPTI with dithiothreitol rapidly and specifically reduced one disulfide bond, the reduction of disulfide bonds in rTAP proceeded at a slower rate and appeared to be nonspecific, reaching a maximum of two disulfides reduced. Reduced rTAP derivatized with either iodoacetic acid or iodoacetamide lost 59% of its inhibitory activity. In contrast, BPTI alkylated with iodoacetic acid inhibited trypsin half as well as the iodoacetamide derivative. Although the arrangement of disulfides in the two inhibitors is the same, their susceptibility to reduction is markedly different.
Journal of Medicinal Chemistry, Oct 31, 2001
Proceedings of the National Academy of Sciences of the United States of America, May 9, 2000
γ-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amy... more γ-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two Aβ peptide isoforms, Aβ40 and Aβ42. Here we report the detergent solubilization and partial characterization of γ-secretase. The activity of solubilized γ-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the β-secretase cleavage site. Cleavage of C100Flag by γ-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Aβ40 or Aβ42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Aβ40 and Aβ42 termini. Recovery of catalytically competent, soluble γ-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized γ-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Aβ40 and Aβ42 in mammalian cells. Upon gel exclusion chromatography, solubilized γ-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 × 106. Anti-PS1 antibody immunoprecipitates γ-secretase activity from the solubilized γ-secretase preparation. These data suggest that γ-secretase activity is catalyzed by a PS1-containing macromolecular complex.
Biochemical Journal, Apr 1, 1975
The porphyrogenic drug allylisopropylacetamide, a potent inducer of 5-aminolaevulinate synthetase... more The porphyrogenic drug allylisopropylacetamide, a potent inducer of 5-aminolaevulinate synthetase, specifically increases nucleoplasmic RNA synthesis in rat liver. The drugmediated increase in nucleoplasmic RNA synthesis is blocked by cycloheximide and haemin, which also inhibit the enzyme induction.
Archives of Biochemistry and Biophysics, Mar 1, 1993
S. Karger AG eBooks, Apr 16, 2015
Applied and Environmental Microbiology, 2000
A tripeptidase (PepT) from a thermophilic dairy starter strain of Lactobacillus helveticus was pu... more A tripeptidase (PepT) from a thermophilic dairy starter strain of Lactobacillus helveticus was purified by four chromatographic steps. PepT appeared to be a trimeric metallopeptidase with a molecular mass of 150 kDa. PepT exhibited maximum activity against hydrophobic tripeptides, with the highest activity for Met-Gly-Gly ( K m , 2.6 mM; V max , 80.2 μmol · min −1 · μg −1 ). Some of the hydrophobic dipeptides were slowly hydrolyzed, distinguishing the Lactobacillus PepT from its counterpart in mesophilic Lactococcus lactis . No activity against tetrapeptides or amino acid p -nitroanilide derivatives was observed. The pepT gene and its flanking regions were isolated by PCR and sequenced by cyclic sequencing. The sequence analyses revealed open reading frames (ORFs) 816 bp (ORF1) and 1,239 bp (ORF2) long. ORF2 encoded a 47-kDa PepT protein which exhibited 53% identity with the PepT from L. lactis . The mRNA analyses indicated that pepT conforms a novel operon structure with an ORF1 lo...