M. Martinez-carrion - Academia.edu (original) (raw)

Papers by M. Martinez-carrion

Journal of Biological Chemistry, 1970

The binding of ar-keto acids and other dicarboxylic acids to the pyridoxal form of the isozymes o... more The binding of ar-keto acids and other dicarboxylic acids to the pyridoxal form of the isozymes of pig heart glutamateaspartate transaminase (EC 2.6.1.1) can be studied spectrophotometrically because the absorption of the resultant complexes differs from that of the free enzymes. Spectroscopic and catalytic rate data indicate a correlation of the inhibition of transamination with the affinity of the dicarboxylic acids for the pyridoxal form of the enzymes. Maleate and succinate are the most effective inhibitors of mitochondrial transaminase, whereas succinate, maleate, glutarate, and adipate inhibit the supernatant isozyme. The amino acid erythro-/3-hydroxy-r.-aspartate forms distinctive binary complexes of unique absorption maxima with either isozyme; the equilibria between these complexes are pH insensitive. Competition of dicarboxylic acids with erythro-/3-hydroxy-Laspartate shows that inhibition by 4-or 5-carbon dicarboxylic acids is accomplished through competition with the amino acid for the pyridoxal (aldimine) and pyridoxamine forms of the supernatant isozyme. In contrast, the mitochondrial isozyme is more highly affected by 4-carbon inhibitors. They compete with the amino acid for the pyridoxal form of the enzyme and also form ternary complexes of the nature enzyme-substrate-dicarboxylic acid. Combination of the keto acid with the enzyme shifts the proton ionization (pK) of the active site chromophore (bound pyridoxal phosphate) to higher pH values in both transaminases. Oxalacetate is much more effective than o(ketoglutarate in the mitochondrial isozyme. Differences for each isozyme in the effect of the pH on the aspartate-arketoglutarate and glutamate-oxalacetate transaminations are explained by preferential binding of oxalacetate to the mitochondrial enzyme and of either a-ketoglutarate or oxalacetate to the supernatant isozyme. Product and substrate inhibition have been used to study functional isozyme variations in an effort to discover the physio

Journal of Biological Chemistry, 1971

Abstract The effects of anions on substrate binding to the mitochondrial and supernatant isozymes... more Abstract The effects of anions on substrate binding to the mitochondrial and supernatant isozymes of aspartate transaminase (EC 2.6.1.1) have been studied by direct spectrophotometric titrations of the active site-bound pyridoxal phosphate. The binding affinity of both isozymes for amino and dicarboxylic acids is dependent on the type and concentration of buffer anions. At high physiological pH, the substrate dissociation constants are proportional to the anion concentration. At low pH, substrate binding to the enzyme involves the displacement of more than one anion. The anions act as competitive inhibitors of the substrates and are thought to occupy the positively charged sites at the isozymes' active center where the substrate(s) carboxyl group(s) bind. Of all the anions studied, the order of inhibition was benzoate g chloride g acetate g phosphate g cacodylate. The comparison of the dissociation constants of the enzyme-substrate complexes for the mitochondrial and supernatant isozymes confirms that, although there is competition between the anions and the substrates or dicarboxylic acid inhibitors for the active center of the enzyme, the anion independent dissociation constants for substrates and inhibitors are intrinsically different in each isozyme. The various buffer anions also affect the pK of the active site-bound pyridoxal phosphate chromophore in different ways. Instead of the pK values of 6.3 previously reported for the mitochondrial and supernatant chromophore in chloride or phosphate buffers (high affinity anions), a pK of 5.4 can be measured for both transaminases with cacodylate buffers (low affinity anions). Thus, the binding of anions results in a pK shift of the chromophore to higher pH. This shift seems to be more pronounced the higher the affinity of the enzyme for the anion. Anions also act as competitive inhibitors for the second half of the transamination, the conversion of the pyridoxamine enzyme to the pyridoxal enzyme. The anion-binding affinities of the pyridoxamine enzyme are higher than those for the pyridoxal enzyme in both supernatant and mitochondrial transminases. Thus, a given anion concentration will affect unequally the apparent affinity of the pyridoxal form for amino acid and of the pyridoxamine form for keto acid. Since in general, the mitochondrial enzyme shows a higher anion affinity than the supernatant isozyme, whether in the pyridoxal or pyridoxamine form, the anion competition with substrates at any given concentration will be greater for the mitochondrial isozyme. The mechanistic schemes of either isozyme of aspartate transaminase must, therefore, include anion effects. Besides the obligatory enzyme substrate intermediates, there must be complexes such as pyridoxal enzyme-anion and pyridoxamine enzyme-anions in equilibrium mixtures of aspartate transaminase, substrate(s), and buffers.

Biochemistry and Molecular Biology of Vitamin B6 and PQQ-dependent Proteins, 2000

The cytosolic molecular chaperone Hsp70 discriminates between the cytosolic (cAAT) and mitochondr... more The cytosolic molecular chaperone Hsp70 discriminates between the cytosolic (cAAT) and mitochondrial (mAAT) aspartate aminotransferase isozymes, recognizing and binding exclusively mAAT. By screening a library of synthetic peptides, we have identified six putative Hsp70binding sites in mAAT. Phylogenetic analyses indicate that these Hsp70-binding sequences show less variability in mAAT than cAAT and contain more fixed differences between the two isozymes than the rest of the sequence. Thus, sequence variation between cAAT and mAAT might direct their selective interaction with molecular chaperones and thereby contribute to their correct localization in the cell.

Journal of Biological Chemistry, 1976

Abnormal lysyl residues can be detected in aspartate transaminase by following the rate of reacti... more Abnormal lysyl residues can be detected in aspartate transaminase by following the rate of reaction of amino groups with KN'%O and the rate of enzymatic inactivation. Peptide isolation subsequent to carbamylation of the apoenzyme produces a peptide which is absent in the carbamylated holoenzyme. The composition of the carbamylated peptide matches that of a tryptic peptide containing the active site Lys-258. The holoenzyme retains full catalytic activity after carbamylation of its NHz-terminal alanine and lysyl residues other than Lys-258, which is protected by aldimine formation with pyridoxal phosphate. Apoenzyme prepared from KNCO-treated holoenzyme (apoenzyme') is susceptible to further carbamylation at Lys-258 with irreversible loss of catalytic activity. Carbamylation of the active site lysyl residue is 25 to 50 times more rapid than that of the other 18 lysyl residues of aspartate transaminase. The kinetics of inactivation by KNCO at different pH values served to determine the pH-independent second order rate constant (k) and the pK of the amino group of Lys-258. These values are pK = 7.98 & 0.08 and k = 146 * 5 M-k', which are similar to the values determined for carbamylation of the NH,terminal groups of human hemoglobin (

J Biol Chem, Sep 1, 1965

Jornalismo e representações sociais: algumas considerações RESUMO Este artigo tem como objetivo e... more Jornalismo e representações sociais: algumas considerações RESUMO Este artigo tem como objetivo estabelecer aproximações entre a teoria das representações e os estudos da notícia. Consideramos que as conexões entre os dois campos oferecem possibilidades teóricas para as pesquisas que se preocupam como, na atividade diária, os jornalistas contribuem para a construção de representações da cultura, da economia e da política. Em resumo, investigações que tratam o campo jornalístico como um lugar de construção do real.

Biochemistry, Apr 19, 1977

The hydrocarbon-chain orientational order parameters of membranes of Acholeplasma laidlawii B enr... more The hydrocarbon-chain orientational order parameters of membranes of Acholeplasma laidlawii B enriched with straight-chain saturated, methyl iso-branched, methyl anteiso-branched, or trans-unsaturated fatty acids have been determined via fluorine-19 nuclear magnetic resonance spectroscopy (I9F NMR). A theoretical description of the I9F NMR spectral line shape is presented that permits the determination of the orientational order parameters associated with specifically monofluorinated palmitic acid probes biosynthetically incorporated into membrane glycerolipids. Membrane orientational order profiles determined by 19F NMR in the case of straight-chain saturated fatty acid enrichment were qualitatively similar to profiles obtained by ?H NMR. The methyl iso-branch and methyl anteiso-branch structural substituents induced a local ordering while the trans double bond substituent induced a local disordering evident from alterations to the character of the orientational order T e hydrocarbon milieu of biological and model membranes has been the subject of intensive investigation via nuclear magnetic resonance (NMR) techniques [for reviews, see

The Journal of biological chemistry, 1984

The enzyme, aspartate aminotransferase, is a dimer consisting of two identical subunits which con... more The enzyme, aspartate aminotransferase, is a dimer consisting of two identical subunits which contain overlapping subunit regions ( Eichele , G., Ford, G.C., Glor , M., Jansonius , J.N., Mavrides , C., and Christen , P. (1979) J. Mol. Biol. 133, 161-180), suggesting the possibility of subunit interactions. The structurally similar cytosolic isozyme exhibits noncooperative binding of pyridoxal 5'-phosphate ( Boettcher , M., and Martinez -Carrion, M. (1975) Biochemistry 14, 4528-4531; Relimpio , A., Iriarte , A., Chlebowski , J.F., and Martinez -Carrion, M. (1981) J. Biol. Chem. 256, 4478-4488) in which the apoenzyme/holoenzyme hybrid dimer shows a distinctive thermal stability. Using a nonequilibrium isoelectric focusing technique, it can be shown that mitochondrial aspartate aminotransferase also binds cofactor in a noncooperative random fashion. However, differential scanning calorimetry (DSC) thermograms show different characteristics from the cytosolic form. These differences...

Journal of Biological Chemistry, 1988

Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoboro... more Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar

Journal of Biological Chemistry, 1972

Journal of Biological Chemistry, 1993

The mitochondrial isozyme of aspartate aminotransferase (mAspAT), a dimeric pyridoxal phosphate (... more The mitochondrial isozyme of aspartate aminotransferase (mAspAT), a dimeric pyridoxal phosphate (PLP)-dependent enzyme, is encoded by the nuclear genome and synthesized in the cytoplasm as a precursor protein (pmAspAT) containing a 29-residue amino-terminal signal peptide which is essential for its targeting and import into mitochondria. In the cytosolic-like environment of rabbit reticulocyte lysate, newly synthesized rat liver pmAspAT has been found to slowly fold and bind PLP (Mattingly, J. R., Jr., Youssef, J., Iriarte, A. and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 3925-3937). On the other hand, isolated mammalian (pig) mAspAT, when denatured with guanidine hydrochloride, seems unable to refold to a catalytically active state (West, S. M., and Price, N. C. (1990) Biochem. J. 265, 45-50). With the availability of rat liver recombinant precursor and mature forms of mAspAT as homogeneous, stable preparations, an assessment of the influence of the signal peptide on the in vitro refolding of this protein can be made. Following unfolding induced by guanidine hydrochloride, we have investigated the refolding process of this complex, dimeric coenzyme-dependent protein system by activity, fluorescence, and circular dichroism. Both mAspAT and pmAspAT can be efficiently renatured after rapid dilution of the denaturing agent at low protein concentrations. The equilibrium unfolding/refolding transitions and the kinetics of folding are protein concentration-independent and identical for both protein forms. Binding of coenzyme into the active site pocket seems to occur at a late step in the folding process of both mAspAT and pmAspAT, suggesting that in these proteins the coenzyme does not direct the folding of the polypeptide chain. These results indicate that the in vitro refolding of mAspAT is not regulated or influenced by the presence of the amino-terminal signal peptide. On the other hand, in vitro refolding in buffer is significantly faster than the folding of newly synthesized precursor protein in reticulocyte lysate examined in our previous report (reference above), pointing at the likely influence of cytosolic factors in modulating folding in the cell.

Journal of Biological Chemistry, 1983

The environment of the phosphate group of pyridoxal-P bound at the active site of cytosolic serin... more The environment of the phosphate group of pyridoxal-P bound at the active site of cytosolic serine hydroxymethyltransferase has been investigated by 3LP NMR spectroscopy. In the holoenzyme, the pyridoxal-P chemical shift is pH-dependent with a p& of 6.45. The chemical shift of the bound pyridoxal-P is shifted upfield about 0.3 ppm from the signal for free pyridoxal-P. Saturation of the active site with the substrates Lserine, glycine, and tetrahydrofolate does not alter the chemical shift or the pK, of the phosphate group. The addition of these substrates does, however, alter the absorption and circular dichroism spectra of the bound coenzyme, reflecting environmental changes of the pyridine ring-Schiff s base system. We conclude from these studies that the phosphate group of the bound coenzyme is exposed to the solvent. The reorientation and conformational changes of the pyridoxal-P ring which take place during the formation of enzyme-substrate complexes do not appear to change the environment of the phosphate moiety of the coenzyme. Cytosolic serine hydroxymethyltransferase (EC 2.1.2.1) catalyzes the formation of glycine and 5,lO-methylenetetrahydrofolate from serine and tetrahydrofolate (1). The enzyme is a

Journal of Biological Chemistry, 1969

Abstract A detailed comparison of the interactions of substrates with the mitochondrial and super... more Abstract A detailed comparison of the interactions of substrates with the mitochondrial and supernatant isozymes of glutamateaspartate transaminase (EC 2.6.1.1) has been made by direct spectrophotometric titrations of the natural reporter, bound pyridoxal phosphate, at the active site. Spectroscopic methods are presented for the analysis of the dissociation constants of the aspartate-oxalacetate and glutamate-α-ketoglutarate equilibria of mitochondrial glutamate-aspartate transaminase. Mathematical treatment of the spectrophotometric data is based on the assumption that the mechanism involves only binary complexes. The agreement and linearity of the graphical results of these methods rule out site-site interactions or the formation of ternary complexes, such as enzyme-amino acid-keto acid. The pyridoxal form of the mitochondrial isozyme has a higher affinity for aspartate (K1 = 0.47 mm) than for glutamate (K1 = 12.4 mm). It binds oxalacetate (K3 = 46 mm) and α-ketoglutarate (K3 = 620 mm) as inactive abortive complexes. In the supernatant enzyme these values are aspartate, K1 = 4 mm; glutamate, K1 = 14 mm; oxalacetate, K3 = 100 mm; and α-ketoglutarate, K3 = 50 mm. The pyridoxamine form of both isozymes binds oxalacetate (K2 = 14 µm, mitochondrial; K2 = 20 µm, supernatant) better than α-ketoglutarate (K2 = 0.7 mm, mitochondrial; K2 = 0.4 mm, supernatant), but it has a low affinity for glutamate and aspartate. The substrate analogue, erythro-β-hydroxyaspartate, forms a complex(es) with the mitochondrial aminotransferase which absorbs at 497 mµ. The affinity of the pyridoxal enzyme for this analogue (K = 0.2 mm) is comparable to that of aspartate and is lower than in the supernatant enzyme (K = 0.4 mm). The pyridoxamine form of each isozyme has a higher affinity for erythro-β-hydroxyaspartate than for either glutamate or aspartate. Radioactive exchange studies indicate that transamination can occur between the substrate amino acid and its analogous keto acid. Dissociation constants obtained by this method agree with those of steady state equilibrium spectrophotometric data. A comparison of the intermediates of the mitochondrial and supernatant glutamate-aspartate transaminase indicates that the mitochondrial isozyme has a high affinity for 4-carbon substrates, whereas the supernatant enzyme has no distinctive preference for either 4- or 5-carbon substrates.

Journal of Biological Chemistry, 1980

Sodium b~ro[~H]hydride treatment of holoaspartate aminotransferase results in the reduction of th... more Sodium b~ro[~H]hydride treatment of holoaspartate aminotransferase results in the reduction of the Schiff's base formed between pyridoxal phosphate and Lys 258. Treatment of the reduced enzyme with papain followed by acid hydrolysis liberates e-N-[3H]pyridoxyl lysine which is degraded to [3HJpyridoxamine diHCl and stereochemically analyzed with apoaspartate aminotransferase. Sodium b~ro[~HJhydride treatment of active site carbamylated aspartate aminotransferase reconstituted with pyridoxyl phosphate and sodium aspartate results in the trapping of an enzyme. substrate complex through the reduction of the Schiffs base formed between pyridoxal phosphate and aspartate. Active site bound N-[3H]pyridoxyl aspartate is liberated by treatment with papain and degraded to [3H]pyridoxamine diHCl for stereochemical analysis. Borohydride reduction of the holoenzyme occurs from the re face of the pyridoxal phosphate Lys 258 Schiff's base. Similarly, reduction of active site carbamylated enzyme substrate complex occurs from the re face of the pyridoxal phosphate-aspartate Schiff s base. These results indicate that when active site carbamylated enzyme binds substrate to pyridoxal phosphate it does so stereospecifically and without changing the face of the Schiff base that is available for reduction as compared to native enzyme. ~ ~~~ Stereochemical studies of aspartate aminotransferase as well as other pyridoxal phosphate-dependent enzymes have led to a detailed understanding of the role played by pyridoxal phosphate in the mechanism of catalysis by those enzymes. Dunathan (1) suggested that one of the primary catalytic functions of pyridoxal phosphate enzymes is to orient the cofactorsubstrate complex in a reactive conformation. Such an orientation has the breaking and forming bonds orthogonal to the plane of the conjugated n system so as to stabilize the developing charge. Also, the cofactor. substrate complex would be rapidly bound by these enzymes on a single face, thereby forcing all of the breaking and making of bonds to occur on the opposite side. In aspartate aminotransferase, the C-a-H bond is broken and the C-4'-H bond formed through a cis process occurring on the si face of the cofactor substrate complex (Scheme I). Recent studies (2-4) have shown that in at least two other pyridoxal phosphate enzymes, reduction of enzyme-bound Schiff s base intermediates is an effective means of determining the exposed "solvent side" of cofactor. substrate com-* This work was supported by Grants GM 24885 and HL 22265 from the National Institutes of Health. 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.

Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds As Cofactors, 1991

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1994

Three mAbs, mAbs 249E, 370, and 383C, directed against the alpha-bungarotoxin (alpha BgTx) bindin... more Three mAbs, mAbs 249E, 370, and 383C, directed against the alpha-bungarotoxin (alpha BgTx) binding site of the acetylcholine receptor (AChR) induce a hyperacute form of experimental autoimmune myasthenia gravis (EAMG), characterized by death within hours of mAb injection. To analyze the mechanisms of this effect, purified AChR-mAb complexes were investigated for their ability to bind the cholinergic agonist carbamoylcholine and to undergo agonist-induced activation of the cholinergic ionophore. The three mAbs inhibited carbamylcholine binding, and, conversely, their binding to AChR was inhibited by carbamylcholine. All three completely inhibited carbamylcholine-induced T1+ influxes to AChR-rich vesicles. These data indicate that the severe hyperacute EAMG induced by these mAbs results from blockage of AChR function and that the role of such potent Abs (even if present in small amounts) in the pathogenesis of human myasthenia gravis deserves further investigation.

Progress in clinical and biological research, 1984

Biochemistry of Vitamin B6, 1987

A method has been devised for the selective labeling of cytoplasmic domains of transmembrane prot... more A method has been devised for the selective labeling of cytoplasmic domains of transmembrane proteins with pyridoxal phosphate (PLP). Sealed vesicles are loaded with 3H pyridoxine phosphate (PNP) and pyridoxine phosphate oxidase (PNPoxidase) by freezing-thawing (FT) and PLP is generated intravesicularly by enzymatic oxidation. Results obtained using acetylcholine receptor (AcChR) vesicles as a model are summarized.

Journal of Biological Chemistry, 1998

The partially homologous mitochondrial (mAAT) and cytosolic (cAAT) aspartate aminotransferase hav... more The partially homologous mitochondrial (mAAT) and cytosolic (cAAT) aspartate aminotransferase have nearly identical three-dimensional structures but differ in their folding rates in cell-free extracts and in their affinity for binding to molecular chaperones. In its native state, each isozyme is protease-resistant. Using limited proteolysis as an index of their conformational states, we have characterized these proteins (a) during the early stages of spontaneous refolding; (b) as species trapped in stable complexes with the chaperonin GroEL; or (c) as newly translated polypeptides in cellfree extracts. Treatment of the refolding proteins with trypsin generates reproducible patterns of large proteolytic fragments that are consistent with the formation of defined folding domains soon after initiating refolding. Binding to GroEL affords considerable protection to both isozymes against proteolysis. The tryptic fragments are similar in size for both isozymes, suggesting a common distribution of compact and flexible regions in their folding intermediates. cAAT synthesized in cellfree extracts becomes protease-resistant almost instantaneously, whereas trypsin digestion of the mAAT translation product produces a pattern of fragments qualitatively akin to that observed with the protein refolding in buffer. Analysis of the large tryptic peptides obtained with the GroEL-bound proteins reveals that the cleavage sites are located in analogous regions of the N-terminal portion of each isozyme. These results suggest that (a) binding to GroEL does not cause unfolding of AAT, at least to an extent detectable by proteolysis; (b) the compact folding domains identified in AAT bound to GroEL (or in mAAT fresh translation product) are already present at the early stages of refolding of the proteins in buffer alone; and (c) the two isozymes seem to bind in a similar fashion to GroEL, with the more compact C-terminal portion completely protected and the more flexible N-terminal first 100 residues still partially accessible to proteolysis.

Journal of Biological Chemistry, 2003

The refolding of mitochondrial aspartate aminotransferase (mAAT; EC 2.6.1.1) has been studied fol... more The refolding of mitochondrial aspartate aminotransferase (mAAT; EC 2.6.1.1) has been studied following unfolding in 6 M guanidine hydrochloride for different periods of time. Whereas reactivation of equilibriumunfolded mAAT is sigmoidal, reactivation of the short term unfolded protein displays a double exponential behavior consistent with the presence of fast and slow refolding species. The amplitude of the fast phase decreases with increasing unfolding times (k Ϸ 0.75 min ؊1 at 20°C) and becomes undetectable at equilibrium unfolding. According to hydrogen exchange and stoppedflow intrinsic fluorescence data, unfolding of mAAT appears to be complete in less than 10 s, but hydrolysis of the Schiff base linking the coenzyme pyridoxal 5-phosphate (PLP) to the polypeptide is much slower (k Ϸ 0.08 min ؊1). This implies the existence in short term unfolded samples of unfolded species with PLP still attached. However, since the disappearance of the fast refolding phase is about 10-fold faster than the release of PLP, the fast refolding phase does not correspond to folding of the coenzyme-containing molecules. The fast refolding phase disappears more rapidly in the pyridoxamine and apoenzyme forms of mAAT, both of which lack covalently attached cofactor. Thus, bound PLP increases the kinetic stability of the fast refolding unfolding intermediates. Conversion between fast and slow folding forms also takes place in an early folding intermediate. The presence of cyclophilin has no effect on the reactivation of either equilibrium or short term unfolded mAAT. These results suggest that proline isomerization may not be the only factor determining the slow refolding of this cofactor-dependent protein.