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PubMed1995

Biochemical characterization of gapB-encoded erythrose 4-phosphate dehydrogenase of Escherichia coli K-12 and its possible role in pyridoxal 5'-phosphate biosynthesis.

One step in de novo pyridoxine (vitamin B6) and pyridoxal 5'-phosphate biosynthesis was predicted to be an oxidation catalyzed by an unidentified D-erythrose-4-phosphate dehydrogenase (E4PDH). To help identify this E4PDH, we purified the Escherichia coli K-12 gapA- and gapB-encoded dehydrogenases to homogeneity and tested whether either uses D-erythrose-4-phosphate (E4P) as a substrate. gapA (gap1) encodes the major D-glyceraldehyde-3-phosphate dehydrogenase (GA3PDH). The function of gapB (gap2) is unknown, although it was suggested that gapB encodes a second form of GA3PDH or is a cryptic gene. We found that the gapB-encoded enzyme is indeed an E4PDH and not a second GA3PDH, whereas gapA-encoded GA3PDH used E4P poorly, if at all, as a substrate under the in vitro reaction conditions used in this study. The amino terminus of purified E4PDH matched the sequence predicted from the gapB DNA sequence. Purified E4PDH was a heat-stable tetramer with a native molecular mass of 132 kDa. E4PDH had an apparent Km value for E4P [Kmapp(E4P)] of 0.96 mM, an apparent kcat catalytic constant for E4P [kcatapp(E4P)] of 200 s-1, Kmapp(NAD+) of 0.074 mM, and kcatapp(NAD+) of 169 s-1 in steady-state reactions in which NADH formation was determined. From specific activities in crude extracts, we estimated that there are at least 940 E4PDH tetramer molecules per bacterium growing in minimal salts medium plus glucose at 37 degrees C. Thin-layer chromatography confirmed that the product of the E4PDH reaction was likely the aldonic acid 4-phosphoerythronate. To establish a possible role of E4PDH in pyridoxal 5'-phosphate biosynthesis, we showed that 4-phosphoerythronate is a likely substrate for the 2-hydroxy-acid dehydrogenase encoded by the pdxB gene. Implications of these findings in the evolution of GA3PDHs are also discussed. On the basis of these results, we propose renaming gapB as epd (for D-erythrose-4-phosphate dehydrogenase).

Alcohol Oxidoreductases↗
PubMed1995

Tissue B-6 vitamer concentrations in rats fed excess vitamin B-6.

Diets containing 1, 10, 100, 175 or 250 times the NRC recommended level of pyridoxine HCl (7 mg/kg) were fed to rats (218 g, 12 per group) to evaluate the effects on tissue B-6 vitamer concentrations. After 10 wk, food intake and body weights did not differ among groups. Overt toxicity was not observed. Tissues were taken from five rats of each group after overnight food deprivation (unfed rats); the remaining seven rats in each group were allowed access to food (fed rats). In plasma of unfed rats, 4-pyridoxic acid and pyridoxal concentrations increased significantly (P < 0.05) with increasing dietary pyridoxine; pyridoxal phosphate was not affected by dietary pyridoxine. Concentrations of pyridoxal phosphate and pyridoxal increased significantly with increasing dietary pyridoxine in erythrocytes of unfed rats. Excretion of urinary B-6 vitamers and 4-pyridoxic acid in a 24-h period increased with dietary pyridoxine in fed rats. As dietary pyridoxine was increased, kidney pyridoxal concentrations increased significantly in fed rats only. Dietary pyridoxine did not affect vitamer concentration in muscle and liver of either unfed or fed rats, or in brain of unfed rats. Muscle glycogen phosphorylase, which contains pyridoxal phosphate, was not affected by dietary pyridoxine. There was a marginally significant (P = 0.058) increase in erythrocyte alanine, but not in aspartate, aminotransferase activity with increasing dietary pyridoxine. Plasma concentration of pyridoxal phosphate, which is used as a measure of vitamin B-6 status, did not reflect intake of pyridoxine in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

A structural and mechanistic comparison of pyridoxal 5'-phosphate dependent decarboxylase and transaminase enzymes.

Stereochemical studies of three pyridoxal phosphate dependent decarboxylases and serine hydroxymethyltransferase have allowed the dispositions of conjugate acids that operate at the C alpha and C-4' positions of intermediate quinoids to be determined. Kinetic work with the decarboxylase group has determined that two different acids are involved, a monoprotic acid and a polyprotic acid. The use of solvent kinetic isotope effects allowed the resolution of chemical steps in the reaction coordinate profile for decarboxylation and abortive transamination and pH-sensitivities gave the molecular pKa of the monoprotic base. Thus the epsilon-ammonium group of the internal aldimine-forming lysine residue operates at C-4'-si-face of the coenzyme and the imidazolium side chain of an active site histidine residue protonates at C alpha from the 4'-si-face. Histidine serves two other functions, as a base in generating nitrogen nucleophiles during both transaldimination processes and as a binding group for the alpha-carboxyl group of substrates. The latter role for histidine was determined by comparison of the sequences for decarboxylase active site tetrapeptides (e.g. -S-X-H-K-) with that for aspartate aminotransferase (e.g. -S-X-A-K-) where it was known, from X-ray studies, that the serine and lysine residues interact with the coenzyme. By using the Dunathan Postulate, the conformation of the external aldimine was modified, and without changing the tetrapeptide conformation, the alanine residue was altered to a histidine. This model for the active site of a pyridoxal dependent decarboxylase was consistent with all available stereochemical and mechanistic data.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Hemoglobin-based blood substitutes: characterization of five pyridoxal 5'-phosphate derivatives of hemoglobin.

Six fractions (I-VI) of pyridoxal 5'-phosphate (PLP) hemoglobin (Hb), prepared by the method of De Venuto and Zegna [J. Surg. Res., 34 (1983) 205] have been isolated and purified by anion-exchange high-performance liquid chromatography (HPLC). Total phosphate analyses indicate that I is HbA, II and III are double-labelled, IV and V are tetra-labelled and VI contains 6 mol of phosphorus per mol of hemoglobin. The purified components have been resolved into their alpha and beta chains by preparative reversed-phase HPLC using a macroporous C4 support. Phosphate analyses indicate that the beta chains of II, III and IV each contain one phosphate per chain while the beta chains of V and VI each contain two phosphates. The alpha chains of IV and VI were found to be monophosphate-labelled. Reversed phase HPLC analysis of the tryptic peptides of the beta chains indicates that the label is bound exclusively to the 1-valine residue in II, III and IV while both the 1-valine and the 82-lysine are labelled in V and VI. Similarly, modification of the 1-valine residue of the alpha chains of IV and VI was detected. Components II and III have the same molecular formula. Evidence is presented which shows that they are interconvertible and that they correspond to the PLP2-Hb species and component V is Benesch's PLP4-Hb [J. Biol. Chem., 257 (1983) 1320 and references cited therein]. Component IV is III with one additional PLP per alpha chain and similarly VI is V with monolabelled alpha chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Substitutes↗
PubMed1983

Interaction of pyridoxal-5'-phosphate and black-eye pea trypsin and chymotrypsin inhibitor.

Pyridoxal-5'-phosphate (pyridoxal-P) reacts with lysine residues of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) at pH 7.4 and 30 degrees C, forming a Schiff-base which was stabilized by NaBH4 reduction. The interaction of pyridoxal-P and BTCI was accompanied by a difference spectrum presenting a negative peak at 382 nm and a positive peak at 435 nm which was shifted to 325 nm by reduction with NaBH4. The kinetics of interaction indicated that a noncovalent protein-pyridoxal-P complex was formed prior to Schiff-base formation. The number of pyridoxal-P (or pyridoxal) groups incorporated per protein molecule after NaBH4 reduction was determined spectrophotometrically as a function of the reagent/protein molar ratio. A saturation value of about 0.75 pyridoxal-P residues per protein molecule was found for an 80-100 molar excess of reagent over the protein. In the case of pyridoxal, the incorporation of reagent into the protein was much less than that of pyridoxal-P. The results obtained indicate that lysine residues in BTCI (5 per molecule) are in unfavourable environments for binding pyridoxal-P. The trypsin and chymotrypsin inhibitory activities of the pyridoxal-P-treated, NaBH4-reduced BTCI are slightly reduced.

Chemical Phenomena↗
PubMed1975

[Activity studies of an iron-vitamin B6 preparation for euteral treatment of iron deficiency anemia].

In 17 respectively 15 children with iron deficiency anemia the effect of a combined orally applicated iron-vitamin B6 therapy and iron therapy only was studied. Pyridoxal phosphate, activities of pyridoxal kinase and red cell GOT, and excretion of 4-pyridoxic acid in urine were measured as indices of vitamin B6 nutriture before therapy was started, on the 3rd and 6th day with therapy and on the 1st and 4th day after therapy was stopped. Red cells, concentration of hemoglobin, reticulocytes and hematokrit were simultaneously counted, whereas serum iron had been measured once only before therapy. A group of 22 hematologically healthy children was studied as controls. After iron therapy a decrease of vitamin B6 nutriture occured as a consequence of an increased requirement for pyridoxal phosphate for heme synthesis. Additional vitamin B6 was followed by a normal vitamin B6 nutriture and a significantly accelerating effect on heme synthesis.

Administration, Oral↗
PubMed1995

Resolution of pyridoxal 5'-phosphate from O-acetylserine sulfhydrylase from Salmonella typhimurium and reconstitution of apoenzyme with cofactor and cofactor analogues as a probe of the cofactor binding site.

A procedure has been developed to prepare the apoenzyme of O-acetylserine sulfhydrylase (apoOASS) by first converting the native enzyme to the alpha-aminoacrylate intermediate and dialyzing against 5 M guanidinium chloride. Aposulfhydrylase is stable for at least a month in buffers containing phosphate or phosphate analogues. Reconstitution of aposulfhydrylase with pyridoxal 5'-phosphate (PLP), 2'-methyl PLP (2'-MePLP), and pyridoxal 5'-deoxymethylenephosphonate (PDMP) results in enzymatically competent proteins. Pyridoxal in the absence and presence of phosphate and pyridoxal 5'-phosphate monomethyl ester are unable to form a Schiff base with apoOASS. The reconstitution of apoOASS with PLP is highly cooperative judged by the initial rate of activity regained and shows no evidence of saturation with PLP. The reconstituted enzymes have been studied using 31P NMR spectroscopy. The 31P NMR of the aposulhydrylase reconstituted with PLP exhibits a chemical shift of 5.2 ppm, identical to that of native enzyme. The latter has been interpreted in terms of a strong ionic interaction between enzyme and the 5'-phosphate of PLP (P. F. Cook, S. Hara, S. Nalabolu, and K. D. Schnackerz, 1992, Biochemistry 31, 2298-2303). Reconstitution with 2'-MePLP gives a lower chemical shift of 4.95 ppm, suggesting a weaker ionic interaction at the 5'-phosphate when compared to native enzyme. The PDMP-reconstituted enzyme gives a chemical shift of 23.7 ppm, consistent with the monoanionic form of the bound phosphonate. All of the chemical shifts are pH independent. The apoenzyme has also been reconstituted with pyridoxal 5'-sulfate. Although the resulting enzyme is not active in the overall reaction, it forms the external Schiff base. The PDMP- and 2'-MePLP-reconstituted enzymes have also been studied in the presence of amino acid reactants and analogues, and results are discussed in terms of the mechanism of OASS.

Apoenzymes↗
PubMed1975

Reversible modification of pig heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate.

1. Pig heart mitochondrial malate dehydrogenase incubated with pyridoxal 5'-phosphate at pH 8.0 and 25 degrees C gradually loses activity. Such inactivation can be largely reversed by dialysis or by addition of L-lysine or L-cysteine, and can be made permanent by NaBH4 reduction. 2. Modification of malate dehydrogenase with pyridoxal 5'-phosphate at 35 degrees C involves two phases, an initial inactivation which is reversible and a slower irreversible second stage. 3. The initial reaction between pyridoxal 5'-phosphate and malate dehydrogenase appears to involve reversible formation of a Schiff base with the epsilon-amino group of a lysine residue. 4. Inactivation of malate dehydrogenase by pyridoxal 5'-phosphate at 10 degrees C involves only the reversible reaction. 5. At 10 degrees C repeated cycles of treatment with pyridoxal 5'-phosphate and NaBH4 reduction lead to a stepwise decline in residual activity. 6. Apparent Km values for malate and NAD+ are unaltered in the partially inactivated enzyme. 7. NAD+ and NADH give only partial protection against pyridoxal 5'-phosphate inactivation. Substrates give no effect.

Borohydrides↗
PubMed1991

Separation and quantification of the B6 vitamers in plasma and 4-pyridoxic acid in urine of adolescent girls by reversed-phase high-performance liquid chromatography.

The vitamin B6 status of seemingly healthy adolescent girls was determined using several accepted and proposed parameters in an effort to establish guidelines for status evaluation. High-performance liquid chromatography-derived plasma B6 vitamers (pyridoxal phosphate, PLP; pyridoxine phosphate. PNP; pyridoxamine phosphate, PMP; pyridoxal, PL; pyridoxine, PN; and pyridoxamine, PM) and 4-pyridoxic acid (4-PA) concentrations and urinary 4-PA levels of 28 white adolescent females, 12-15 years, having radiomonitored plasma PLP concentrations and coenzyme stimulation of erythrocyte alanine aminotransferase activities indicative of adequate status were determined. Mean vitamin B6 and protein intakes were 1.48 mg and 78.3 g. Ranges for plasma B6 vitamer and 4-PA concentrations (nmol/l) were: PLP, 40.9-122.2; PNP, non-detectable (ND)-16.1; PMP, ND-8.1; PL, ND-15; PN, ND-21.9; PM, ND-17.8; and 4-PA, ND-55.7. PLP was the only vitamer found in plasma of all subjects. Urinary 4-PA concentrations ranged from 0.11 to 2.50 mumol/mmol of creatinine. B6 vitamer values of these girls should be of use in the establishment of normal ranges for vitamin B6 status parameters.

Adolescent↗
PubMed1987

Active-site-directed inactivation of wheat-germ aspartate transcarbamoylase by pyridoxal 5'-phosphate.

Treatment of 1 microM wheat-germ aspartate transcarbamoylase with 1 mM-pyridoxal 5'-phosphate caused a rapid loss of activity, concomitant with the formation of a Schiff base. Complete loss of activity occurred within 10 min when the Schiff base was reduced with a 100-fold excess of NaBH4. Concomitantly, one amino group per chain was modified. No further residues were modified in the ensuing 30 min. The kinetics of inactivation were examined under conditions where the Schiff base was reduced before assay. Inactivation was apparently first-order. The pseudo-first-order rate constant, kapp., showed a hyperbolic dependence upon the concentration of pyridoxal 5'-phosphate, suggesting that the enzyme first formed a non-covalent complex with the reagent, modification of a lysine then proceeding within this complex. Inactivation of the enzyme by pyridoxal was 20 times slower than that by pyridoxal 5'-phosphate, indicating that the phosphate group was important in forming the initial complex. Partial protection against pyridoxal phosphate was provided by the leading substrate, carbamoyl phosphate, and nearly complete protection was provided by the bisubstrate analogue, N-phosphonoacetyl-L-aspartate, and the ligand-pair carbamoyl phosphate plus succinate. Steady-state kinetic studies, under conditions that minimized inactivation, showed that pyridoxal 5'-phosphate was also a competitive inhibitor with respect to the leading substrate, carbamoyl phosphate. Pyridoxal 5'-phosphate therefore appears to be an active-site-directed reagent. A sample of the enzyme containing one reduced pyridoxyl group per chain was digested with trypsin, and the labelled peptide was isolated and shown to contain a single pyridoxyl-lysine residue. Partial sequencing around the labelled lysine showed little homology with the sequence surrounding lysine-84, an active-centre residue of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli, whose reaction with pyridoxal 5'-phosphate shows many similarities to the results described in the present paper. Arguably the reactive lysine is conserved between the two enzymes whereas the residues immediately surrounding the lysine are not. The same conclusion has been drawn in a comparison of reactive histidine residues in the two enzymes [Cole & Yon (1986) Biochemistry 25, 7168-7174].

Amino Acid Sequence↗
PubMed1976

13C NMR spectroscopy of labeled pyridoxal 5'-phosphate. Model studies, D-serine dehydratase, and L-glutamate decarboxylase.

Pyridoxal 5'-phosphate labeled to the extent of 90% with 13C in the 4' (aldehyde) and 5' (methylene) positions has been synthesized. 13C NMR spectra of this material and of natural abundance pyridoxal 5'-phosphate are reported, as well as 13C NMR spectra of the Schiff base formed by reaction of pyridoxal 5'-phosphate with n-butylamine, the secondary amine formed by reduction of this Schiff base, the thiazolidine formed by reaction of pyridoxal 5'-phosphate with cysteine, the hexahydropyrimidine formed by reaction of pyridoxal 5'-phosphate with 1,3-diaminobutane, and pyridoxamine 5'-phosphate. The range of chemical shifts for carbon 4' in these compounds is more than 100 ppm, and thus this chemical shift is expected to be a sensitive indicator of structure in enzyme-bound pyridoxal 5'-phosphate. The chemical shift of carbon 5', on the other hand, is insensitive to these structure changes. 13C NMR spectra have been obtained at pH 7.8 and 9.4 for D-serine dehydratase (Mr = 46,000) containing natural abundance pyridoxal 5'-phosphate and containing 13C-enriched pyridoxal 5'-phosphate. The enriched material contains two new resonances not present in the natural abundance material, one at 167.7 ppm with a linewidth of approximately 24 Hz, attributed to carbon 4' of the Schiff base in the bound coenzyme, and one at 62.7 Hz with a linewidth of approximately 48 Hz attributed to carbon 5' of the bound Schiff base. A large number of resonances due to individual amino acids are assigned. The NMR spectrum changes only slightly when the pH is raised to 9.4. The widths of the two enriched coenzyme resonances indicate that the coenzyme is rather rigidly bound to the enzyme but probably has limited motional freedom relative to the protein. 13C NMR spectra have been obtained for L-glutamate decarboxylase containing natural abundance pyridoxal 5'-phosphate and 13C-enriched pyridoxal 5'-phosphate. Under conditions where the two enriched 13C resonances are clearly visible in D-serine dehydratase, no resonances are visible in enriched L-glutamate decarboxylase, presumably because the coenzyme is rigidly bound to the protein and the 300,000 molecular weight of this enzyme produces very short relaxation times for the bound coenzyme and thus very broad lines.

Binding Sites↗
PubMed1985

Active-site modification of mammalian pyruvate dehydrogenase by pyridoxal 5'-phosphate.

The pyruvate dehydrogenase multienzyme complex from bovine kidney and heart is inactivated by treatment with pyridoxal 5'-phosphate and sodium cyanide or sodium borohydride. The site of this inhibition is the pyruvate dehydrogenase (E1) component of the complex. Inactivation of E1 by the pyridoxal phosphate-cyanide treatment was prevented by thiamin pyrophosphate. Equilibrium binding studies showed that E1 contains two thiamin pyrophosphate binding sites per molecule (alpha 2 beta 2) and that modification of E1 increased the dissociation constant (Kd) for thiamin pyrophosphate about 5-fold. Incorporation of approximately 2.4 equiv of 14CN per mole of E1 tetramer in the presence of pyridoxal phosphate resulted in about a 90% loss of E1 activity. Radioactivity was incorporated predominantly into the E1 alpha subunit. Radioactive N6-pyridoxyllysine was identified in an acid hydrolysate of the E1-pyridoxal phosphate complex that had been reduced with NaB3H4. The data are interpreted to indicate that in the presence of sodium cyanide or sodium borohydride, pyridoxal phosphate reacts with a lysine residue at or near the thiamin pyrophosphate binding site of E1. This binding site is apparently located on the alpha subunit.

Animals↗
PubMed2004

Analysis of antibody reactivity against cysteine sulfinic acid decarboxylase, a pyridoxal phosphate-dependent enzyme, in endocrine autoimmune disease.

The structurally related group II pyridoxal phosphate (PLP)-dependent amino acid decarboxylases glutamic acid decarboxylase (GAD), aromatic L-amino acid decarboxylase (AADC), and histidine decarboxylase (HDC) are known autoantigens in endocrine disorders. We report, for the first time, the prevalence of serum autoantibody reactivity against cysteine sulfinic acid decarboxylase (CSAD), an enzyme that shares 50% amino acid identity with the 65- and 67-kDa isoforms of GAD (GAD-65 and GAD-67), in endocrine autoimmune disease. Three of 83 patients (3.6%) with autoimmune polyendocrine syndrome type 1 (APS1) were anti-CSAD positive in a radioimmunoprecipitation assay. Anti-CSAD antibodies cross-reacted with GAD-65, and the anti-CSAD-positive sera were also reactive with AADC and HDC. The low frequency of anti-CSAD reactivity is in striking contrast to the prevalence of antibodies against GAD-65, AADC, and HDC in APS1 patients, suggesting that different mechanisms control the immunological tolerance toward CSAD and the other group II decarboxylases. Moreover, CSAD may be a useful mold for the construction of recombinant chimerical antigens in attempts to map conformational epitopes on other group II PLP-dependent amino acid decarboxylases.

Addison Disease↗
PubMed2001

Crystal structure of histidinol phosphate aminotransferase (HisC) from Escherichia coli, and its covalent complex with pyridoxal-5'-phosphate and l-histidinol phosphate.

The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants. The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5'-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9). Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5'-phosphate (PMP) at 1.5 A resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 A resolution. This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine.HisC is a dimeric enzyme with a mass of approximately 80 kDa. Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an alpha/beta/alpha topology, and a smaller domain. An N-terminal arm contributes to the dimerization of the two monomers. The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes. Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases. The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis. Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure. Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups. We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site.

Amino Acid Sequence↗
PubMed1989

The modification of essential lysine residues for actin binding of myosin subfragment-1 by pyridoxal-5'-phosphate.

Myosin subfragment-1 was modified with pyridoxal-5'-phosphate, a lysine modifying agent. Approximately two lysines could be blocked with a concomitant decrease in the actin-activated Mg2(+)-ATP-ase activities of S-1 remained unchanged. This selective inhibition of actin-activated Mg2(+)-ATP-ase activity of S-1 by pyridoxal-5'-phosphate was further characterized by kinetic studies. The double reciprocal plot revealed no change in the Vmax, while KM increased from 15 microM to 36 microM indicating that the modification reduced the actin affinity of S-1. The effects of pyridoxylation of S-1 were compared to those of 2, 4, 6 trinitrobenzene-sulfonate modification of S-1. The two types of lysine modification are strikingly different. The reactive lysine residue (Lys 83) remained unmodified after pyridoxylation of S-1 thus the effects of trinitrophenylation could be revealed independently in the double-modified sample. Furthermore after trinitrophenylation the S-1 fragments were found to be protected against partial tryptic digestion in the presence of nucleotides.

Actins↗
PubMed1978

Mode of binding of pyridoxal 5'-phosphate in rat liver ornithine aminotransferase.

1. Pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate were effective for the association of apo-form II of ornithine aminotransferase [EC 2.6.1.13]; whereas other B6 derivatives, such as pyridoxal, pyridoxamine, pyridoxine and pyridoxine 5'-phosphate, had no effect on form II of this apoenzyme. 2. The pyridoxal 5'-phosphate contents of the native enzyme, and reconstituted forms I and II were determined by two different methods to be 1.5 moles, 2.5 moles and 3.3 moles of pyridoxal 5'-phosphate/mole of enzyme, respectively.

Animals↗
PubMed1989

Pyridoxal phosphate-dependent reactions in water pools.

Pyridoxal 5'-phosphate, its Schiff base with L-alanine, and cytosolic aspartate aminotransferase were dissolved in isooctane solutions containing reverse micelles of the surfactant di-2-ethylhexylsodium sulfosuccinate and water. The physico-chemical properties of these compounds in the new environment have been studied.

Alanine↗