The reaction of amino-oxyacetate with pyridoxal phosphate-dependent enzymes.

The carbonyl reagent amino-oxyacetate is frequently used in metabolic studies to inhibit individual pyridoxal phosphate enzymes. The reaction of this compound with three such enzymes, aspartate transaminase, 4-aminobutyrate transaminase and dopa (3,4-dihydroxyphenylalanine) decarboxylase, was studied to determine the extent to which the inhibition is reversible and the rates at which it takes place. Reactions were followed by observing changes in the absorption spectra of the bound coenzyme and by measuring loss of enzyme activity. The reactions with aspartate transaminase and aminobutyrate transaminase were not rapidly reversible and had second-order rate constants (21 degrees C) of 400 M-1.s.1 and 1300 M-1.s-1 respectively and all all concentrations studied showed the kinetics of a simple bimolecular reaction. The reaction with 4-aminobutyrate transaminase could not be reversed and that with aspartate transaminase could only be reversed significantly by addition of cysteinesulphinate to convert the enzyme into its pyridoxamine form. The first-order rate constant (21 degrees C) for the reverse reaction was 4 X 10(-5)s-1. Dopa decarboxylase inhibition by amino-oxyacetate was more rapid and more readily reversible, but measurements of rate and equilibrium constants were not obtained for this enzyme.     

Polyamine biosynthesis and vitamin B6 deficiency Evidence for pyridoxal phosphate as coenzyme for S-adenosylmethionine decarboxylase

Extracts of liver from vitamin B₆-deficient rats had only 50% of the S-adenosylmethionine decarboxylase activity of extracts of liver from control rats when assayed with no exogenous pyridoxal phosphate. When pyridoxal phosphate was included in the reaction mixture, both extracts exhibited the same activity, indicating that pyridoxal phosphate is the coenzyme for S-adenosylmethionine decarboxylase. There was no similar decreased activity in extracts of brain from vitamin B₆-deficient rats.The activity of the pyridoxal phosphate-dependent enzyme, ornithine decarboxylase, was increased in extracts of liver from vitamin B₆-deficient rats: 1.6-fold when assayed with no pyridoxal phosphate and 4-fold when assayed with pyridoxal phosphate.The concentrations of putrescine and spermidine were decreased 50% in liver of vitamin B₆-deficient animals, but only putrescine was decreased in brain. Putreanine was barely detectable in liver of vitamin B₆-deficient animals, but was unchanged in brain.     

Effect of pyridoxal phosphate on gamma-aminobutyric acid metabolism in different sections of the brain in irradiated animals]

A study was made of the effect of X-rays (4,5 Gy) and pyridoxal phosphate (3 mg/kg, v/v) on the activity of pyridoxal enzymes of GABA metabolism (e.g. glutamate decarboxylase, E.C. 4.1.1.15) and aminobutyrate aminotransferase (GABA-T, E.C. 2.6.1.19), as well as on GABA and glutamate content of the hemisphere cortex, brain stem and cerebellum of rabbits 6 and 10 days following irradiation and injection of a coenzyme. The height of the radiation sickness in rabbits was characterized by the manifest changes in glutamate decarboxylase and GABA-T activity, as well as in GABA and glutamate content of various brain parts differing in the structural and functional functions. The administration of pyridoxal phosphate produced pronounced activation of glutamate decarboxylase, particularly 6 days after irradiation and administration of the co-enzyme, and, to a lesser extent, influenced GABA-T function. Pyridoxal phosphate favored maintaining the GABA level above the control level in the hemisphere cortex and brain stem 6 and 10 days after exposure. The injection of pyridoxal phosphate did not normalize the glutamate content of the brain parts 6 days after exposure, but favored the normalization of GABA-T activity on day 10.     

Role of pyridoxal phosphate in mammalian polyamine biosynthesis. Lack of requirement for mammalian S-adenosylmethionine decarboxylase activity.

1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of methylglyoxal bis(guanylhydrazone) (1,1'-[(methylethanediylidine)dinitrilo]diguanidine) to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity or ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.     

Pyridoxal phosphate-unrelated inhibition of hippocampal glutamic acid decarboxylase by convulsant pyridoxal sulphate

Previous studies from this laboratory have shown that pyridoxal-5-sulphate, the synthetic analogue of pyridoxal phosphate, causes epileptic seizures including tonic-clonic convulsions. These seizure activities are prevented or reversed by GABA or muscimol. In an attempt to delineate the biochemical basis of these seizure processes further, we have studied and shown that pyridoxal sulphate is a competitive inhibitor of glutamic acid decarboxylase. In addition, the chronic administration of pyridoxal sulphate was shown to reduce the concentration of pyridoxal phosphate in the cerebellum, the cerebrum, and basal ganglion, but not in the hippocampus. The activity of hippocampal glutamic acid decarboxylase was reduced after 1, 3, and 5 days of chronic application of pyridoxal sulphate. The inhibition was demonstrated, whether glutamic acid decarboxylase was assayed in the presence or absence of its coenzyme pyridoxal phosphate. Unlike findings in the hippocampus, the activity of glutamic acid decarboxylase in other brain regions was unaffected following chronic application of pyridoxal sulphate. The selective toxic effects of pyridoxal sulfate to the hippocampus, a brain area well known for its high susceptibility to seizure discharges, deserve additional indepth investigation.     

FORMATION OF GABA FROM PUTRESCINE IN THE BRAIN OF FISH (SALMO IRIDEUS GIBB.)

Abstract— It was demonstrated after intraperitoneal and intracerebral injections of [1,4-¹⁴C]-putrescine.2 HCl that GABA is formed in vivo in the trout brain via a pathway in which glutamic acid is not an intermediate. Intraperitoneal and intracerebral injections of both thiosemicarbazide and 3-mercaptopropionic acid had no measurable effects on GABA concentration, transformation of glutamic acid into GABA in vivo , or on glutamate de-carboxylase activity in the brain within the first 3 h after the application of the inhibitors. Only a small decrease in concentration of pyridoxal phosphate was noticed in the fish brain after thiosemicarbazide administration. The relatively high concentrations of pyridoxal phosphate in the trout brain may be one of the reasons for the ineffectiveness of thiosemicarbazide in inhibiting glutamate decarboxylase in vivo. After intracerebral injections of [1-¹⁴C]GABA, a half-life of 7 h was determined for GABA. The slow turnover rate of GABA in trout brain, which can be assumed from this observation, may give a further explanation of the ineffectiveness of the glutamate decarboxylase inhibitors in lowering the GABA content ot fish brain within a few hours.     

Evaluations of tyrosine apodecarboxylase assays for pyridoxal phosphate

The alternate procedures used in the tyrosine apodecarboxylase assays for pyridoxal 5'-phosphate were evaluated to determine optimal conditions. Two preparations of tyrosine apodecarboxylase from Streptococcus faecalis were used: a cell suspension and a partially purified cell-free form. The activity of the decarboxylase was measured in two different assays using [14C]tyrosine or [3H]tyrosine as substrate. The presence of serum proteins caused greater inhibition of the assay for serum pyridoxal phosphate using [14C]tyrosine as substrate than the assay with [3H]tyrosine. In contrast, addition of deproteinized serum extract did not appear to inhibit either assay. The rate of reconstitution of the apodecarboxylase in the cell suspension was at least four times slower than that of the cell-free enzyme. The rate of reconstitution of the cell-free enzyme was faster in acetate than in citrate buffer. Inorganic sulfate or phosphate, at normal plasma concentrations, did not alter either the reconstitution rate of tyrosine decarboxylase or the final activity obtained in the assays using either substrate. The tyrosine apodecarboxylase assay for pyridoxal phosphate can be optimized by using deproteinized sera or plasma and incubating the cell-free apoenzyme with the coenzyme in acetate buffer for a time sufficient to obtain maximum reconstitution.     

Function of the phosphate group of pyridoxal 5'-phosphate in the glycogen phosphorylase reaction

To understand the catalytic mechanism of glycogen phosphorylase (EC 2.4.1.1), pyridoxal(5')phospho(1)-beta-D-glucose was synthesized and examined as a hypothetical intermediate in the catalysis. Pyridoxal phosphoglucose bound stoichiometrically to the cofactor site of rabbit muscle phosphorylase b in a similar mode of binding to the natural cofactor, pyridoxal 5'-phosphate. The rate of binding of pyridoxal phosphoglucose was only 1/100 compared with that of pyridoxal phosphate. The enzyme reconstituted with pyridoxal phosphoglucose showed no enzymatic activity at all even after prolonged incubation of the enzyme with substrates and activator. The present data would contradict participation of the phosphate group of pyridoxal phosphate in a covalent glucosyl-enzyme intermediate even if the covalent intermediate was formed during the catalysis.     

Interrelationships between pyridoxal phosphate and pyridoxal kinase in rabbit brain

—An inverse relationship was demonstrable between the concentration of pyridoxal phosphate and the activity of pyridoxal kinase in rabbit brain. The administration of pyridoxine elevated the concentration of pyridoxal phosphate and decreased the activity of pyridoxal kinase. Conversely, the administration of deoxypyridoxine decreased the concentration of pyridoxal phosphate and increased the activity of pyridoxal kinase. The increase in the activity of pyridoxal kinase by deoxypyridoxine was blocked by actinomycin D or puromycin. These results were interpreted to indicate that the tissue availability of pyridoxal phosphate regulated the activity of pyridoxal kinase.     

Seizure susceptibility in the developing mouse and its relationship to glutamate decarboxylase and pyridoxal phosphate in brain.

The relationship between the susceptibility to convulsions, the content of pyridoxal 5'-phosphate and the activity of pyridoxal kinase (EC 2.7.1.35) and glutamate decarboxylase (EC 4.1.1.15) in brain, was studied in the developing mouse. Seizures were induced by pyridoxal phosphate-gamma-glutamyl hydrazone (PLPGH), a drug previously reported to reduce the levels of pyridoxal 5'-phosphate and as a consequence to inhibit the activity of glutamate decarboxylase in brain of adult mice. It was found that the seizure pattern, as well as the time of appearance of convulsions, differed between 2- and 5-day old mice and 10-day old or older mice, indicating a progressive increase in seizure susceptibility during development. In brain, pyridoxal kinase activity and pyridoxal 5'-phosphate levels were decreased by the administration of PLPGH at all ages studied, whereas glutamate decarboxylase activity was inhibited less than 25% in 2- and 5-day old mice, and about 50% thereafter. Parallelly, the activation of glutamate decarboxylase by pyridoxal 5'-phosphate added in vitro to control homogenates was less in 2- and 5-day old mice than in older animals. It is concluded that the increase in the susceptibility to seizures induced by PLPGH during development is probably related to the increase observed in the sensitivity of glutamate decarboxylase in vivo to a decrease of pyridoxal 5'-phosphate levels. The correlation between pyridoxal 5'-phosphate, glutamate decarboxylase, and seizure susceptibility seems to be established at about 10 days of age.     

Seizure susceptibility in the developing mouse and its relationship to glutamate decarboxylase and pyridoxal phosphate in brain

The relationship between the susceptibility to convulsions, the content of pyridoxal 5′-phosphate and the activity of pyridoxal kinase (EC 2.7.1.35) and glutamate decarboxylase (EC 4.1.1.15) in brain, was studied in the developing mouse. Seizures were induced by pyridoxal phosphate-σ-glutamyl hydrazone (PLPGH), a drug previously reported to reduce the levels of pyridoxal 5′-phosphate and as a consequence to inhibit the activity of glutamate decarboxylase in brain of adult mice. It was found that the seizure pattern, as well as the time of appearance of convulsions, differed between 2- and 5-day old mice and 10-day old or older mice, indicating a progressive increase in seizure susceptibility during development. In brain, pyridoxal kinase activity and pyridoxal 5′-phosphate levels were decreased by the administration of PLPGH at all ages studied, whereas glutamate decarboxylase activity was inhibited less than 25% in 2- and 5-day old mice, and about 50% thereafter. Parallelly, the activation of glutamate decarboxylase by pyridoxal 5′-phosphate added in vitro to control homogenates was less in 2- and 5-day old mice than in older animals. It is concluded that the increase in the susceptibility to seizures induced by PLPGH during development is probably related to the increase observed in the sensitivity of glutamate decarboxylase in vivo to a decrease of pyridoxal 5′-phosphate levels. The correlation between pyridoxal 5′-phosphate, glutamate decarboxylase, and seizure susceptibility seems to be established at about 10 days of age.     

The oxidation of Schiff bases of pyridoxal and pyridoxal phosphate with amino acids by manganous ions and peroxidase

1. Oxygen was taken up rapidly when pyridoxal or pyridoxal phosphate was added to mixtures of pea-seedling extracts and Mn(2+) ions. 2. The increases in total oxygen uptake were proportional to the pyridoxal or pyridoxal phosphate added and were accompanied by the disappearance of these compounds. 3. In addition to Mn(2+) ions, the reactions depended on two factors in the extracts, a thermolabile one in the non-diffusible material and a thermostable one in the diffusate; these factors could be replaced in the reactions by horse-radish peroxidase (donor-hydrogen peroxide oxidoreductase, EC 1.11.1.7) and amino acids respectively. 4. When pyridoxal phosphate was added to mixtures of amino acids and Mn(2+) ions oxygen uptake was rapid after a lag period of 30-90min.; the lag period was shortened to a few minutes by peroxidase, particularly in the presence of traces of p-cresol, or by light. 5. When pyridoxal replaced pyridoxal phosphate relatively high concentrations were required and peroxidase had only a small activating effect. 6. Pyridoxal or pyridoxal phosphate disappeared during the reactions and carbon dioxide and ammonia were formed. 7. With phenylalanine as the amino acid present, benzaldehyde was identified as a reaction product. 8. It is suggested that the reactions are oxidations of the Schiff bases formed between pyridoxal or pyridoxal phosphate and amino acids, mediated by a manganese oxidation-reduction cycle, and resulting in oxidative decarboxylation and deamination of the amino acids.     

Effect of Ro 4-1284 on audiogenic seizure susceptibility and intensity in epilepsy-prone rats

The effects of CNS monoamine depletion on audiogenic seizure (AGS) susceptibility and intensity were studied in two types of Sprague-Dawley derived rats: (1) the progeny of a nonsusceptible strain (controls); and (2) the nonsusceptible progeny of epilepsy-prone (audiogenic seizure susceptible) parents (NSPSP). Forty-five minutes after injection of the benzoquinolizine Ro 4-1284, a significant fraction of the NSPSP developed AGS susceptibility, whereas the incidence in controls was not significant. AGS intensity was also significantly elevated 45 minutes and 19 days following Ro 4-1284 in NSPSP. In controls, there was a smaller, but significant, elevation of seizure intensity only at the earlier time period. Both types of rats exhibited a marked depletion of norepinephrine (NE) and 5-hydroxytryptamine (5-HT) in all of the six different areas of the CNS which were examined. In the NSPSP, a significant incidence of seizure susceptibility was retained as long as 19 days after Ro 4-1284 administration, despite the complete repletion of monoamine stores. These observations suggest that CNS monoaminergic neurons function as determinants of AGS susceptibility and intensity in animals which also carry some other genetically determined susceptibility factor(s). A deficiency in monoaminergic transmission is insufficient to cause susceptibility in animals not carrying the other trait(s). Also, although a monoaminergic deficit may initially cause the appearance of susceptibility, the presence of the deficit may not be necessary for the continuation of susceptibility once an animal has actually sustained an AGS in the presence of the monoaminergic deficit.     

STUDY ON THE INHIBITION OF BRAIN GLUTAMATE DECARBOXYLASE BY PYRIDOXAL PHOSPHATE OXIME-O-ACETIC ACID

The kinetics of the inhibition of mouse brain glutamate decarboxylase by pyri-doxaI-5′-phosphate oxime-O-acetic acid (PLPOAA) was studied. The inhibition was noncompetitive with regard to glutamic acid; it could be partially reversed by pyridoxal phosphate, but only when the concentration of the latter in the incubation medium was higher than that of pyridoxal-5′-phosphate oxime-O-acetic acid. The inhibition produced by aminooxyacetic acid, which is remarkably greater than that produced by PLPOAA, was also partially reversed only when an excess of pyridoxal phosphate was added. Both in the presence and in the absence of a saturating concentration of pyridoxal phosphate, the activity of the enzyme was decreased by PLPOAA at a 10^?4m concentration to a value of about 50 per cent of the control value obtained without added coenzyme. This activity could not be further reduced even when PLPOAA concentration was increased to 5 × 10^?3m . This same minimal activity of glutamate decarboxylase was obtained after dialysis of the enzymic preparation, or after incubation with glutamic acid in the cold followed by filtration through Sephadex G-25. The addition of pyridoxal phosphate to the dialysed or glutamic acid-treated enzyme restored the activity to almost the control values. PLPOAA did not affect the activity of glutamate decarboxylase from E. coli or that of DOPA decarboxylase and GABA transaminase from mouse brain. To account for the results obtained it is postulated that brain glutamate decarboxylase has two types of active site, one with firmly bound, non-dialysable pyridoxal phosphate and the other with loosely bound, dialysable coenzyme; PLPOAA behaves as a weak inhibitor probably because it can combine mainly with the loosely bound coenzyme site, while aminooxyacetic acid is a potent inhibitor probably because it can block both the ‘loosely bound coenzyme’ and the ‘firmly bound coenzyme’ sites.     

Inactivation of rhodanese by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate and other aromatic aldehydes inactivate rhodanese. The inactivation reaches higher extents if the enzyme is in the sulfur-free form. The identification of the reactive residue as an amino group has been made by spectrophotometric determination of the 5'-phosphorylated pyridoxyl derivative of the enzyme. The inactivation increases with pyridoxal 5'-phosphate concentration and can be partially removed by adding thiosulfate or valine. Prolonged dialysis against phosphate buffer also leads to the enzyme reactivation. The absorption spectra of the pyridoxal phosphate - rhodanese complex show a peak at 410 nm related to the Schiff base and a shoulder in the 330 nm region which is probably due to the reaction between pyridoxal 5'-phosphate and both the amino and thiol groups of the enzyme that appear reasonably close to each other. The relationship betweenloss of activity and pyridoxal 5'-phosphate binding to the enzyme shows that complete inactivation is achieved when four lysyl residues are linked to pyridoxal 5'-phosphate.     

Some aspects of pyridoxal phosphokinase in chick brain

Abstract— The activity of pyridoxal phosphokinase (EC 2.7.1.35) has been studied in two brain areas of the White Leghorn chick during post-hatch development. Activities of this enzyme were approximately the same in both forebrain and cerebellum at 2 days of age but when maximum activity was reached, at 20-25 days, the enzyme activity in forebrain was considerably higher than in the cerebellum. In homogenates, the activity of pyridoxal phosphokinase (as measured by pyridoxal phosphate formation) was inhibited by a particulate-bound inhibitor. In the forebrain of the 15-day-old chick, this inhibitor was detected in concentrations 3- to 5-fold greater than in the cerebellum. The inhibitor appeared to be an atypical ATPase which approached adult levels in the chick forebrain by two days after hatching. The possible physiological significance as well as the possible artifactual nature of this pyridoxal phosphokinase inhibitor in the in vitro assay system has been considered.     

A novel intermediate in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase.

An unusual intermediate bound to the enzyme was detected in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase. This intermediate had absorbance maxima at 464 and 440 nm. Such spectra are characteristic of resonance stabilized intermediates detected in the interaction of substrates and quasi-substrates with pyridoxal phosphate enzymes. An intermediate of this kind has not been detected in the interaction of thiosemicarbazide with other pyridoxal phosphate enzymes. This intermediate was generated slowly (t 1/2 = 4 min) following the addition of thiosemicarbazide (200 microM) to sheep liver serine hydroxymethyltransferase (5 microM). It was bound to the enzyme as evidenced by circular dichroic bands at 464 and 440 nm and the inability to be removed upon Centricon filtration. The kinetics of interaction revealed that thiosemicarbazide was a slow binding reversible inhibitor in this phase with a k(on) of 11 M-1 s-1 and a k(off) of 5 x 10(-4) s-1. The intermediate was converted very slowly (k = 4 x 10(-5) s-1) to the final products, namely the apoenzyme and the thiosemicarbazone of pyridoxal phosphate. A minimal kinetic mechanism involving the initial conversion to the intermediate absorbing at longer wavelengths and the conversion of this intermediate to the final product, as well as, the formation of pyridoxal phosphate-thiosemicarbazone directly by an alternate pathway is proposed.     

THE CONVULSANT ACTION OF HYDRAZIDES AND REGIONAL CHANGES IN CEREBRAL γ-AMINOBUTYRIC ACID AND PYRIDOXAL PHOSPHATE CONCENTRATIONS

Abstract— Regional changes in the concentration of GABA and pyridoxal phosphate were determined in rat brain after i.p. administration of convulsant doses of methyldithiocarbazinate (11 mg/kg), isonicotinic acid hydrazide (250 mg/kg) and thiosemicarbazide (25 mg/kg). At 15 and 30 min after methyldithiocarbazinate GABA concentrations were reduced in all brain regions (except ventral mid-brain). After 30 min the largest decrease was in the cerebellum (41%) and the smallest decrease in the hypothalamus (20%). Pyridoxal phosphate concentrations were decreased by 39-57%. After isonicotinic acid hydrazide. the regional decreases in GABA concentration were smaller and of slower onset than those seen after methyldithiocarbazinate. The pons-medulla was the first region to show a decrease (at 15 min) whereas a decrease was not seen in the frontal cortex until 45 min. Regional decreases in pyridoxal phosphate were smaller than those seen after methyldithiocarbazinate. After thiosemicarbazide, small regional decreases in GABA concentration were observed only in the hypothalamus, cerebellum, pons-medulla and posterior cortex (13-18%) and there was no apparent correlation between regional decreases in pyridoxal phosphate and regional decreases in GABA.     

The synthesis and subcellular distribution of pyridoxal phosphate in rat and bovine retinas

In the absence of any known studies dealing with status of vitamin B₆ metabolism in mammalian retinas, the concentration of pyridoxal phosphate and the activity of its synthesizing enzyme pyridoxal kinase were determined in rat retina and bovine retina and its subcellular compartments. In bovine retina, the highest concentration of pyridoxal phosphate (148 pmol/mg protein) was present in pellet 2 fraction containing synaptosomes comparable to those isolated from brain. The second highest concentration of pyridoxal phosphate (91 pmol/mg protein) was present in pellet 1 fraction containing large synaptosomes resembling photoreceptor cell terminals. The concentrations of pyridoxal phosphate in pellets 1 and 2 fractions were approx 3- to 6-fold higher than that found in the whole retina. The concentration of pyridoxal phosphate and the activity of pyridoxal kinase in the rat retina were considerably higher than those observed in the bovine retina. In general, no apparent correlation existed between the concentrations of pyridoxal phosphate and the activities of pyridoxal kinase in bovine retina and its subcellular compartments.     

Observations on the pyridoxal 5'-phosphate inhibition of DNA polymerases.

Pyridoxal 5'-phosphate at concentrations greater than 0.5 mM inhibits polymerization of deoxynucleoside triphosphate catalyzed by a variety of DNA polymerases. The requirement for a phosphate as well as aldehyde moiety of pyridoxal phosphate for inhibition to occur is clearly shown by the fact that neither pyridoxal nor pyridoxamine phosphate are effective inhibitors. Since the addition of nonenzyme protein or increasing the amount of template primer exerted no protective effect, there appears to be specific affinity between pyridoxal phosphate and polymerase protein. The deoxynucleoside triphosphates, however, could reverse the inhibition. The binding of pyridoxal 5'-phosphate to enzyme appears to be mediated through classical Schiff base formation between the pyridoxal phosphate and the free amino group(s) present at the active site of the polymerase protein. Kinetic studies indicate that inhibition by pyridoxal phosphate is competitive with respect to substrate deoxynucleoside triphosphate(s).