Purification and properties of deoxyadenosine kinase from rat liver mitochondria. I. Purification and physical properties

Deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76, AdR kinase) from rat liver mitochondria has been partially purified and compared with partially purified AdR kinase from the cytosol of the same biological material. Some physical properties of both enzymes, including molecular weight, gel electrophoresis and gel isoelectric focusing were investigated and considerable differences between these data for mitochondrial and cytosol AdR kinase were found.     

Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.     

Purification and properties of a phosphoprotein phosphatase from rat liver.

A phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been partially purified from rat liver homogenates by (NH4)2SO4 and ethanol precipitations followed by DEAE-cellulose and Sepharose 6B chromatography. The phosphoprotein phosphatase is capable of cleaving [32P]phosphate from radiolabelled phosphopyruvate kinase (type L) (EC 2.7.1.40), phosphohistones, and phosphoprotamine. However, it did not detectably dephosphorylate ATP, ADP, DL-phosphorylserine or beta-glycerophosphate. Dephosphorylation of [32P]phosphopyruvate kinase was stimulated by divalent cations and inhibited by ATP, ADP, Fru-1,6-P2, and orthophosphate. Divalene cations could reverse inhibition induced by ADP or ATP. At least one function of the phosphoprotein phosphatase may be to remove phosphate groups from the phosphorylated form of pyruvate kinase in the liver.     

Adenosine kinase from human liver

Adenosine kinase (ATP: adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified to homogeneity from human liver. The yield was 55% of the initial activity with a final specific activity of 6.3 mumol/min per mg protein. The molecular weight was estimated as about 40 000 by Sephadex G-100 gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme catalyzed the phosphorylation of adenosine, deoxyadenosine, arabinoadenosine, inosine and ribavirin. The activity of deoxyadenosine phosphorylation was 18% of that of adenosine. The pH optimum profile was biphasic; a sharp pH optimum at pH 5.5 and a broad optimum at pH 7.5--8.5. The Km value for adenosine was 0.15 micrometer, and the activity was strongly inhibited at higher concentrations than 0.5 micrometer. ATP, dATP, GTP and dGTP were proved to be effective phosphate donors. Co2+ was more effective than Mg2+, and Ca2+, Mn2+, Fe2+ and Ni2+ showed about 50% of the activity for Mg2+. Some difference in structure between the adenosine kinase from human liver and that from rabbit or rat tissue, was observed by amino acid analysis and peptide mapping analysis.     

cDNA cloning, overproduction and characterization of rat adrenodoxin reductase.

We isolated a full-length cDNA clone for rat adrenodoxin reductase (AdR). The precursor of rat AdR was predicted to consist of 34 amino-terminal residues of extrapeptide for transport into mitochondria and the following 460 residues of the mature peptide region. The deduced amino acid sequence was 70.8 and 61.8% homologous to those of bovine and human AdRs in the extrapeptide region, respectively, and 88.5% homologous to both the sequences of bovine and human AdRs in the mature peptide region. The predicted mature form of rat AdR was directly expressed in Escherichia coli, using cDNA, and was purified with a yield of 32 mg/l of culture. The purified recombinant rat AdR showed an absorption spectrum characteristic of a flavoprotein with peaks at 270, 378 and 450 nm and shoulders at 280, 425 and 474 nm. The extinction coefficient was estimated to be 10.9 mM(-1) cm(-1) at 450 nm. The absorbance ratio at 270 nm/450 nm was 7.1. From the θ(208) value in the circular dichroism spectrum, the alpha-helix content in the rat AdR was calculated to be 30%. In NADPH-cytochrome c reductase activity reconstituted with adrenodoxin (Ad), the apparent K(m) value of rat AdR for NADPH was 0.32 microM, a value significantly lower than that of bovine AdR (1.4 microM). The rat AdR showed a higher affinity to the heterologous redox partner (bovine Ad, K(m)=9.3 nM) than to the native partner (rat Ad, K(m)=16.7 nM), whereas the affinity of bovine AdR was slightly higher to the native partner (bovine Ad, K(m)=37.1 nM) than to the heterologous partner (rat Ad, K(m)=46.8 nM). The K(m) values showed a reverse correlation to the difference of pI values between the redox partners. These results indicate that AdR binds to Ad mainly by ionic interaction.     

Partial purification and properties of a cyclic 3',5'-AMP-independent protein kinase from rat liver.

1. A cyclic 3',5'-AMP-independent protein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from rat liver cytosol was partially purified and characterized. Purification by (NH4)2SO4 precipitation, DEAE-cellulose, Bio Gel A-0.5 m and cellulose phosphate chromatography increased the specific activity about 700-fold. 2. An endogenous protein substrate was closely associated with the protein kinase and was not separable from this enzyme up to the cellulose phosphate stage. After phosphorylation, chromatography with Bio Gel A-0.5 m partially separated this endogenous phosphoprotein from the enzyme activity; this dissociation had no apparent effect on kinase activity with casein or phosvitin as substrates, or on the apparent molecular weight of the enzyme (approx. 158,000). 3. This protein kinase with casein, phosvitin, or the endogenous substrate was totally insensitive to the thiol reagents, p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), iodoacetamide, and N-ethylmaleimide. The enzyme was also unaffected by cyclic 3',5'-AMP, heat-stable protein kinase inhibitor, and the regulatory subunit of a cyclic 3',5'-AMP-dependent protein kinase.     

Histidine decarboxylase. Purification from fetal rat liver, immunologic properties, and histochemical localization in brain and stomach

Histidine decarboxylase from fetal rat liver was purified to near-homogeneity. The purified enzyme has a molecular weight of 210,000, and appears to contain two subunits with molecular weights of 145,000 and 66,000, respectively. The enzyme is inhibited by heavy metals such as Hg2+ and Zn2+ and sulfhydryl-reactive compounds such as 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme is partially dependent on exogenous pyridoxal phosphate. Extensive dialysis results in 50% loss of enzyme activity which can be fully recovered by adding pyridoxal phosphate. Affinity of pyridoxal phosphate for the apoenzyme is 0.1 microM at pH 6.8. Antibody against purified histidine decarboxylase was raised in rabbits. The antibody has been employed in immunohistochemical studies to visualize histidine decarboxylase containing cells and neuronal processes in rat stomach and brain, respectively. Immunologic studies indicate that histidine decarboxylase from brain, gastric mucosa, and fetal rat liver share common antigenic properties.     

Kinase activator protein mediates longer-term effects of starvation on activity of pyruvate dehydrogenase kinase in rat liver mitochondria.

Starvation of rats for 48 h increased the activity of PDH (pyruvate dehydrogenase) kinase 2.2-fold in extracts of liver mitochondria, 2.9-fold in PDH complex partially purified therefrom by fractional precipitation, and 5-fold in PDH complex partially purified by gel filtration on Sephacryl S-300. A protein fraction was separated from PDH complex in extracts of rat liver mitochondria by gel filtration or fractional precipitation, which increased the activity of PDH kinase in rat liver and pig heart PDH complexes. The activity of this protein fraction was increased approx. 2.5-fold by 48 h starvation of rats. With highly purified pig heart PDH complex it was shown that the protein fraction increased the Vmax. of the PDH kinase reaction 35-fold (fraction from fed rats) or 82-fold (fraction from starved rats); starvation had no effect on the concentration of protein fraction required to give 0.5 Vmax. Evidence is given that the increase in PDH kinase activity effected in extracts of liver mitochondria by starvation is due to increased activity of kinase activator protein, which is tightly bound by rat liver PDH complex and not removed by a single gel filtration. With pig heart PDH complex, increased PDH kinase activity was retained after gel filtration of an admixture with kinase activator protein from starved rats, but was restored to the control value by a second gel filtration; the alterations in PDH kinase activity were associated with obvious changes in protein bands in SDS gels.     

Deoxyadenosine/deoxycytidine kinase from Bacillus subtilis. Purification, characterization, and physiological function

Three different deoxyribonucleoside kinases with specificities toward thymidine, deoxyguanosine, and deoxyadenosine/deoxycytidine, respectively, are identified in Bacillus subtilis. The deoxyadenosin/deoxycytidine kinase is purified 950-fold employing blue Sepharose CL-6B column chromatography. The two deoxyribonucleoside kinase activities copurify and are present in the same band after polyacrylamide gel electrophoresis. The molecular weight is determined by gel filtration to be 47,000. Cytidine, adenosine, arabinosylcytosine, and arabinosyladenine are substrates for the enzyme. The activities toward these substrates are less than 20% of the activities obtained with deoxyadenosin and deoxycytidine. The deoxycytidine and deoxyadenosine saturation curves are hyperbolic with Km values for both nucleosides around 5 microM. The maximal velocities for the two deoxyribonucleosides are nearly identical with GTP as phosphate donor. GTP is the best donor showing hyperbolic saturation curves and Km values around 150 microM depending on the deoxyribonucleoside concentration. dATP and dCTP are inhibitors when GTP is the phosphate donor. They may both act as phosphate donors themselves. A divalent metal ion is required, Mg2+ giving the highest activity. A spontaneous mutant, selected as resistant to 5-fluorodeoxycytidine, lacks both deoxycytidine and deoxyadenosine kinase activity, while it retains normal activities toward deoxyguanosine, deoxyuridine, and thymidine.     

Choline kinase and ethanolamine kinase are separate, soluble enzymes in rat liver.

Choline kinase and ethanolamine kinase are located in the cytosol from rat liver and have been copurified more than 500-fold by affinity chromatography [P. J. Brophy and D. E. Vance (1976) FEBS Lett. 62, 123-125]. Kinetic properties of the two activities were determined. Choline kinase had a Km for choline of 0.033 mM and ethanolamine was a competitive inhibitor (Ki = 6.2 mM). Ethanolamine kinase had a Km for ethanolamine of 7.7 mM and choline was a 'mixed' type of inhibitor with a Ki of 0.037 mM. Both enzymes activities responded in a similar fashion to the adenylate energy charge. Betaine and choline phosphate partially inhibited both kinases with a 93% inhibition of the ethanolamine kinase by 5 mM choline phosphate. CTP and ethanolaminephosphate partially inhibited the ethanolamine kinase, but not the choline kinase. Other metabolites tested had negliglible effects on both kinases. The affinity-column-purified enzyme was analyzed by disc gel electrophoresis which resolved the two activities. Hence, although many of the properties of the two activities are similar, choline kinase and ethanolamine kinase must be separate enzymes. Analysis of rat liver cytosol by disc gel electrophoresis indicated four isoenzymes for choline kinase and ethanolamine kinase.     

In vitro phosphorylation and inactivation of rat liver acetyl-CoA carboxylase purified by avidin affinity chromatography

Acetyl-CoA carboxylase (EC 6.4.1.2) has been isolated from rat liver by an avidin-affinity chromatography technique. This preparation has a specific activity of 1.17 +/- 0.06 U/mg and appears as a major (240,000 dalton) and minor (140,000 dalton) band on SDS-polyacrylamide gel electrophoresis. Enzyme isolated by this technique can incorporate 1.09 +/- 0.07 mol phosphate per mol enzyme (Mr = 480,000) when incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase at 30 degrees C for 1 h. The associated activity loss under these conditions is 57 +/- 4.0% when the enzyme is assayed in the presence of 2.0 mM citrate. Less inactivation is observed when the enzyme is assayed in the presence of 5.0 mM citrate. The specific protein inhibitor of the cyclic AMP-dependent protein kinase blocks both the protein kinase stimulated phosphorylation and inactivation of acetyl-CoA carboxylase. The phosphorylated, inactivated rat liver carboxylase can be partially dephosphorylated and reactivated by incubation with a partially purified protein phosphatase. Preparations of acetyl-CoA carboxylase also contained an endogenous protein kinase(s) which incorporated 0.26 +/- 0.11 mol phosphate per mol carboxylase (Mr = 480,000) accompanied by a 26 +/- 9% decline in activity. We have additionally confirmed that the rat mammary gland enzyme, also isolated by avidin affinity chromatography, can be both phosphorylated and inactivated upon incubation with the cyclic AMP-dependent kinase.     

Complete purification of choline kinase from rat kidney and preparation of rabbit antibody against rat kidney choline kinase

Choline kinase was purified from rat kidney to apparent homogeneity with respect to both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme showed a minimum molecular weight of 42,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the other hand, the molecular size of 75,000-80,000 was estimated through Sephadex G-150 gel filtration, indicating that the enzyme in rat kidney exists most likely in a dimeric form. Specific antibody was raised in rabbit against the highly purified rat kidney choline kinase protein, then immunochemical cross-reactivity was investigated between rabbit antiserum and choline kinase preparations from various rat tissues. The antiserum inhibited choline kinase activity almost completely in the crude preparation not only from kidney but also from lung, intestine, and normal untreated liver cytosol, but it could inhibit only partially the activity from either 3-methylcholanthrene- or carbon tetrachloride-induced rat liver cytosol. The overall results demonstrated that, although choline kinase protein appears to exist in multiple forms in rat tissues, most of them are immunochemically identical, and that either 3-methylcholanthrene- or carbon tetrachloride-inducible form(s) of choline kinase in rat liver could be quite different from a form or forms existing in normal untreated rat liver cytosol.     

Calmodulin-dependent glycogen synthase kinase: Identification in liver of normal and phosphorylase kinase-deficient rats

We have purified a calmodulin-dependent glycogen synthase kinase from livers of normal and phosphorylase kinase-deficient (gsd/gsd) rats. No differences between normal and gsd/gsd rats were apparent in either (a) the ability of liver extracts to phosphorylate exogenous glycogen synthase in a Ca²⁺- and calmodulin-dependent manner or (b) the purification of the calmodulin-dependent synthase kinase. Although extracts from rat liver, when compared to rabbit liver extracts, had a significantly reduced ability to phosphorylate exogenous synthase, the calmodulin-dependent synthase kinase could be purified from rat liver using a protocol identical to that described for rabbit liver. Moreover, the synthase kinase purified from rat liver had properties very similar to those of the rabbit liver enzyme. The enzyme was completely dependent on calmodulin for activity against glycogen synthase, was unable to phosphorylate phosphorylase b, catalyzed the rapid incorporation of 0.4 mol phosphate/mol of glycogen synthase subunit, selectively phosphorylated sites 1b and 2 in the glycogen synthase molecule, had a Stokes' radius of about 70 Å, and appeared to be composed of subunits of M_r 56,000 and 57,000. These observations led us to conclude that (1) calmodulin-dependent glycogen synthase kinase is distinct from other kinases previously described and (2) the rat liver kinase and the rabbit liver kinase are very similar enzymes.     

Proteolytic activation of protein kinase C by membrane-bound protease in rat liver plasma membrane

Incubation of rat liver plasma membrane produced histone phosphorylating activity at 75 mM Mg2+ in the soluble fraction. The release of the kinase activity was inhibited by leupeptin and bovine pancreatic trypsin inhibitor, suggesting the involvement of membrane-bound protease. When partially purified protein kinase C from rat liver cytosol was treated with the trypsin-like protease purified from rat liver plasma membrane, histone phosphorylating kinase which was independent of Ca2+ and phospholipids, produced with a molecular weight of about 5 X 10(4). These results suggest that membrane-bound, trypsin-like protease activates protein kinase C in plasma membrane and the activated kinase is released from the membrane to the soluble fraction.     

Phosphorylation and inactivation of liver glycogen synthase by liver protein kinases

A rapid method for purifying glycogen synthase a from rat liver was developed and the enzyme was tested as a substrate for nine different protein kinases, six of which were isolated from rat liver. The enzyme was phosphorylated on a 17-kDa CNBr fragment to approximately 1 phosphate/87-kDa subunit by phosphorylase b kinase from muscle or liver with a decrease in the activity ratio (-Glc-6-P/+Glc-6-P) from 0.95 to 0.6. Calmodulin-dependent glycogen synthase kinase from rabbit liver produced a similar phosphorylation pattern, but a smaller activity change. The catalytic subunit of beef heart cAMP-dependent protein kinase incorporated greater than 1 phosphate/subunit initially into a 17-kDa CNBr peptide and then into a 27-30-kDa CNBr peptide, with an activity ratio decrease to 0.5. Glycogen synthase kinases 3, 4, and 5 and casein kinase 1 were purified from rat liver. Glycogen synthase kinase 3 rapidly phosphorylated liver glycogen synthase to 1.5 phosphate/subunit with incorporation of phosphate into 3 CNBr peptides and a decrease in the activity ratio to 0.3. Glycogen synthase kinase 4 produced a pattern of phosphorylation and inactivation of liver synthase which was very similar to that caused by phosphorylase b kinase. Glycogen synthase kinase 5 incorporated 1 phosphate/subunit into a 24-kDa CNBr peptide, but did not alter the activity of the synthase. Casein kinase 1 phosphorylated and inactivated liver synthase with incorporation of phosphate into a 24-kDa CNBr peptide. This kinase and glycogen synthase kinase 4 were more active against muscle glycogen synthase. Calcium-phospholipid-dependent protein kinase from brain phosphorylated liver and muscle glycogen synthase on 17- and 27-kDa CNBr peptides, respectively. However, there was no change in the activity ratio of either enzyme. The following conclusions are drawn. 1) Liver glycogen synthase a is subject to multiple site phosphorylation. 2) Phosphorylation of some sites does not per se control activity of the enzyme under the assay conditions used. 3) Liver contains most, if not all, of the protein kinases active on glycogen synthase previously identified in skeletal muscle.     

Induction of choline kinase by polycyclic aromatic hydrocarbons in rat liver. I. A comparison of choline kinases from normal and 3-methylcholanthrene-induced rat liver cytosol

Choline kinase in rat liver has been shown to be induced up to 2-fold by the administration of polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene and 3,4-benzo[a]pyrene (Ishidate, K., Tsuruoka, M. and Nakazawa, Y., (1980) Biochem. Biophys. Res. Commun. 96, 946-952). In order to characterize the nature of choline kinase induction by these carcinogens, the 3-methylcholanthrene-induced form as well as the normal form of choline kinase were partially purified from rat liver cytosol through acid treatment, (NH4)2SO4 precipitation and DEAE-cellulose column chromatography with linear KCl-gradient elution, and the catalytic properties were compared between the two preparations. Both enzyme activities were purified about 17-fold with a yield of 50% through the purification steps and there appeared no detectable difference in the elution pattern from either DEAE-cellulose column or Sephadex G-200 gel filtration. On the other hand, some differences were observed in catalytic properties between the two enzyme preparations; (1) the induced form showed a higher apparent Km value for choline (0.19 mM) when compared to the normal form (0.11 mM) and (2) the addition of polyamines caused a considerable increase in the maximum reaction velocity for the normal form whereas no remarkable change for the induced form, when the activities were plotted as a function of choline concentration. The overall results suggest that the 3-methylcholanthrene-induced form of choline kinase in rat liver could be different from the normal form, or that there exist several isoenzymes of choline kinase in rat liver, and one or some of them are inducible by the administration of polycyclic aromatic hydrocarbons.     

Estimation of acyldihydroxyacetone phosphate and lysophosphatidate in animal tissues

Chemical and enzymatic methods have been developed to measure small quantities (10(-8) - 10(-10) mol) of acyldihydroxyacetone phosphate in animal tissues. Lipids extracted from tissue samples with acidic CHCl3/methanol were subjected to solvent partitioning at two different pH values for partial purification of this keto-lipid from other lipids. This lipid was then estimated radiometrically either by chemical reduction with NaB3H4 or by enzymatic reduction with [4B-3H]NADPH using a partially purified acyldihydroxyacetone-phosphate reductase (EC 1.1.1.101). Thin-layer chromatography revealed the presence of a number of 3H-labeled lipids in the NaB3H4-reduced product and further purification of the product was necessary to estimate the amount of acyl[2-3H]glycerol 3-phosphate formed. The enzymatic reduction was very specific for acyl/alkyldihydroxyacetone phosphate. The amounts (nmol/g) of these keto-lipids estimated in different tissues by the enzymatic method were 10.06 +/- 0.64 (guinea pig liver), 4.3 +/- 0.15 (rat liver), 2.1 (rat testis), 1.5 (rad kidney) and 1.2 (rat brain). Monoacylglycerol 3-phosphate, i.e., lysophosphatidic acid, which was co-purified with acyldihydroxyacetone phosphate, was found to be present in relatively larger amounts in tissues. The amounts (nmol/g) of this lipid, estimated by enzymatically measuring the amounts of sn-glycerol 3-phosphate released after alkaline methanolysis of the partially purified lipid extracts, were 143 (guinea pig liver), 58 (rat liver), 53 (rat kidney) and 92 (rat brain). Stearic acid (18:0) was found to be the major (65%) fatty acid present in the lysophosphatidate purified from guinea pig liver.     

Partial purification and characterization of monomethylethanolamine kinase and dimethylethanolamine kinase activities from rat liver.

Monomethylethanolamine (MEA) kinase and dimethylethanolamine (DEA) kinase activities were purified 950 and 750 fold respectively from rat liver by conventional procedures. Certain properties of the partially purified enzyme preparation suggest that they are different from both choline kinase activity and ethanolamine kinase activity and differ from one another. This is based upon the following observations: 1. The heat stabilities of MEA kinase and DEA kinase activities are significantly different from one another and are different from the stability of choline kinase and ethanolamine kinase activities. 2. K+ in the presence of Mg2+ increases MEA kinase activity by 100% but has no effect on DEA kinase activity. 3. Different Ki values and the types of inhibition by several structurally related amino alcohols were found for MEA kinase and DEA kinase activities. 4. The purification fold of MEA kinase and DEA kinase are different from each other and from that of choline kinase and ethanolamine kinase.     

T-lymphoblast-specific nucleoside kinase: characterization and comparison with deoxycytidine kinase

The results of Sephadex G-100 gel filtration and sucrose density gradient centrifugation showed that T-lymphoblast-specific nucleoside kinase (TSK) purified from MOLT 4FT cell extract has a molecular weight of 26,500, while deoxycytidine kinase (dCK) 56,000. The pI value of TSK (pH 8.2) is quite different from that of dCK (4.8). TSK phosphorylated deoxycytidine, deoxyadenosine, deoxyguanosine and arabinocytidine, similar to dCK, but the respective kinetic properties were quite different. In the phosphate donor specificity and metal ion requirement, some differences were observed between TSK and dCK. dTTP was a good phosphate donor for dCK but no effect at all as phosphate donor for TSK. dCK was inhibited at very low concentration of dCTP, but TSK at much higher concentration of dCTP.     

Regulation of aspartate aminotransferase activity associated with change of pyridoxal phosphate level.

The activities of aspartate aminotransferase (EC 2.6.1.1) in the cytosol fractions of the liver and kidney of rats fed pyridoxine-deficient or control diet for 3 weeks were determined. In the absence of pyridoxal phosphate, the activities in the liver and kidney preparations of deficient rats were both abnormally low. The activity in the kidney fraction of deficient rats was restored to almost the control level by addition of pyridoxal phosphate, whereas that of the liver was only partially restored. The antigen activity, however, measured using anti-aspartate aminotransferase, was similar in liver fractions from deficient and control rats. These findings suggest the existence of a form of transaminase with little or no activity in the liver of deficient rats. The properties of the crude enzymes from deficient and control rats were indistinguishable by immunodiffusion, and the enzymes had the same subunit size and heat stability under the conditions tested. However, purified enzyme from deficient rat liver had a different specific activity and absorption spectrum from purified enzyme from normal liver.