Glucose-6-Phosphate Dehydrogenase in Plastids from Developing Castor Bean Seeds

Glucose-6-phosphate dehydrogenase, together with the other enzymesof pentose phosphate pathway, was found in the cytosol as wellas in the plastid from developing castor bean (Ricinus communisL.) seeds. The plastid enzyme was found in both the matrix andthe membrane. The plastid enzyme has a sharp pH profile withthe optimum at 8.5, while the cytosolic enzyme has a broad pHprofile, optimum at 7.5. The plastid enzyme was inactivatedby storage at 0°C and by detergents such as Triton X-100,Brij and Nonidet, but the cytosolic enzyme was not. Slab geldisc electrophoresis indicated that three isoenzymes of glucose-6-phosphatedehydrogenase were found in the plastid but one enzyme in thecytosol of developing castor bean seed. From the presence ofglucose-6-phosphate dehydrogenase in the plastid, the operationof whole pentose phosphate pathway in this organelle of developingcastor bean seeds is suggested. (Received September 21, 1982; Accepted January 17, 1983)     

Plastid and cytosolic phosphofructokinases from the developing endosperm of ricinus communis: II. Comparison of the kinetic and regulatory properties of the isoenzymes

A steady-state kinetic analysis of plastid phosphofructokinase at pH 8.2 is consistent with the enzyme having a sequential reaction mechanism. Cytosolic phosphofructokinase probably has a similar mechanism. At pH 7.0 plastid phosphofructokinase shows cooperative binding of fructose 6-phosphate and is inhibited by higher concentrations of ATP. In contrast cytosolic phosphofructokinase shows normal kinetics at both pH 8.2 and 7.0 with respect to fructose 6-phosphate and is not inhibited by ATP. In the case of plastid phosphofructokinase the affinity for fructose 6-phosphate increases as the pH is raised from 7 to 8.2 whereas cytosolic phosphofructokinase is affected in an opposite manner. Phosphate is the principal activator of plastid phosphofructokinase since the cooperative kinetics toward fructose 6-phosphate are shifted toward Michaelis-Menten kinetics by 1 mm sodium phosphate and this concentration of phosphate relieves the inhibition by ATP. Both isoenzymes are inhibited by phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate at pH 7.2. Plastid phosphofructokinase is most strongly inhibited by phosphoenol pyruvate with the I_0.5 value varying from 0.08 to 0.5 μm depending on substrate concentrations; phosphate reverses this inhibition. In contrast cytosolic phosphofructokinase is much less inhibited by phosphoenolpyruvate with an I_0.5 approximately 1000-fold higher. Cytosolic phosphofructokinase is powerfully inhibited by 3-phosphoglycerate with an I_0.5 value of 60 μm and this appears to be the principal regulator of this isoenzyme. The two isoenzymes of phosphofructokinase in the endosperm appear, therefore, to be regulated differently. Plastid phosphofructokinase is inhibited by phosphoenolpyruvate and ATP and is activated by phosphate; whereas the cytosolic enzyme is inhibited principally by 3-phosphoglycerate and this inhibition is only partially relieved by phosphate. Some of the differences reported previously for phosphofructokinases from different plant tissues may, therefore, be due to varying ratios of the cytosolic and plastid isoenzymes.     

Kinetic Properties of the ATP-Dependent Phosphofructokinase Isoenzymes from Cucumber Seeds

Two isoenzymes of ATP:D-fructose-6-phosphate 1-phosphotransferase(phosphofructokinase) are present in germinating cucumber seeds,one in the plastids and the other in the cytosol. Both isoenzymeswere purified and some of their kinetic properties studied.These two isoenzymes differ kinetically, the pH optimum of thecytosolic isoenzyme being 7.2 and that of the plastid isoenzymebeing 8.0. Both isoenzymes are activated by phosphate althoughthe concentration required for activation is much lower forthe plastid isoenzyme than cytosolic isoenzyme. Phosphate increasesthe affinity of the isoenzymes for fructose-6-phosphate andalso changes the sigmoidal kinetics of the plastid isoenzymefor this substrate to hyperbolic kinetics at pH 7.2. The fructose-6-phosphatesaturation kinetics of the cytosolic isoenzyme becomes moresigmoidal with an increase in pH while the opposite is truefor the plastid isoenzyme. The cytosolic isoenzyme has a higheraffinity for fructose-6-phosphate at pH 7.2 than pH 8.0 whilethe affinity of the plastid isoenzyme for fructose-6-phosphateis highest at pH 8.0. Both isoenzymes are inhibited by ATP andthe extent of inhibition is pH dependent. The cytosolic isoenzymeis more sensitive to ATP inhibition at pH 8.0 than pH 7.2 whilethe opposite holds for the plastid isoenzyme. Magnesium alleviatesthe ATP inhibition of the plastid isoenzyme suggesting thatfree ATP is the inhibitory form. In contrast the ATP inhibitionof the cytosolic isoenzyme apparently appears to be caused bythe magnesium-ATP complex. (Received May 19, 1987; Accepted January 18, 1988)     

Plant Pyruvate Kinase

Pyruvate kinase is an important enzyme of glycolytic pathway that also functions in providing carbon skeleton for fatty acid biosynthesis. It has been purified to near homogeneity from Ricinus communis, Selenastrum minutum, Cynodon dactylon, Brassica campestris and B. napus, and characterised. Partially purified preparations are reported from several other sources. A phosphoenolpyruvate (PEP) phosphatase accompanies pyruvate kinase. In plants, two isozymes of pyruvate kinase are reported, namely cytosolic and plastidic. Isoforms of cytosolic pyruvate kinase have also been reported from spinach. In most cases pyruvate kinase is a tetrameric protein and the molecular mass lies between 200 to 250 kDa. The pH optimum is in the range of 6.2 to 7.5. It requires both Mg²⁺ and K⁺ for maximum activity. ATP, citrate, and oxalate inhibit pyruvate kinase in most cases. A sequential compulsory ordered mechanism of binding of substrates to the enzyme has been proposed.     

Plastid 3-hydroxy-3-methylglutaryl coenzyme A reductase has distinctive kinetic and regulatory features: Properties of the enzyme and positive phytochrome control of activity in pea seedlings

Plastid 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (mevalonate:NADP oxidoreductase [acylating CoA] EC 1.1.1.34) differs from the cytosolic (microsomal) reductase in pH optimum and apparent K_m for RS-HMG-CoA. Values for the plastid and cytosolic enzyme (brackets) are: pH optimum 7.9 (6.9); apparent K_mRS-HMG-CoA, 0.77 μm (160 μm). Hence the plastid and cytosolic enzymes appear to be different species and not simply compartmented forms of the same protein. The plastid reductase is membrane bound, optimally active only in the presence of dithiothreitol, and specifically requires NADPH; in these respects it is similar to the cytosolic enzyme. In dark-grown seedlings irradiated with red light plastid reductase activity increases to 139% of controls after 20 min, approximately double after 1.75 h, and subsequently declines to a new steady state higher than controls. Far-red reversal studies indicate phytochrome (Pfr) mediation. Reversal can only be demonstrated with very brief (1.5 min) red irradiation followed immediately by far red. It is concluded that Pfr does not act by binding to the enzyme, but that the regulatory mechanism is closely linked to the primary action of Pfr. The rapid Pfr stimulation indicates that this is an early event in the phytochrome control of chloroplast development. The response time and light effects on plastid isoprenoids (photosynthetic and hormonal) also suggest that the regulation of this enzyme is associated with the coordinate control of chloroplast and leaf development by phytochrome. The present positive Pfr control of the plastid reductase contrasts with the previously reported negative Pfr control of the cytosolic reductase.     

Light-dependent development of single cell C4 photosynthesis in cotyledons of Borszczowia aralocaspica (Chenopodiaceae) during transformation from a storage to a photosynthetic organ

BACKGROUND AND AIMS: Previous work has shown that Borszczowia aralocaspica (Chenopodiaceae) accomplishes C4 photosynthesis in a unique, polarized single-cell system in leaves. Mature cotyledons have the same structure as leaves, with chlorenchyma cells having biochemical polarization of dimorphic chloroplasts and C4 functions at opposite ends of the cell. KEY RESULTS: Development of the single-celled C4 syndrome in cotyledons was characterized. In mature seeds, all cell layers are already present in the cotyledons, which contain mostly lipids and little starch. The incipient chlorenchyma cells have a few plastids towards the centre of the cell. Eight days after germination and growth in the dark, small plastids are evenly distributed around the periphery of the expanding cells. Immunolocalization studies show slight labelling of Rubisco in plastids in seeds, including chlorenchyma, hypodermal and water storage, but not epidermal, cells. After imbibition and 8 d of growth in the dark labelling for Rubisco progressively increased, being most prominent in chlorenchyma cells. There was no immunolabelling for the plastid C4 enzyme pyruvate, Pi dikinase under these conditions. Cotyledons developing in light show formation of chlorenchyma tissue, induction of the cytosolic enzyme phosphoenolpyruvate carboxylase and development of dimorphic chloroplasts at opposite ends of the cells. Proximal chloroplasts have well-developed grana, store starch and contain Rubisco; those located distally have reduced grana, lack starch and contain pyruvate, Pi dikinase. CONCLUSIONS: The results show cotyledons developing in the dark have a single structural plastid type which expresses Rubisco, while light induces formation of dimorphic chloroplasts from the single plastid pool, synthesis of C4 enzymes, and biochemical and structural polarization leading to the single-cell C4 syndrome.     

NADP-malic enzyme from plants.

NADP-malic enzyme functions in plant metabolism as a decarboxylase of malate in the chloroplast or cytosol. It serves as a source of CO2 for photosynthesis in the bundle sheath chloroplasts of C4 plants and in the cytosol of Crassulacean acid metabolism plants, and as a source of NADPH and pyruvate in the cytosol of various tissues. Mg2+ or Mn2+ is required as a cofactor. The enzyme has a high specificity and low Km for NADP+. It exists as a tetramer which may undergo changes in oligomerization and exhibit hysteresis. Its kinetic properties vary depending on the compartmentation and function of the enzyme. The chloroplast form in C4 plants has a high pH optimum (pH 8) under high malate, which favours the tetramer, whereas lower pH (pH 7) favours the dimer form. Generally, other forms of the enzyme, which are thought to be cytosolic, have lower pH optima than the chloroplast enzyme. In a number of cases these forms have been shown to have allosteric properties with malate as a substrate. Chemical modifications of the plant enzyme suggest sulphydryl, histidine and arginine residues are required for catalysis. Primary sequence studies on the chloroplastic enzyme from C4 plants show significant similarities to cytosolic NADP-ME in plants and animals, including a sequence motif which is indicative of the NADP+ binding site. The possible origin of the chloroplast form of the enzyme is discussed.     

A high affinity pyruvate decarboxylase is present in cottonwood leaf veins and petioles: A second source of leaf acetaldehyde emission?

Considerable evidence indicates that acetaldehyde is released from the leaves of a variety of plants. The conventional explanation for this is that ethanol formed in the roots is transported to the leaves where it is converted to acetaldehyde by the alcohol dehydrogenase (ADH) found in the leaves. It is possible that acetaldehyde could also be formed in leaves by action of pyruvate decarboxylase (PDC), an enzyme with an uncertain metabolic role, which has been detected, but not characterized, in cottonwood leaves. We have found that leaf PDC is present in leaf veins and petioles, as well as in non-vein tissues. Veins and petioles contained measurable pyruvate concentrations in the range of 2 mM. The leaf vein form of the enzyme was purified approximately 143-fold, and, at the optimum pH of 5.6, the K_m value for pyruvate was 42 μM. This K_m is lower than the typical millimolar range seen for PDCs from other sources. The purified leaf PDC also decarboxylates 2-ketobutyric acid (K_m = 2.2 mM). We conclude that there are several possible sources of acetaldehyde production in cottonwood leaves: the well-characterized root-derived ethanol oxidation by ADH in leaves, and the decarboxylation of pyruvate by PDC in leaf veins, petioles, and other leaf tissues. Significantly, the leaf vein form of PDC with its high affinity for pyruvate, could function to shunt pyruvate carbon to the pyruvate dehydrogenase by-pass and thus protect the metabolically active vascular bundle cells from the effects of oxygen deprivation.     

Characterization and kinetics of isoenzymes of pyruvate kinase from developing castor bean endosperm

Isozymes of pyruvate kinase (PK) have been isolated from developing castor bean endosperm. One isozyme, PK_c, is localized in the cytosol, and the other, PK_p, is in the plastid. Both isozymes need monovalent and divalent cations for activity, requirements which can be filled by K⁺ and Mg²⁺. Both isozymes are inhibited by citrate, pyruvate, and ATP. PK_c has a much broader pH profile than PK_p and is also more stable. Both have the same K_m (0.05 millimolar) for PEP, but PK_p has a 10-fold higher K_m (0.3 millimolar) for ADP than PK_c (0.03 millimolar). PK_c also has a higher affinity for alternate nucleotide substrates than PK_p. The two isozymes have different kinetic mechanisms. Both have an ordered sequential mechanism and bind phosphoenolpyruvate before ADP. However, the plastid isozyme releases ATP first, whereas pyruvate is the first product released from the cytosolic enzyme. The properties of the two isozymes are similar to those of their counterparts in green tissue.     

PURIFICATION AND CHARACTERIZATION OF PYRUVATE KINASE FROM THE GREEN ALGA CHLAMYDOMONAS REINHARDTII1

A single form of pyruvate kinase was isolated from the green alga Chlamydomonas reinhardtii Dang. (Chlorophyta) and partially purified over twentyfold, yielding a final specific activity of 2.68 μmol pyruvate produced-min^-1.mg^-1 protein. Studies of its physical characteristics reveal that the pyruvate kinase is heat stable, is partially inactivated by sulfhydryl reagent N-ethylmaleimide, and has a pH optimum at 6.8 and a native molecular mass of 224 kDa. Immunological precipitation and western blotting, using antibodies raised against Selenastrum minutum Naeg. (Chlorophyta) cytosolic pyruvate kinase, reveal that C. reinhardtii pyruvate kinase possesses a subunit molecular mass of 57 kDa, indicating a homo-tetrameric structure. This enzyme exhibits an absolute requirement for a divalent cation that can be fulfilled, by Mg²⁺. The monovalent cation K⁺ acts as a strong activator. The K_m values for phosphoenolpyruvate and adenosine diphosphate (ADP) are 0.16 mM and 0.18 mM, respectively. The enzyme is capable of using other nucleotides with V_max for UDP, GDP, IDP, and CDP of 70%, 55%, 53%, and 25% of that with ADP, respectively. Dihydroxyacetone phosphate, ribulose 1,5-bisphosphate, adenosine monophosphate (AMP), ribose-5-phosphate, and glyceraldehyde-3-phosphate are activators, whereas glutamate, orthophosphate, adenosine triphosphate (ATP), citrate, isocitrate, malate, oxalate, phosphoglycolate, and 2,3-diphosphoglycerate are potent inhibitors of this enzyme. Dihydroxyacetone phosphate can reverse the inhibition by glutamate and phosphate. These properties are discussed in light of pyruvate kinase regulation during anabolic and catabolic respiration. Substrate interaction and product inhibition studies indicate that ADP is the first substrate bound to the enzyme and pyruvate is the last product released (Ordered Bi Bi mechanism).     

Regulation of glycolysis in Neurospora crassa. Kinetic properties of pyruvate kinase.

Pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40), extracted from the mycelium of Neurospora crassa has been purified 560-fold by precipitation with ammonium sulphate, chromatography with DEAE-Sephadex, and gel filtration with Sephadex G-200. Potassium and magnesium are required for enzyme activity. Fructose, 1,6-diphosphate is the only physiological activator found for the enzyme. In decreasing order of potency, citrate, oxalacetate, calcium, and ATP are inhibitors. Phosphoenolpyruvate is cooperatively bound by the enzyme and the cooperatively is reduced by ATP and completely eliminated by fructose-1,6-diphosphate. Lowering of pH from 7-5 to 5-5 changes the Hill coefficient from 2-7 to 1-0. Substitution of ADP by other nucleotides reduces enzyme activity. Manganese can substitute for the cofactor magnesium, but the reaction velocity is then reduced. MgADP- is cooperatively bound by the enzyme and inhibition of the enzyme occurs only when either magnesium or ADP is in excess of the other beyond the optimum concentration. These kinetics properties of pyruvate kinase are compatible with the role of a regulator of glycolysis in Neurospora crassa.     

Purification and characterization of pyruvate kinase from Trypanosoma brucei

Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been partially purified by carboxymethylcellulose chromatography, and gel filtration. The enzyme is unstable in aqueous solution and requires the presence of a thiol protecting reagent as well as glycerol for the maintenance of activity. Dithiothreitol activates as well as stabilizes the enzyme. Phosphoenolpyruvate allosterically activates trypanosome pyruvate kinase whereas hyperbolic kinetics are found when ADP is the variable substrate. Mg²⁺ or Mn²⁺ ions and a monovalent cation are essential for enzyme activity. Fructose 1,6-diphosphate acts as a heterotropic allosteric activator, markedly decreasing the S_0.5 value for phosphoenolpyruvate from 1.34 to 0.25 mm at 1 mm fructose 1,6-diphosphate and transforms the phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form. The enzyme has a pH optimum of 6.5–7.0 and a molecular weight of 270,000 ± 27,000 as estimated by gel chromatography. Purine nucleotides are the preferred coenzymes for the reaction, having much lower K_m values than the pyrimidine nucleotides. The possible role of pyruvate kinase in the regulation of glycolysis in T. brucei is discussed.     

Characterization of purified leaf cytosolic pyruvate kinase from the C4 plant Cynodon dactylon

Cytosolic pyruvate kinase (EC 2.7.1.40) from leaves of the C₄ plant Cynodon dactylon (L.) Pers. was purified 56-fold to apparent homogeneity by polyethylene glycol fractionation and column chromatography including Q-Sepharose anion exchanger, ADP-Agarose and gel filtration. Nondenaturing PAGE of the final preparation resulted in a single protein band that co-migrated with the pyruvate kinase activity. Gel filtration and SDS-PAGE (± DTT) showed that this enzyme has a molecular mass of 200 kDa and is a homotetramer with a subunit molecular mass of 50 kDa. The subunits are not associated to each other with S-S bonds. The enzyme has a pH optimum of 6.2 and is heat stable. Typical Michaelis-Menten kinetics was obtained for both substrates, PEP and ADP, with K_m values of 64 and 235 μ M , respectively. Initial velocity studies indicated a sequential binding of the substrates to the enzyme.     

Purification and properties of an asparagine aminotransferase from Pisum sativum leaves

The enzyme responsible for the transamination of L-asparagine in pea leaves has been partially purified. It appears to be the same protein as the serine-glyoxylate aminotransferase. It is able to use serine or asparagine as amino donors and pyruvate or glyoxylate as amino acceptors. The reaction is reversible but the equilibrium is toward glycine or alanine production. The favored substrates are serine and glyoxylate: serine shows competitive inhibition toward asparagine, as does pyruvate toward glyoxylate. Substrate interaction and product inhibition patterns are consistent with a ping-pong mechanism. The enzyme has a pH optimum at 8.1. Gel filtration indicates a Mr of 105,000. Inhibition was caused by aminoxyacetate and hydroxylamine, but the enzyme was unaffected by isonicotinic acid hydrazide. The apoenzyme was resolved and was inactive: addition of pyridoxal 5'-phosphate restored 85% of the original activity.     

Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells

Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation.     

Subcellular location of delta-pyrroline-5-carboxylate reductase in root/nodule and leaf of soybean

The expression of Δ¹-pyrroline-5-carboxylate reductase (P5CR) gene was found to be higher in soybean root nodules than in leaves and roots, and its expression in roots appeared to be osmoregulated (AJ Delauney, DPS Verma [1990] Mol Gen Genet 221: 299-305). P5CR was purified to homogeneity as a monomeric protein of 29 kilodaltons by overexpression of a soybean P5CR cDNA clone in Escherichia coli. The pH optimum of the purified P5CR was altered by increasing the salt concentration, and maximum enzyme activity was attainable at a lower pH under high salt (0.2-1 molar NaCl). Kinetic studies of the purified enzyme suggested that nicotinamide adenine dinucleotide phosphate⁺ inhibited P5CR activity, whereas nicotinamide adenine dinucleotide⁺ did not. Subcellular fractionation and antibodies raised against purified soybean P5CR were used to investigate location of the enzyme in different parts of soybean as well as in leaves of transgenic tobacco plants synthesizing soybean P5CR. P5CR activity was present in cytoplasm of soybean roots and nodules as well as in leaves, but in leaves, about 15% of the activity was detected in the plastid fraction. The location of P5CR was further confirmed by western blot assay of the proteins from cytosol and plastid fractions of different parts of the plant. Expression of soybean nodule cytosolic P5CR in transgenic tobacco under the control of cauliflower mosaic virus 35S promoter led to the accumulation of this protein exclusively in the cytoplasm, suggesting that the chloroplastic activity may be due to the presence of a plastid form of the enzyme. The different locations of P5CR in root and leaf suggested that proline may be synthesized in different subcellular compartments in root and leaf. Proline concentration was not significantly increased in transgenic plants exhibiting high level P5CR activity, indicating that reduction of P5C is not a rate-limiting step in proline production.     

Isozymes of phosphoglyceromutase from the developing endosperm of Ricinus communis: isolation and kinetic properties

The isozymes of phosphoglyceromutase from the developing endosperm of Ricinus communis have been partially purified. The purified cytosolic and plastid isozymes have specific activities of 622.8 and 83.8 mumol min-1 mg protein-1, respectively. They both have relative molecular masses of approximately 64,000. The cytosolic enzyme has lower Km values for both 2-phosphoglycerate and 3-phosphoglycerate than the plastid enzyme. The Km values for 3-phosphoglycerate are 330 +/- 25 and 430 +/- 48 microM for the cytosolic and plastid isozymes, respectively. The corresponding Km values for 2-phosphoglycerate are 60 +/- 10 and 112 +/- 22 microM. The two isozymes also have different pH optima and heat labilities. Neither isozyme requires 2,3-bisphosphoglycerate or a divalent cation and neither is regulated by metabolites.     

Purification and properties of alanine dehydrogenase from Bacillus sphaericus.

1. The bacterial distribution of alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating, EC 1.4.1.1) was investigated, and high activity was found in Bacillus species. The enzyme has been purified to homogeneity and crystallized from B. sphaericus (IFO 3525), in which the highest activity occurs. 2. The enzyme has a molecular weight of about 230 000, and is composed of six identical subunits (Mr 38 000). 3. The enzyme acts almost specifically on L-alanine, but shows low amino-acceptor specificity; pyruvate and 2-oxobutyrate are the most preferable substrates, and 2-oxovalerate is also animated. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. 4. The enzyme is stable over a wide pH range (pH 6.0--10.0), and shows maximum reactivity at approximately pH 10.5 and 9.0 for the deamination and amination reactions, respectively. 5. Alanine dehydrogenase is inhibited significantly by HgCl2, p-chloromercuribenzoate and other metals, but none of purine and pyrimidine bases, nucleosides, nucleotides, flavine compounds and pyridoxal 5'-phosphate influence the activity. 6. The reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by ammonia and pyruvate, and the products are released in the order of L-ALANINE AND NAD+. The Michaelis constants are as follows: NADH (10 microM), ammonia (28.2 mM), pyruvate (1.7 mM), L-alanine (18.9 mM) and NAD+ (0.23 mM). 7. The pro-R hydrogen at C-4 of the reduced nicotinamide ring of NADH is exclusively transferred to pyruvate; the enzyme is A-stereospecific.