Purification and Some Properties of Adenosine (Phosphate) Deaminase from the Liver of the Squid Todarodes pacificus

An enzyme that catalyzed the deamination of adenosine 3′-phenylphosphonate was purified from squid liver to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 60,000 by SDS-PAGE and 140,000 by Sephadex G-150 gel filtration. The enzyme deaminated adenosine, 2′-deoxyadenosine, 3′-AMP, and 2′,3′-cyclic AMP, but not adenine, 5′-AMP, 3′,5′-cyclic AMP, ADP, or ATP. The apparent K_m and V_max at pH 4.0 for these substrates were comparable (0.11-0.34mM and 179-295 μmol min^?1 mg^?1, respectively). The enzyme had maximum activity at pH 3.5-4.0 for adenosine 3′-phenylphosphonate, at pH 5.5 for adenosine and 2′-deoxyadenosine, and at pH 4.0 for 2′,3′-cyclic AMP and 3′-AMP when the compounds were at concentration of 0.1 mM. The K_m at 4.0 and 5.5 for each substrate varied, but the Vmax were invariant. These results indicated that the squid enzyme was a novel adenosine (phosphate) deaminase with a unique substrate specificity.     

Studies on Metabolic Pathway of NAD in Yeast

Quantitative studies on yeast 5′-nucIeotidase are presented.

K_m values for purine 5′-nucleotides were generally smaller than those for pyrimidine 5′-nucleotides and, among purine series, K_m value for 5′-AMP was the smallest, while their V values were almost same.

The enzyme activity was inhibited in the competitive type by bases, nucleosides, 3′- or 2′-nucleotides, and NMN and in the mixed type by NAD and NADP.

Base-, ribose-, 3′- or 5′-phosphate moiety of nucleoside and nucleotide had some effects on binding with enzyme; especially the structure of base moiety characterizes the K_m or K_i value.

The enzyme activity was accelerated by Ni⁺⁺ or Co⁺⁺, which increases V value but never affects K_m value.

The relationship between the structure of substrate and its affinity towards enzyme is discussed.     

Some properties of choline acetyltransferase isolated from the electric organ ofTorpedo marmorata: Specificity for choline analogues and inhibition by certain amines and amino acids

(1) Choline acetyltransferase ofTorpedo marmorata electric organ was studied by using soluble tissue extracts partially purified by (NH₄)₂SO₄ fractionation. (2) Linear enzymatic rates were observed at 30°C, in the presence of 350 M acetyl-CoA and 50 mM choline, over a 30–40 min incubation period. (3) A number of analogues of choline, including mono-, di-, and triethylcholine and pyrrolcholine were synthesized and theK _m (apparent) andV (maximum velocity) values determined. TheK _m (apparent) for choline (11.5 mM), with theTorpedo enzyme, was high in comparison to values reported for mammalian or invertebrate nervous tissue. TheTorpedo enzyme was also not so specific for choline in comparison with the other choline analogues (based onK _m values) as were other sources of the enzyme. TheV values for choline and mono-, di-, and triethylcholine with theTorpedo enzyme indicated a direct relationship between enzyme activity andN-alkyl substitution. (4) Several amines and amino acids inhibited choline acetyltransferase fromTorpedo. Histamine (15 mM) brought about a 60% inhibition and was found to be a noncompetitive inhibitor with respect to choline.     

Purification and properties of 5′-nucleotidase from alkalophilic Bacillus No. C-3

5′-Nucleotidase (EC 3. 1. 3. 5) from alkalophilic Bacillus no. C-3 was purified to homogeneity. The molecular weight of the enzyme was 80,000 by gel filtration. The optimum pH for the activity was 9.5, and the enzyme was stable at pH 9.5–10.5 in a buffer containing 10 mM 2-mercaptoethanol. Substrate specificity study revealed that the enzyme acted on 5′-AMP strongly, on several 5′-nucleotides and ADP to a certain extent, but not on 3′-nucleotides, 2′-nucleotides, p-nitrophenyl phosphate, or ATP. The K_m value for 5′-AMP was 3.0 × 10⁻⁴ M. The enzyme required no divalent cation for its activity. The enzyme was inhibited by borate and arsenite ions but not by 1 mM EDTA.     

5′-Nucleotidases of retinal rod membranes

5′-Nucleotidase (EC 3.1.3.5) was solubilized from rod membranes with Ammonyx LO and purified by chromatographic methods. A highly sensitive radioassay was developed. The purified enzyme behaved as a homogeneous protein of 75,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as a protein of 79,000 in gel filtration. Thus, the enzyme does not contain subunits. The K_m values obtained were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin. Rabbit muscle G-actin formed a complex with the enzyme and inhibited its activity. The catalytic site of the enzyme was localized on the internal surface of the disk which, in terms of membrane sidedness, corresponds to the cell surface. A soluble 5′-nucleotidase was extracted from rod membranes with Tris buffer (pH 8.0) containing EGTA in the dark; less enzyme was extracted if the membranes had been exposed to light or incubated with Ca²⁺. The extracted enzyme was partially purified. The enzyme was unstable and lost 50% of its activity in 3 days at 3 °C. The K_m values were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by G-actin. A role for the soluble enzyme in the regulation of 5′-GMP in the rod outer segment was suggested.     

1,N6-etheno-2-aza-adenosine 3',5'-monophosphate: a new fluorescent substrate for cycle nucleotide phosphodiesterase

1,N⁶-etheno-2-aza-adenosine 3′,5′-monophosphate (cyclic 2-aza-?-AMP) has been shown to be a sensitive and an efficient substrate for the assay of cyclic-nucleotide phosphodiesterase. The relative activity is 75% compared to cyclic AMP. Two K_m values of 503 and 15 μm were observed with the beef heart enzyme.     

Molecular and functional characterization of choline transporter in rat renal tubule epithelial NRK-52E cells

Homeostatic regulation of the plasma choline concentration depends on the effective functioning of a choline transporter in the kidney. However, the nature of the choline transport system in the kidney is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake in the rat renal tubule epithelial cell line NRK-52E. Choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (K_m) of 16.5 μM and a maximal velocity (V_max) of 133.9 pmol/mg protein/min. The V_max value of choline uptake was strongly enhanced in the absence of Na⁺ without any change in K_m values. The increase in choline uptake under Na⁺-free conditions was inhibited by Na⁺/H⁺ exchanger (NHE) inhibitors. Choline uptake was inhibited by the choline uptake inhibitor hemicholinium-3 (HC-3) and organic cations, and was decreased by acidification of the extracellular medium and by intracellular alkalinization. Collapse of the plasma membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. NRK-52E cells mainly express mRNA for choline transporter-like proteins (CTL1 and CTL2), and NHE1 and NHE8. CTL1 protein was recognized in both plasma membrane and mitochondria. CTL2 protein was mainly expressed in mitochondria. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in NRK-52E cells and is responsible for choline uptake. This choline transport system uses a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE8. Furthermore, the presence of CTL2 in mitochondria provides a potential site for the control of choline oxidation.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain identification of phospholipids as calcium-independent activators.

Phosphatidyl inositol and lysophosphatidyl choline have been identified as activators of a partially purified brain cyclic nucleotide phosphodiesterase previously shown to be regulated in vitro by Ca²⁺ and a Ca²⁺-binding protein. Microgram quantities of either phospholipid produced a linear, immediate and reversible activation of the enzyme in the absence of Ca²⁺ and the Ca²⁺-dependent regulator (CDR). Fatty acids were also found to activate the phosphodiesterase to varying degrees, with oleic acid being the most effective. Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and lysophosphatidyl ethanolamine were not effective as activators. Only sodium dodecyl sulfate, of a variety of nonionic, cationic, and anionic detergents tested, activated the phosphodiesterase. Sodium dodecyl sulfate produced a modest degree of activation over a narrow concentration range, followed by enzyme denaturation at higher concentrations.The interaction of the phosphodiesterase with the phospholipid activators has been compared to its interaction with the Ca²⁺·CDR complex. Both Ca²⁺·CDR and lysophosphatidyl choline decreased the thermal stability of the enzyme to a similar extent. The apparent K_m of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 30 μm with guanosine-3′,5′-monophosphate (cGMP) as substrate and 1 mm with adenosine-3′,5′-monophosphate (cAMP) as substrate. With increasing lysophosphatidyl choline concentration, the apparent K_m for each nucleotide remained unchanged while the V increased. The apparent K_d for Mg²⁺ of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 3 μm and was unaffected by lysophosphatidyl choline concentration. Activation of the phosphodiesterase by lysophosphatidyl choline was characterized by a high degree of positive cooperativity, exhibiting a Hill coefficient of 3.8. Fluphenazine was a competitive inhibitor of both Ca²⁺·CDR and lysophosphatidyl choline activation of the enzyme.     

S1 nuclease of Aspergillus oryzae: characterization of the associated phosphomonoesterase activity

S₁ nuclease (EC 3.1.30.1) of Aspergillus oryzae was found to catalyze the hydrolysis of 2′- or 3′-phosphomonoester groups from several mono- and oligonucleotides. The specificity of the enzyme for mononucleotide substrates was determined by steady-state kinetic measurements at pH 4.5. The values of V were similar for all ribonucleoside 3′-phosphates tested, and they were 50–400 times greater than those for the corresponding deoxyribonucleotides or ribonucleoside 2′-phosphates. Purine nucleotides had lower apparent K_m values than pyrimidine nucleotides. Apparent K_m values of mononucleotides were also strongly dependent on the type of sugar and the positions of phosphoryl groups. Substrate specificity, as expressed by $\text{V}\text{K}{}_{\mathrm{m}}$, occurred in the following order: ribonucleoside 3′,5′-bisphosphate > ribonucleoside 3′-phosphate > deoxyribonucleoside 3′,5'-bisphosphate > deoxyribonucleoside 3′-phosphate ≈ ribonucleoside 2′-phosphate. S₁ nuclease also catalyzed the dephosphorylation of the dinucleotide ApAp at a high rate and the release of PP_i from adenosine 3′-diphosphate 5′-phosphate at a low rate. The phosphomonoesterase activity of the enzyme was competitively inhibited by single-stranded DNA and 5′-nucleotides. Apparent K_i values for adenosine compounds occurred in the order ATP < ADP < AMP ? adenosine. Tests of S₁ nuclease for phosphotransferase activity at pH 4.5 and 7.0 were negative.     

A bioluminescence method for determining adenosine 3'-phosphate 5'-phosphate (PAP) and adenosine 3'-phosphate 5'-sulfatophosphate (PAPS) in biological materials.

A rapid, sensitive, bioluminescence technique for detecting PAPS (adenosine 3′-phosphate 5′-sulfatophosphate) in biological materials is described. PAPS is first hydrolysed in 0.2 n HCl to PAP (adenosine 3′-phosphate 5′-phosphate) and is then assayed by the luciferin-luciferase system of the sea pansy, Renilla reniformis, which is specific for PAP. This bioluminescence system produces light at a rate that is proportional to the amount of PAP present. Light emission is measured in a liquid scintillation spectrometer with the two photomultipliers out of coincidence.Very low amounts of PAPS (10–100 pmoles) have been determined in extracts of yeast and various plant tissues by this method. The production of PAPS in extracts of young wheat leaves is enhanced by including either 5′-AMP or 3′-AMP in the reaction mixture. It is possible that these nucleotides protect PAPS from enzymes that degrade this compound, e.g., a nucleotidase.     

Phosphofructokinase of Solanum tuberosum tuber

Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. K_m for fructose-6-phosphate, ATP and Mg²⁺ was 2·1 × 10^?4 M, 4·5 × 10^?5 M and 4·0 × 10^?4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.     

Isolation and properties of hexokinase from loranthus leaves

Hexokinase was partially purified from the leaves of Dendrophthoe falcata. The optimum pH for the enzyme was 8.5. The enzyme was sensitive to p-CMB and the inhibition could be reversed by 2-mercaptoethanol. The optimum temperature was 40° and energy of activation 6900 cal/mol. The enzyme had an absolute requirement for a divalent metal ion. Although Mg²⁺ was the preferred metal, it could be partially replaced by Mn²⁺ and Ca²⁺. ATP was the most effective phosphoryl donor. Glucose was the best substrate, the K_m values of 0.14 and 0.26 mM were obtained at saturated and sub-saturated ATP concentration. Phosphorylation coefficients show the following order of reactivity of sugars: glucose mannose 2-deoxy D-glucose fructose glucosamine galactose ribose. The K_m value for ATP was 0.16 mM, which increased to 0.35 mM in the presence of 0.5 mM ADP. ADP and 5′-AMP were competitive inhibitors with respect to ATP, and K_i values were 0.4 and 1.2 mM respectively.     

Changes in plasma hydrolase activities in hereditary and streptozotocin-induced diabetes.

The characteristics of the hydrolysis of 5′-adenylylimidodiphosphate [AMP-P(NH)P] by partially purified plasma membranes from rat liver are described. Hydrolysis was less with membranes from fat cells and was poor with a detergent-dispersed preparation from rat cerebellum. The Chromatographic behavior of the principal degradation products suggests that AMP-P(NH)P is first hydrolyzed to 5'?AMP, which is then hydrolyzed further to adenosine. The adenosine is shown to inhibit adenylate cyclase noncompetitively with respect to substrate and in a cation-dependent manner. Sensitivity to inhibition by adenosine was markedly enhanced by agents that stimulated adenylate cyclase. The characteristics of the initial hydrolysis of AMP-P(NH)P fit best those of nucleotide pyrophosphatase and support the conclusion that several of the various phosphatase activities present in membranes may be due to the same enzyme. Under conditions shown to be linear with respect to time and membrane protein concentration, hydrolysis of AMP-P(NH)P exhibited a pH optimum between 9.5 and 10. At pH 9.5, hydrolysis occurred with a K_m of about 20 μm and a V of about 220 nmol (min) ¹ (mg of protein)^?1. The initial hydrolysis of AMP-P(NH)P was inhibited in a linear-competitive manner by ATP, ADP, 5′-AMP, GTP, 5′-guanylylimidodiphosphate, NAD⁺, and p-nitrophenyl-dTMP and in a noncompetitive manner by UDP-glucose. Adenosine 3′:5?cyclic phosphate and guanosine 3′:5′-cyclic phosphate were not inhibitory at concentrations up to 1 mm. ATP, GTP, and 5′-guanylylimidodiphosphate were also hydrolyzed in a manner comparable to that for AMP-P(NH)P. Hydrolysis of AMP-P(NH)P did not require the presence of added metal, and some metals were inhibitory. Activity was inhibited by dithiothreitol (50% at <1 mm) and by EDTA (50% at about 10 mm). Following pretreatment with EDTA or dithiothreitol, the readdition of certain metals, especially Zn or Co, caused some restoration of hydrolytic activity. The evidence suggests that hydrolytic activity involves the participation of bound metal and that the enzyme is a metallo-protein.     

Acetylcholine Synthesizing Enzymes in Frog Skeletal Muscle

Acetylcholine synthesis in homogenates of frog sartorius muscle was measured by a radiometric method with a low blank. Choline acetyltransferase activity was very low (V_max, 2 nmol g¹ h^?1, K_mfor choline, approx. 50 μ, m ). The enzyme was found only in the endplate area and disappeared after denervation; it was inactivated by 4-(1-naphthylvinyl)pyridine. At high substrate concentrations its activity was overshadowed by the acetylcholine-synthesizing activity of a different enzyme not saturated by 10 mm -choline. The non-specific enzyme was present at and away from the endplate area, and it was not affected by denervation.     

Choline fluxes in primary nerve cell cultures. Correlation with the endocellular metabolism of choline

The fluxes of choline across the plasma membrane were measured in primary nerve cell cultures from chick embryo cerebral hemispheres containing neurons and supporting cells.The incubation of cells with exogenous concentrations of choline far below the concentrations present in the growth medium (～30–50 μM) and in the range of the high affinity uptake mechanism (about 0.5 μM) profoundly affected the steady state of the endocellular free choline levels. The kinetics of the uptake were dependent upon the endocellular status of the choline pool since after preincubation in the absence of choline two K_ms are observed (K_m1: 0.8 μM; V_max1: 44.8 pmol/mg protein/2 min; K_m2: 14.3 μM, V_max2: 333.3 pmol/mg protein/2 min) while only one mechanism can be found when the endocellular pool of choline was kept in steady state conditions (K_m: 14.3 μM, V_max: 545.5 pmol/mg protein/2 min). The presence of an homoexchange phenomenon was suspected since choline efflux could be increased by increasing the concentrations of choline in the incubation medium.The results suggest that the movement of choline into nerve cells in culture appears to be mediated by a single mechanism which is regulated by the endocellular status of the choline pool.     

Partial Purification and Characterization of a 3'- Phosphoadenosine 5' -Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum)

A 3′ -phosphoadenosine 5′ -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 ± 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K_m 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K_m 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (K_m 670 micromolar), and desulfoallylglucosinolate (K_m 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl₂, MnCl₂ and reducing agents and inhibited by ZnCl₂, PbNO₃ NiCl₂ and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5′ -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by β-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM THE NERVOUS SYSTEM OF DIFFERENT INVERTEBRATES

—Choline acetyltransferase has been purified from three invertebrate species, namely snail (Helix aspersa), cockroach (Periplaneta americana) and horse shoe crab (Limulus polyphemus.) All three enzymes followed a Theorell-Chance enzyme mechanism with a sequential addition of the substrates. All three enzymes were activated by sodium and potassium chloride and inhibited by high concentrations of magnesium or calcium chloride. The apparent K_m for choline and acetyl-CoA was for snail: K_mch= 370 μm ,K_mAcetyl-CoA= 51μm ; cockroach:K_mCh= 550 μm , K_mAcely-CoA= 16 μm horse shoe crab:K_mCn= 2700 μm K_mAcctyl-coA= 68 μm CoA inhibited the enzymes competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibited the enzymes competitively with respect to choline and non-competitively with respect to acetyl-CoA. All the enzymes were inhibited strongly by 5,5′-dithiobis (2-nitrobenzoate), iodoacetate, acryloylcholine, chloracetylcholine and 3-bromacetonyltrimethyl-ammonium. The enzymes were only weakly inhibited by the styrylpyridine derivatives. The isoelectric points were 5.3 and 5.0 for the horse shoe crab and cockroach enzymes respectively. All three enzymes showed low affinity for a cation-exchanger (CM-Sephadex).     

Purification and properties of the endo-1,4-β-glucanase III from Ruminococcus albus

Endoglucanase III (EGIII) was purified from Ruminococcus albus culture supernatant. An enzyme having a molecular weight of 53,000 was stabilized by mercaptoethanol and inhibited by sulfhydryl group-blocking reagents, and exhibited its highest CMC-degrading activity of pH 5.7 and 55°C. The enzyme hydrolyzed cellobiose (G2) and cellotriose (G3) only negligibly, but significantly hydrolyzed cellotetraose (G4), cellopentaose (G5) and cellohexaose (G6). The major hydrolysis reactions conducted by the enzyme were G4→2G2, G5→G2+G3, G6→G2+G4 and G6→2G3. The V_max values of these reactions increased remarkably while the K_m values decreased significantly with an increase in degree of polymerization of the substrate.     

Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa: Purification by affinity chromatography and physicochemical properties

Pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa was purified 150-fold by affinity chromatography on immobilized 2′-AMP. The binding of the enzyme is pH dependent. Elution was achieved with 2′-AMP, NADP⁺, or NADPH but not with 5′-AMP, NAD⁺, or NADH. The enzyme preparations appeared to be homogeneous in gel chromatography and ultracentrifugation, but only if these procedures were carried out in the presence of 2′-AMP or NADP⁺. With 2′-AMP a sedimentation coefficient of 34 S, a molecular weight of 1.6–1.7 million, and a Stokes' radius of 11.7 nm were determined. In the presence of NADP⁺ the sedimentation coefficient was 42 S and the molecular weight was 6.4 million. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed one kind of subunit with a molecular weight of 54,000. This was consistent with results from amino acid analyses and paper chromatography of peptides. Eight molar urea inactivated the enzyme but did not dissociate it into subunits. Full activity was restored after dialysis against urea-free buffer by mercaptoethanol and flavin-adenine dinucleotide.     

Effect of pH on the Kinetic Parameters of NADP-Malic Enzyme from a C(4)Flaveria (Asteraceae) Species

We have used the pH variation in the kinetic parameters with respect to malate of NADP-malic enzyme purified from the C₄ species, Flaveria trinervia, to compare the pK values of its functional groups with those for the pigeon liver NADP-malic enzyme (MI Schimerlik, WW Cleland [1977] Biochemistry 16: 576-583) and the plant NAD-malic enzyme (KO Willeford, RT Wedding [1987] Plant Physiol 84: 1084-1087). Like the other enzymes, the C₄ enzyme has a group with a pK of about 6.0 (6.6 for the C₄ enzyme), as indicated from plots of the log V_max/K_m (V_max = maximum rate of catalysis) versus pH, which must lose a proton for malate binding and subsequent catalysis. The optimum ionization for the C₄ enzyme-NADP-Mg²⁺ complex occurs at pH 7.1 to 7.5. From pH 7.5 to 8.4, the K_m increases, but V_max remains constant. The log V_max/K_m plot in this pH range indicates a group with a pK of about 7.7. The other malic enzymes exhibit a similar pK. Above pH 8.4, deprotonation leads to a marked increase in K_m and a decrease in V_max for the C₄ enzyme. As in the case of the animal enzyme, the log V_max/K_m plot for the C₄ enzyme appears to approach a slope of two. The curve suggests an average pK of 8.4 for the groups involved, while the animal enzyme exhibits an average pK of 9.0. The NAD-malic enzyme does not exhibit any pK values at these high pK values. We hypothesize that the putative groups with the high pK values may be at least partially responsible for the ability of the C₄ NADP-malic enzyme to maintain high activity at pH 8.0 in illuminated chloroplasts.