Interactions between magnesium ions,pH, glucose-6-phosphate,and NADPH/NADP+ ratios in the modulation of chloroplast glucose-6-phosphate dehydrogenase in vitro

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg²⁺, proton, and substrate concentrations. Mg²⁺ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg²⁺-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP⁺ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg²⁺. Increasing the NADPH/NADP⁺ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg²⁺ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP⁺ ratio, enzyme activity is not affected by the Mg²⁺ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - G-6-PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - PPC pentose phosphate cycle     

Evidence for a cytosolic-dependent light induction of chloroplastic glutamine synthetase during greening of etiolated rice leaves

During the greening of etiolated rice leaves, total glutamine synthetase activity increases about twofold, and after 48 h the level of activity usually observed in green leaves is obtained. A density-labeling experiment with deuterium demonstrates that the increase in enzyme activity is due to a synthesis of the enzyme. The enhanced activity obtained upon greening is the result of two different phenomena: there is a fivefold increase of chloroplastic glutamine synthetase content accompanied by a concommitant decrease (twofold) of the cytosolic glutamine synthetase. The increase of chloroplastic glutamine synthetase (GS₂) is only inhibited by cycloheximide and not by lincomycin. This result indicates a cytosolic synthesis of GS₂. The synthesis of GS₂ was confirmed by a quantification of the protein by an immunochemical method. It was demonstrated that GS₂ protein content in green leaves is fivefold higher than in etiolated leaves.Abbreviations AbH heavy chain of antibodies - AbL light chain of antibodies - AP acid phosphatase - CH cycloheximide - G6PDH glucose-6-phosphate dehydrogenase - GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ chloroplastic glutamine synthetase - LC lincomycin - NAD-MDH NAD malate dehydrogenase - NADP-G3PDH NADP glyceraldehyde-3-phosphate dehydrogenase     

Response of N2-Fixing Cyanobacteria to Salt

The effect of salt on photosynthetic activity, acetylene reduction, and related activities was examined in two species of cyanobacteria, Nostoc muscorum and Calothrix scopulorum. Photosynthesis was more resistant to high salt concentration than was N₂ fixation. The salt resistance of both activities increased after a period of exposure of the cells to salinity. The transfer of electrons via ferredoxin and ferredoxin-nicotinamide adenine dinucleotide phosphate reductase was found to be extremely sensitive to salt. In comparison, the transfer of reducing power by glucose-6-phosphate dehydrogenase, isocitric dehydrogenase, and photosystem 1 was less affected by NaCl, whereas glutamine synthetase exhibited higher tolerance to salt.     

Metabolic changes associated with cyanophage N-1 infection of the cyanobacterium Nostoc muscorum

The development of cyanophage N-1 in the N₂-fixing cyanobacterium Nostoc muscorum is dependent on light. The redox state of thioredoxin m was altered in phage infected cells, with the proportion of reduced thioredoxin increasing during the eclipse period. In one step growth experiments, the specific activity of glucose-6-phosphate dehydrogenase increased transiently during the eclipse period, whereas that of glutamine synthetase increased towards the end of the eclipse period (2–4h after infection) then remained high until the end of the latent period (about 7 h after infection). The rate of respiratory O₂ uptake was maintained until the end of the latent period. In contrast, the specific activity of phosphoribulokinase and the rate of photosynthetic O₂ evolution began to decrease towards the end of the eclipse period and later than the level of extractable protein began to decrease. Nitrogenase activity remained high throughout the eclipse period then decreased rapidly after 5 h. The level of glutamine synthetase protein decreased in parallel with the decrease in total extractable protein, whereas the level of thioredoxin m protein decreased more slowly.     

Response of n(2)-fixing cyanobacteria to salt

The effect of salt on photosynthetic activity, acetylene reduction, and related activities was examined in two species of cyanobacteria, Nostoc muscorum and Calothrix scopulorum. Photosynthesis was more resistant to high salt concentration than was N(2) fixation. The salt resistance of both activities increased after a period of exposure of the cells to salinity. The transfer of electrons via ferredoxin and ferredoxin-nicotinamide adenine dinucleotide phosphate reductase was found to be extremely sensitive to salt. In comparison, the transfer of reducing power by glucose-6-phosphate dehydrogenase, isocitric dehydrogenase, and photosystem 1 was less affected by NaCl, whereas glutamine synthetase exhibited higher tolerance to salt.     

Inhibition by Catalase of Dark-mediated Glucose-6-Phosphate Dehydrogenase Activation in Pea Chloroplasts

Dark activation of light-inactivated glucose-6-phosphate dehydrogenase was inhibited by catalase in a broken pea chloroplast system. Partially purified glucose-6-phosphate dehydrogenase from pea leaf chloroplasts can be inactivated in vitro by dithiothreitol and thioredoxin and reactivated by H₂O₂. The in vitro activation by H₂O₂ was not enhanced by horseradish peroxidase, and dark activation in the broken chloroplast system was only slightly inhibited by NaCN. These results indicate that the dark activation of glucose-6-phosphate dehydrogenase may involve oxidation by H₂O₂ of SH groups on the enzyme which were reduced in the light by the light effect mediator system.     

Role of glutamine synthetase in citric acid fermentation byAspergillus niger

The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6·0–6·5, when the pH of the external medium was varied between 2·3–7·0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn²⁺ ions partially protected the enzyme against this inactivation. Mn²⁺-dependent glutamine synthetase activity was higher at acidic pH (6·0) compared to Mg²⁺-supported activity. While the concentration of Mg²⁺ required to optimally activate glutamine synthetase at pH 6·0 was very high (≥ 50 mM), Mn²⁺ was effective at 4 mM. Higher concentrations of Mn²⁺ were inhibitory. The inhibition of both Mn²⁺ and Mg²⁺-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn²⁺ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis     

Stress response in two strains of the aquatic hyphomycete <Emphasis Type="BoldItalic">Heliscus lugdunensis</Emphasis> after exposure to cadmium and copper ions

Biochemical responses to cadmium (Cd²⁺) and copper (Cu²⁺) exposure were compared in two strains of the aquatic hyphomycete (AQH) Heliscus lugdunensis. One strain (H4-2-4) had been isolated from a heavy metal polluted site, the other (H8-2-1) from a moderately polluted habitat. Conidia of the two strains differed in shape and size. Intracellular accumulation of Cd²⁺ and Cu²⁺ was lower in H4-2-4 than in H8-2-1. Both␣strains synthesized significantly more glutathione (GSH), cysteine (Cys) and γ-glutamylcysteine (γ-EC) in the presence of 25 and 50 μM Cd²⁺, but quantities and rates of synthesis were different. In H4-2-4, exposure to 50 μM Cd²⁺ increased GSH levels to 262% of the control; in H8-2-1 it increased to 156%. Mycelia of the two strains were analysed for peroxidase, dehydroascorbate reductase, glutathione reductase and glucose-6-phosphate dehydrogenase. With Cd²⁺ exposure, peroxidase activity increased in both strains. Cu²⁺ stress increased dehydroascorbate reductase activity in H4-2-4 but not in H8-2-1. Dehydroascorbate reductase and glucose-6-phosphate dehydrogenase activities progressively declined in the presence of Cd²⁺, indicating a correlation with Cd²⁺ accumulation in both strains. Cd²⁺ and Cu²⁺ exposure decreased glutathione reductase activity.     

Turbellaria Phagocata sibirica: Some Enzymes of Carbohydrate and Energy Metabolisms

Activities and properties of some enzymes of carbohydrate and energy metabolisms in free-living turbellaria Phagocata sibirica are studied. The enzymes are studied in various subcellular fractions. A high activity of hexokinase is accompanied by high activity of glucose-6-phosphate dehydrogenase (G6PDG). The level of pyruvate kinase activity is sufficient to provide dissimilation of phosphoenolpyruvate with formation of pyruvate. P. sibirica has highly-active lactate dehydrogenase (LDH) and malate dehydrogenase (MDH); a predominance of MDH activity over LDH and a low activity of phosphoenolpyruvate carboxykinase is revealed. NADP-dependent isocitrate dehydrogenase is found, which is activated by Mn²⁺ and Mg²⁺ and inhibited by salts of heavy metals and p-chloromercuribenzoate. Activities and properties of -ketoglutarate dehydrogenase, succinate dehydrogenase (SDH), and fumarate reductase are studied, and it is concluded that in P. sibirica there is the system of succinate oxidation, whereas the system of fumarate reduction into succinate is absent. Mitochondrial and microsomal fractions from P. sibirica had Mg²⁺- and Ca²⁺-dependent adenosine triphosphatases.     

Evidence for adenylylation/deadenylylation control of the glutamine synthetases of Rhodospirillum tenue and Rhodocyclus purpureus

The phylogenetically related phototrophic bacteria Rhodospirillum tenue and Rhodocyclus purpureus modulate activity of their glutamine synthetases by adenylylation/deadenylylation. Evidence for covalent modification includes the inhibitory effect of Mg²⁺ on the activity of glutamine synthetase extracted from cells of either species grown on excess ammonia, and the lack of Mg²⁺ inhibition of activity of the enzyme isolated from N₂-(R. tenue) or glutamine (R. purpureus)-grown cells. In addition, snake venom phosphodiesterase treatment of glutamine synthetase from either species grown on excess ammonia relieved Mg²⁺ inhibition of the enzyme (as measured via the -glutamyl transferase assay), and changed the cation specificity from Mn²⁺ to Mg²⁺ (in the biosynthetic assay).     

Studies onAspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grownAspergillus niger was increased 3–5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH ₄ ⁺ , and further, the enzyme is repressed by increasing concentrations of NH ₄ ⁺ . In contrast to other micro-organisms, theAspergillus niger enzyme was neither specifically inactivated by NH ₄ ⁺ or L-glutamine nor regulated by covalent modification. Glutamine synthetase fromAspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn²⁺ or Mg²⁺-dependent synthetase and Mn²⁺-dependent transferase activity. Aspergillusniger glutamine synthetase was completely inactivated by two mol of phenyl-glyoxal and one mol of N-ethylmaleimide with second order rate constants of 3.8 M^-1 min^-1 and 760 M^-1 min^-1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH ₄ ⁺ , Mn²⁺ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.     

Effect of photosynthetic intermediates on the magnesium inhibition of oxygen evolution by barley chloroplasts

Millimolar concentrations of Mg²⁺ inhibited CO₂-dependent O₂ evolution by barley (Hordeum vulgare L.) chloroplasts and also prevented the activation of NADP-glyceraldehyde-3-phosphate dehydrogenase, ribulose-5-phosphate kinase, and fructose-1,6-diphosphatase by light in intact chloroplasts. When added in the dark, 3-phosphoglycerate prevented the inhibition of O₂ evolution by Mg²⁺ and reduced the Mg²⁺ inhibition of enzyme activation by light. Fructose 1,6-diphosphate and ribulose 5-phosphate also prevented the inhibition of O₂ evolution by Mg²⁺ whereas glucose 1-phosphate, glucose 6-phosphate, ribulose 1,5-diphosphate, and citrate had no effect. Phosphoenolpyruvate gave an intermediate response. Metabolites that prevented the Mg²⁺ inhibition of O₂ evolution shortened the lag phase of CO₂-dependent O₂ evolution in the absence of M²⁺. Loading chloroplasts in the dark with 3-phosphoglycerate reduced both the lag phase of O₂ evolution and the inhibition of O₂ evolution by Mg²⁺. The results suggested that Mg²⁺ inhibition was lessened either by external metabolites that compete with inorganic phosphate for transport into the chloroplast or by a high concentration of internal metabolites.     

Metabolic activators of spinach leaf nitrate reductase: Effects on enzymatic activity and dephosphorylation by endogenous phosphatases

Nitrate reductase (NR; EC 1.6.6.1) in spinach (Spinacia oleracea L.) leaves was inactivated in the dark and reactivated by light in vivo. When extracted from dark leaves, NR activity was lower and more strongly inhibited by Mg²⁺ relative to the enzyme extracted from leaves harvested in the light. When dark extracts were desalted at pH 6.5 and preincubated at 25° C prior to assay, enzyme activity (assayed either in the presence or absence of Mg²⁺) remained essentially constant, i.e. there was no spontaneous reactivation in vitro. However, addition of certain metabolites resulted in a time- and concentration-dependent activation of NR in vitro. Effective activators included inorganic phosphate (Pi), 5-AMP, and certain of its derivatives such as FAD and pyridine nucleotides (both oxidized and reduced forms). All of the activators increased NR activity as assayed in the absence of Mg²⁺, whereas some activators (e.g. Pi, 5-AMP and FAD) also reduced Mg²⁺ inhibition. The reduction of Mg²⁺ inhibition was also time-dependent and was almost completely prevented by a combination of okadaic acid plus KF, suggesting the involvement of dephosphorylation catalyzed by endogenous phosphatase(s). In contrast, the activation of NR (assayed minus Mg²⁺) was relatively insensitive to phosphatase inhibitors, indicating a different mechanism was involved. Compounds that were not effective activators of NR included sulfate, ribose-5-phosphate, adenosine 5-monosulfate, coenzyme A, ADP and ATP. We postulate that NR can exist in at least two states that differ in enzymatic activity. The activators appear to interact with the NR molecule at a site distinct from the NADH active site, and induce a slow conformational change (hysteresis) that increases NR activity (assayed in the absence of Mg²⁺). Possibly as a result of the conformational change caused by certain activators, the regulatory phospho-seryl groups are more readily dephosphorylated by endogenous phosphatases, thereby reducing sensitivity to Mg²⁺ inhibition. Preliminary results suggest that light/dark transitions in vivo may alter the distribution of NR molecules between the low- and high-activity forms.Abbreviations AP₅A P¹, P⁵-di(adenosine-5)pentaphosphate - DTT dithiothreitol - Mops 3-(N-morpholino)propanesulfonic acid - NR NADH:nitrate reductase - NRA nitrate reductase activity Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643. This work was also supported in part by grants from the U.S. Department of Energy (Grant DE-AIO5-91 ER 20031) and USDA-NRI (Grant 93-373-5-9231). The authors thank Dr. W.M. Kaiser (Lehrstuhl Botanik I der Universität, Würzburg, Germany) for discussions and Dr. C. Lillo (Rogaland University Center, Stavanger, Norway) for sharing results prior to publication.     

Relationships between nitrogenase,glutamine synthetase,glutamine, and energy charge in Azotobacter vinelandii

When continuous cultures of Azotobacter vinelandii were supplied with ammonium or nitrate in amounts, which just repressed nitrogenase synthesis completely, both the intracellular glutamine level and the degree of adenylylation of the glutamine synthetase (GS) increased only slightly (from 0.45–0.50 mM and from 2 to 3 respectively), while the total GS level remained unaffected. Higher amounts of ammonium additionally inhibited the nitrogenase activity, caused a strong rise in the intracellular glutamine concentration and adenylylation of the GS, but caused no change in the ATP/ADP ratio. These results are considered as evidence that in A. vinelandii the regulation of nitrogenase synthesis is not linked to the adenylylation state of the GS and to the intracellular glutamine level, and that the inhibition of the nitrogenase activity as a consequence of a high extracellular ammonium level is not mediated via a change in the energy charge.Abbreviations GS glutamine synthetase - GS-S(Mg) Mg²⁺ dependent synthetic activity of GS - GS-T(Mn) Mn²⁺ dependent transferase activity of GS     

Characterization and Regulatory Properties of Glucose-6-Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The K_m value for glucose-6-phosphate is 1.6 × 10^?4 and 6.3 × 10^?4M at low (1.0–6.0 × 10^?4M) and high (6.0–30.0 × 10^?4M) concentrations of the substrate, respectively. The K_m value for NADP⁺ is 1.4 × 10^?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg²⁺, and Mn²⁺. In the presence of NADPH, the plot of activity vs. NADP⁺ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg²⁺ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.     

Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases,and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP⁺-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP⁺. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP⁺ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.Abbreviations Cyt cytochrome - Glc6P glucose-6-phosphate - Glc6PDH glucose-6-phosphate dehydrogenase - MVH reduced methyl viologen - NiR nitrite reductase - 6PG 6-phosphogluconate - 6PGDH 6-phosphogluconate dehydrogenase     

Dark modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase in the chloroplast

The properties of the system which reverses light modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in pea chloroplasts were examined. A factor catalyzing dark modulation of these enzymes was found. This factor cochromatographed with thioredoxin in all systems used (Sephacryl S-200, Sephadex G-75, DEAE-cellulose). Inhibition of dithiothreitol-dependent modulation and of dark reversal by antibody against Escherichia coli thioredoxin further suggest that the dark factor is in fact thioredoxin. It appears that the reaction is the reverse of the previously described dithiothreitol-dependent thioredoxin-catalyzed modulation of enzymes. The limiting step in vitro seems to be the oxidation of thioredoxin during the dark period.     

Phloem unloading following reactivation in predarkened mature maize leaves

Mature leaf blades of 48-h predarkened maize plants (Zea mays L. cv. Prior) were excised, and treated apically as the source (light, normal air) and basally as the sink (light or dark, air without CO₂). After providing the source portion with ¹⁴CO₂, the sink portions were harvested after 2, 7 or 14 h by freezing with liquid nitrogen, grinding, and freeze-drying. Extracts, fractionated by ionexchange resins into neutral, basic and acid fractions, were chromatographed on thin cellulose layers, and autoradiographed. Identification of labeled compounds was carried out by co-chromatography with authentic labeled substances. Activities of enzymes pertaining to the metabolism of sucrose were checked. Results show that the source supplies sucrose to the sink, where it is unloaded and metabolized by acid invertase (EC 3.2.1.26) in both the light and the dark. Starch appearing in the sink only in the light, after 7 h of re-illumination, yields labeled glucose upon hydrolysis. Although sucrose-phosphate synthetase (EC 2.4.1.14) is active in sinks and in isolated vascular-bundle fragments, it remains questionable whether sucrose unloaded from sieve tubes is metabolized by a method other than inversion. Sucrose synthetase (EC 2.4.1.13) was found to be inactive. Obviously, the main metabolite of unloaded sucrose is glucose-6-phosphate, giving access to the glycolytic pathway. The main difference between the sinks in the light and the dark is the lack of labeled glycine and serine in the dark. This indicates that in the light decarboxylation of glycine yields CO₂, which is recycled photosynthetically.Abbrevations Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - TLC thin-layer chromatography - UDPGlc uridine 5-diphosphate glucose     

Azospirillum brasilense glutamine synthetase: influence of the activating metal ions on the enzyme properties

The kinetic properties of the Mg²⁺-activated and Mn²⁺-activated glutamine synthetase (GS) of Azospirillum brasilense in the biosynthetic reaction were studied. The Mg²⁺-supported and Mn²⁺-supported GSs in an average state of adenylylation varied in pH optimum, maximum activity, saturation functions for ammonium and glutamate, affinity to substrates, and in the Me²⁺-ATP ratio required for the optimal enzyme activity. Seventeen other cations were tested for the maintenance of GS activity. The level of the latter and the kinetic behavior of the GS in A.brasilense is suggested to depend essentially on the concentrations of Mg²⁺, Mn²⁺ and Co²⁺, as well as on their ratio     

The cytochemical assay of NAD+ kinase activity in the follicular cells of guinea-pig thyroid gland

Summary A quantitative cytochemical assay for NAD⁺ kinase-like activity in the guinea-pig thyroid gland is described. The NADP⁺ produced by the activity of the kinase was used to drive the NADP⁺-dependent enzyme glucose-6-phosphate dehydrogenase which is endogenous to the tissue. The activity of glucose-6-phosphate dehydrogenase is greatly in excess of that of the kinase and was unaffected by the constituents of the kinase incubation medium (ATP, Mg²⁺ and NAD⁺) either alone or in combination. Kinase activity was dependent both on ATP and Mg²⁺, with maximal activity seen when the Mg-ATP ratio was between 1:1 and 4:1. Free ATP inhibited the activity of the enzyme. Enzyme activity was exhibited over a broad pH range (7–9) with a peak at pH 8.2. The sulphhydryl-blocking agents,p-chloromercuribenzoate, iodoacetate and iodoacetamide (at 1 mM), completely abolished kinase activity but were without effect on glucose-6-phosphate dehydrogenase activity.N-ethylmaleimide and citrate (both at 1 mM) had no effect on either kinase or glucose-6-phosphate dehydrogenase activities.