Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.     

Identification and functional characterization of sorbitol-6-phosphate dehydrogenase protein from rice and structural elucidation by in silico approach

The sorbitol-6-phosphate dehydrogenase (S6PDH) is a key enzyme for sorbitol synthesis and plays an important role in the alleviation of salinity stress in plants. Despite the huge significance, the structure and the mode of action of this enzyme are still not known. In the present study, sequence analysis, cloning, expression, activity assays and enzyme kinetics using various substrates (glucose-6-phosphate, sorbitol-6-phosphate and mannose-6-phosphate) were performed to establish the functional role of S6PDH protein from rice (Oryza sativa). For the structural analysis of the protein, a comparative homology model was prepared on the basis of percentage sequence identity and substrate similarity using the crystal structure of human aldose reductase in complex with glucose-6-phosphate and NADP⁺ (PDB ID: 2ACQ) as a template. Molecular docking was performed for studying the structural details of substrate binding and possible enzyme mechanism. The cloned sequence resulted into an active recombinant protein when expressed into a bacterial expression system. The purified recombinant protein was found to be active with glucose-6-phosphate and sorbitol-6-phosphate; however, activity against mannose-6-phosphate was not found. The K _m values for glucose-6-phosphate and sorbitol-6-phosphate were found to be 15.9 ± 0.2 and 7.21 ± 0.5 mM, respectively. A molecular-level analysis of the active site of OsS6PDH provides valuable information about the enzyme mechanism and requisite enantioselectivity for its physiological substrates. Thus, the fundamental studies of structure and function of OsS6PDH could serve as the basis for the future studies of bio-catalytic applications of this enzyme.     

Importance of glucose-6-phosphate dehydrogenase activity in cell death

The intracellular redox potential plays an important role incell survival. The principal intracellular reductant NADPH is mainlyproduced by the pentose phosphate pathway by glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme, and by6-phosphogluconate dehydrogenase. Considering the importance of NADPH,we hypothesized that G6PDH plays a critical role in cell death. Ourresults show that 1) G6PDHinhibitors potentiatedH₂O₂-inducedcell death; 2) overexpression ofG6PDH increased resistance toH₂O₂-induced cell death; 3) serum deprivation, astimulator of cell death, was associated with decreased G6PDH activityand resulted in elevated reactive oxygen species (ROS);4) additions of substrates for G6PDHto serum-deprived cells almost completely abrogated the serumdeprivation-induced rise in ROS; 5)consequences of G6PDH inhibition included a significant increase inapoptosis, loss of protein thiols, and degradation of G6PDH; and6) G6PDH inhibition caused changesin mitogen-activated protein kinase phosphorylation that were similarto the changes seen withH₂O₂.We conclude that G6PDH plays a critical role in cell death by affectingthe redox potential.     

Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae.

The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD+ oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saccharomyces cerevisiae strain H44-3D by affinity- and ion-exchange chromatography. SDS-PAGE indicated that the enzyme had a molecular mass of approximately 42,000 (+/- 1,000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer. The pH optimum for the reduction of dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pI of 7.4. NADPH will not substitute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Km values obtained were 0.023 and 0.54 mM for NADH and DHAP, respectively. NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity. Ki values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.     

Chloroplast glucose-6-phosphate dehydrogenase: Km shift upon light modulation and reduction

Illumination of intact chloroplasts and treatment of chloroplast stroma with dithiothreitol (DTT) both inactivate glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) to less than 10% apparent activity when assayed under standard conditions. Illumination of intact protoplasts and incubation of leaf extract with DTT inactivate about 25-35% of the total G6PDH activity. In the leaf extract, however, further loss of activity is observed if NADP is absent. Light- and DTT-inactivated chloroplast G6PDH can be reactivated by oxidation with sodium tetrathionate or the thiol oxidant diamide. Chloroplast G6PDH is as sensitive toward reductive enzyme modulation in a stromal extract as are other light/dark modulated enzymes, e.g., NADP-malate dehydrogenase. Also, glutathione, provided it is kept reduced, is sufficient to cause inactivation. Light- and DTT-induced inactivation are shown to be due to a Km shift with respect to glucose-6-phosphate (G6P) from 1 to 35 and 43 mM, respectively, and with respect to NADP from 10 to 50 microM without any significant change of the Vmax. NADPH competitively (NADP) inhibits the enzyme (Ki = 8 microM). Reactivation by oxidation can be explained by an enhanced affinity of the oxidized enzyme toward G6P and NADP. The pH optimum of the reduced enzyme is more in the alkaline region (pH 9-9.5) as compared to that of the oxidized form (pH 8.0). The presence of 30 mM phosphate causes a shift of 0.5 to 1.0 pH unit into the alkaline region for both forms.     

Nonreductive interaction of vanadate with an enzyme containing a thiol group in the active site: glycerol-3-phosphate dehydrogenase

The inhibitory effects of vanadium(V) were determined on the oxidation of glycerol 3-phosphate (G3P) catalyzed by glycerol-3-phosphate dehydrogenase (G3PDH), an enzyme with a thiol group in the active site. G3PDH from rabbit muscle was inhibited by vanadate, and the active inhibiting species were found to be the vanadate dimer and/or tetramer. The dimer was a sufficiently weak inhibitor at pH 7.4 with respect to G3P; the tetramer could account for all the observed inhibition. The tetramer was a competitive inhibitor with respect to G3P with a Ki of 0.12 mM. Both the dimer and tetramer were noncompetitive inhibitors at pH 7.4 with respect to NAD with Ki's of 0.36 mM and 0.67 mM. G3PDH inhibited by vanadate was reactivated when EDTA complexed the vanadate. The reactivation occurred even after extended periods of incubation of G3PDH and vanadate, suggesting that the inhibition is reversible despite the thiol group in the active site. Analogous reactivation is also observed with glyceraldehyde-3-phosphate dehydrogenase (Gly3PDH). Gly3PDH is an enzyme that previously had been reported to undergo redox chemistry with vanadate. The work described in this paper suggests vanadate will not necessarily undergo redox chemistry with enzymes containing thiol groups exposed on the surface of the protein.     

Characterization of mannitol metabolism in the mangrove red algaCaloglossa leprieurii (Montagne) J.Agardh

A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheK _m values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD⁺. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aK _m value of 1.2 mM and a high requirement of Mg²⁺ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedK _m values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD⁺. For the non-specific HK, only theK _m values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.Abbreviations F6P fructose-6-phosphate - HK hexokinase - Mt1P mannitol-1-phosphate - Mt1PDH mannitol-1-phosphate dehydrogenase - Mt1Pase mannitol-1-phosphatase - MtDH mannitol dehydrogenase     

Light-induced ATP Formation from Acetyl Phosphate and ADP by Broken Spinach Chloroplasts

Spinach chloroplasts catalyzed ATP formation from acetyl phosphateand ADP when exposed to light. No ATP formation was detectablein the dark. In the absence of ADP, chloroplasts did not hydrolyzeacetyl phosphate in the light or dark. Neither high-energy phosphatessuch as creatine phosphate and phosphoenol pyruvate nor inhibitorsof photophosphorylation competitive with P_i, such as ß-naphthylmonophosphate, phenyl phosphate and pyridoxal 5-phosphate, couldsubstitute for acetyl phosphate as a P_i donor. The apparentK_m values for acetyl phosphate and P_i were 0.81 mM and 0.25mM, respectively. The maximal rate of ATP formation with acetylphosphate and P_i were 331 and 521 µmol ATP formed mg chl^–1hr^–1, respectively. The optimum pH value for acetyl phosphate-dependentATP formation was about 8.0. NH₄Cl, dicyclohexylcarbodiimideand triphenyltin chloride inhibited the acetyl phosphate-dependentATP formation. Acid-base transition also could induce subsequentATP formation from acetyl phosphate and ADP. These results suggestthat the acetyl phosphate-dependent ATP formation requires theformation and the utilization of a proton-motive force as ordinaryphotophosphorylation does. ¹ This work was supported in part by Grants-in-Aid for ScientificResearch from the Ministry of Education, Science and Culture,Japan to H. S. Part of this work was reported at the 1981 AnnualMeeting of the Japanese Society of Plant Physiologists (Sapporo,May 8, 1981). (Received August 25, 1981; Accepted November 1, 1981)     

Multiple enzyme forms of glyceraldehyde-3-phosphate dehydrogenase in Pseudomonas aeruginosa PAO

Both NAD- and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (EC 1.2.1.12) activities were detected in glucose-grown cells of Pseudomonas aeruginosa strain PAO. After growth on gluconeogenic substrates such as citrate, the activity of the NAD-G3PDH was reduced severalfold in contrast to little change for the NADP-G3PDH. The two G3PDH activities could be separated by ammonium sulphate fractionation. PAGE revealed the presence of two G3PDH isoenzymes of 140 (NADP-specific) and 315 (NAD-specific) kDa. Slight differences were observed in the thermostabilities and pH optima of the two enzymes whereas the regulation of their activities by various compounds varied strongly. The NADP-G3PDH enzyme was activated by ATP, reduced NAD, and fructose 6-phosphate. It was inhibited by fructose 1,6-diphosphate and 6-phosphogluconate. The NAD-G3PDH enzyme was inhibited by ATP, reduced NAD, and 6-phosphogluconate; it was slightly activated by reduced NADP. The possible roles of these isoenzymes in the control of hexose catabolism and gluconeogenesis in P. aeruginosa are discussed.     

Purification and Properties of NAD-Dependent Sorbitol Dehydrogenase from Apple Fruit

This is the first report of the purification of NAD-dependentsorbitol dehydrogenase (NAD-SDH) from a plant source. The enzymewas extracted from apple (Malus domestica cv. Ourin) fruit andpurified until it appeared as a single polypeptide chain ona gel after SDS-PAGE. From the apparent molecular mass of 62kDa obtained by SDS-PAGE and that of 120 kDa by gel filtration,the enzyme appeared to be a homodimer. Maximum rates of oxidationof sorbitol and reduction of fructose were observed at pH 9.6and pH 6.0, respectively. The K_m for oxidation of sorbitol was40.3 mM and that for reduction of fructose was 215 mM. The maximumrate of oxidation of sorbitol was about 10 times higher thanthat of the reduction of fructose. The results of the kineticanalysis strongly suggest that in vivo the enzyme would favorthe conversion of sorbitol to fructose over the reverse reaction.None of the divalent cations tested had any effect on the oxidationof sorbitol by NAD-SDH. The reaction catalyzed by NAD-SDH wasnot specific to sorbitol and other substrates could also beoxidized. Among the tested substrates, ethyl alcohol had a particularlyhigh affinity for the enzyme. (Received February 2, 1994; Accepted May 31, 1994)     

Enzymes of mannitol metabolism in the human pathogenic fungus Aspergillus fumigatus--kinetic properties of mannitol-1-phosphate 5-dehydrogenase and mannitol 2-dehydrogenase, and their physiological implications

The human pathogenic fungus Aspergillus fumigatus accumulates large amounts of intracellular mannitol to enhance its resistance against defense strategies of the infected host. To explore their currently unknown roles in mannitol metabolism, we studied A. fumigatus mannitol-1-phosphate 5-dehydrogenase (AfM1PDH) and mannitol 2-dehydrogenase (AfM2DH), each recombinantly produced in Escherichia coli, and performed a detailed steady-state kinetic characterization of the two enzymes at 25 °C and pH 7.1. Primary kinetic isotope effects resulting from deuteration of alcohol substrate or NADH showed that, for AfM1PDH, binding of D-mannitol 1-phosphate and NAD(+) is random, whereas D-fructose 6-phosphate binds only after NADH has bound to the enzyme. Binding of substrate and NAD(H) by AfM2DH is random for both D-mannitol oxidation and D-fructose reduction. Hydride transfer is rate-determining for D-mannitol 1-phosphate oxidation by AfM1PDH (k(cat) = 10.6 s(-1)) as well as D-fructose reduction by AfM2DH (k(cat) = 94 s(-1)). Product release steps control the maximum rates in the other direction of the two enzymatic reactions. Free energy profiles for the enzymatic reaction under physiological boundary conditions suggest that AfM1PDH primarily functions as a D-fructose-6-phosphate reductase, whereas AfM2DH acts in D-mannitol oxidation, thus establishing distinct routes for production and mobilization of mannitol in A. fumigatus. ATP, ADP and AMP do not affect the activity of AfM1PDH, suggesting the absence of flux control by cellular energy charge at the level of D-fructose 6-phosphate reduction. AfM1PDH is remarkably resistant to inactivation by heat (half-life at 40 °C of 20 h), consistent with the idea that formation of mannitol is an essential component of the temperature stress response of A. fumigatus. Inhibition of AfM1PDH might be a useful target for therapy of A. fumigatus infections.     

Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca²⁺-activated K⁺ channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current (I_VNSCC) and a relatively high-threshold voltage-activated (L-type) Ca²⁺ current (I_L). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect I_VNSCC or I_L in dialyzed cells. ATP (1 mM) increased I_VNSCC and decreased I_L in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on I_VNSCC. ADP decreased I_L but had no effect on I_VNSCC. The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on I_VNSCC. ATP stimulation of I_VNSCC was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y₄ is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates I_VNSCC and depresses I_L via binding of P2Y₄ receptors and stimulation of the phospholipase C/PKC pathway. inhibitory junction potentials; smooth muscle; enteric nervous system     

Purification, Kinetic and Regulatory Properties of Phosphofructokinase from Chiorella pyrenoidosa

Phosphofructokinase was purified 585-fold from Chlorella pyrenoidosaby using a combination of ammonium sulphate fractionation, filtrationthrough Sepharose 4B and chromatography on DEAE-Sephacel. Enzymestability was maintained by the presence of 50 mM P_i at pH 6.6.The optimum pH for activity was 7.7. Concentrations of substratesrequired to achieve half maximal velocity in the standard assaywere 9 µM (ATP) and 0.2 mM (fructose-6-P). ATP above 0.5mM was inhibitory. Enzyme activity was inhibited by high concentrations(10–100 mM) of P_i but lower concentrations (1–5mM) were effective in relieving the influence of other inhibitorssuch as P enolpyruvate. Inhibition by P-enolpyruvate was greaterat lower pH and with less P_i in reaction mixtures: 50% inhibitioncould be attained with 0.1 mM P-enolpyruvate. Fructose-2,6-bisphosphate,which was shown to be present in Chlorella, had no effect onthe phosphofructokinase. Chlorella appeared to contain onlyone form of phosphofructokinase, possibly in the chloroplast.No pyrophosphate :D-fructose-6-P 1-phospho transferase activitycould be detected. (Received February 20, 1984; Accepted December 5, 1984)     

Purification of Ribulose 5-phosphate Kinase and Minor Polypeptides of Pyrenoid from the Green Alga Bryopsis maxima

Ribulose 5-phosphate (Ru5P) kinase (ATP:D-ribulose 5-phosphate1-phosphotrans- ferase; EC 2.7.1.19 [EC] ), an enzyme in the reductivepentose phosphate cycle, was purified from the green alga Bryopsismaxima and its activity and peptide composition were studied.The specific activity of purified Ru5P kinase was 20 µmoleRuBP formed (mg protein)^–1 min^–1 corresponding toa 490-fold purification from the supernatant of chloroplasts.The K_m values of Ru5P kinase for ATP and Ru5P were 69 µMand 330 µM, respectively. The molecular size of Ru5P kinase was estimated as 90 kDa bygel filtration and that of its polypeptide as 41 kDa by SDS-polyacrylamidegel electrophoresis. A small portion of the Ru5P kinase wasfound in a large molecular state (500 kDa) which was consideredto be an inactive form of the enzyme. Ru5P kinase activity has been reported in the pyrenoid of Eremosphaeraviridis as well as ribulose 1,5-bisphosphate carboxylase-oxygenase(RuBisCO) and ribose 5-phosphate isomerase activity (Holdsworth1971). In Bryopsis maxima, among the pyrenoid polypeptides otherthan that of RuBisCO, we found a polypeptide of 42 kDa, similarto that of Ru5P kinase in molecular size and ratio to RuBisCO.A peptide map of the 42 kDa pyrenoid polypeptide, however, showedthat it differed from that of Ru5P kinase. In conclusion, Ru5Pkinase may be not involved in the pyrenoid of this alga. (Received January 19, 1985; Accepted May 15, 1985)     

Epidermal growth factor receptor tyrosine kinase phosphorylation of glucose-6-phosphate dehydrogenase in vitro

The specific tyrosine phosphorylation of glucose-6-phosphate dehydrogenase (G6PDH) by the epidermal growth factor (EGF) receptor in vitro is demonstrated. The Km values of the substrate G6PDH and of ATP for the receptor tyrosine kinase were ca. 1 and 10 microM, respectively. The rate of phosphorylation was EGF dependent, with a four-fold increase in Vmax in the presence of EGF. The phosphorylation was stimulated maximally by 0.2 microM or greater EGF, with an ED50 of ca. 20 nM which is consistent with the affinity of the solubilized receptor for EGF. Using conditions of 5 microM G6PDH, 100 microM ATP, 5 mM Mg2+, and 1 mM Mn2+, up to 0.3 mol phosphate was incorporated into 1 mol of the 55-kDa subunit of Baker's yeast G6PDH. Tryptic peptide mapping revealed several unique phosphopeptides for both Baker's yeast and bovine adrenal G6PDH. The patterns of phosphopeptides for a given enzyme were identical for basal and EGF-stimulated phosphorylation.     

苹果S6PDH基因克隆与原核表达

6-磷酸山梨醇脱氢酶(sorbitol-6-phosphate dehydrogenase,S6PDH)是蔷薇科植物中合成山梨醇的重要酶。以苹果叶片为材料,利用RT-PCR法克隆到S6PDHcDNA全长,将其与大肠杆菌表达载体pET-32a( )构建原核表达载体pET-S并转化大肠杆菌BL21,经IPTG诱导表达后,SDS-PAGE检测结果表明该基因表达了1个约54kD的蛋白,为进一步研究目的蛋白的结构和功能提供了试验基础。     

Hysteretic behaviour of glucose-6-phosphate dehydrogenase of pea chloroplasts

Glucose-6-phosphate dehydrogenase (G6PDH) from pea chloroplasts has at least two interconvertible kinetic states which differ from one another in their catalytic activities (‘hyperactive’ and ‘hypoactive’ forms). Preincubation of chloroplast extracts with 10 mM glucose-6-phosphate (G6P) led to the accumulation of a ‘hyperactive’ G6PDH form which exhibited a burst of activity at the start of the assay; steady state was reached after a period of several minutes. Preincubation of the pea chloroplast extracts in the absence of G6P resulted in the formation of a ‘hypoactive’ enzyme from which exhibited a lag during the assay. Steady state was reached after several minutes. The enzyme activity in the steady state was the same for both forms. The length of the lag (τ) was inversely related to the concentration of G6DH and substrate concentration. These results show that the G6PDH of pea chloroplasts, like the enzyme of cyanobacteria, behaves as a hysteretic enzyme.     

Malate dehydrogenase and glucose-6-phosphate dehydrogenase, key markers for studying the genetic diversity of Actinobacillus actinomycetemcomitans

Abstract Cell-free extracts of strains belonging to the 5 serotypes of A. actinomycetemcomitans were screened for several enzymes. Enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt and the TCA cycle were present. Of these glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) were the most readily detected and stable. MDH and G6PDH retained more than 50% of their activities at alkaline pHs (10–11) for up to 6 h and 3 h at 25°C, respectively, while at pH 6.5, 50% of their activities were lost within 2–3 h. The K _m for malate oxidation catalysed by MDH was 5.8×10⁻⁴ M while that for glucose-6-phosphate oxidation was 2.0×10⁻⁴ M. The pH optima for MDH and G6PDH oxidation activities were 10 and 9.5, respectively. Among the 5 designated serotypes of A. actinomycetemcomitans three groups were delineated by multilocus enzyme electrophoresis using MDH and G6PDH.     

Microphotometric determination of enzymes in brain sections. I. Hexokinase

A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM D-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN3, 10 mM MgCl2, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 microns. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.