Addition Of Glucose 6-phosphate Research Articles

Glucose-6-phosphate reduces calcium accumulation in rat brain endoplasmic reticulum

Brain cells expend large amounts of energy sequestering calcium (Ca2+), while loss of Ca2+ compartmentalization leads to cell damage or death. Upon cell entry, glucose is converted to glucose-6-phosphate (G6P), a parent substrate to several metabolic major pathways, including glycolysis. In several tissues, G6P alters the ability of the endoplasmic reticulum (ER) to sequester Ca2+. This led to the hypothesis that G6P regulates Ca2+ accumulation by acting as an endogenous ligand for sarco-endoplasmic reticulum calcium ATPase (SERCA). Whole brain ER microsomes were pooled from adult male Sprague-Dawley rats. Using radio-isotopic assays, 45Ca2+ accumulation was quantified following incubation with increasing amounts of G6P, in the presence or absence of thapsigargin, a potent SERCA inhibitor. To qualitatively assess SERCA activity, the simultaneous release of inorganic phosphate (Pi) coupled with Ca2+ accumulation was quantified. Addition of G6P significantly and decreased Ca2+ accumulation in a dose-dependent fashion (1–10 mM). The reduction in Ca2+ accumulation was not significantly different that seen with addition of thapsigargin. Addition of glucose-1-phosphate or fructose-6-phosphate, or other glucose metabolic pathway intermediates, had no effect on Ca2+ accumulation. Further, the release of Pi was markedly decreased, indicating G6P-mediated SERCA inhibition as the responsible mechanism for reduced Ca2+ uptake. Simultaneous addition of thapsigargin and G6P did decrease inorganic phosphate in comparison to either treatment alone, which suggests that the two treatments have different mechanisms of action. Therefore, G6P may be a novel, endogenous regulator of SERCA activity. Additionally, pathological conditions observed during disease states that disrupt glucose homeostasis, may be attributable to Ca2+ dystasis caused by altered G6P regulation of SERCA activity.

Oocytes Survival Requires Metabolic Activity

Vertebrate females produce their full complement of oocytes during embryogenesis and, over time, these are either released (and fertilized or not) or undergo cell death. Oocyte death ultimately leads to sterility as the animals age and conditions that accelerate death of the eggs cause premature infertility. Nutt et al. used unfertilized Xenopus eggs and egg extracts that recapitulate many of the cell death events to investigate the molecular mechanisms that control oocyte survival. Caspase-2 was known to regulate mouse oocyte survival, and Nutt et al. show that caspase-2 activity is inhibited in Xenopus oocytes by NADPH, which is produced as a by-product of metabolic flux through the pentose-phosphate pathway. Addition of glucose-6-phosphate (G6P) or other intermediates in the pentose-phosphate pathway, but not the glycolytic pathway, inhibited activation of caspase-2 and caspase-3, cytochrome c release, and oocyte cell death. Inhibition of G6P dehydrogenase by dehydroisoandrosterone (DHEA), which inhibits the pentose-phosphate pathway, promoted oocyte cell death, increased the rate at which caspase activity rose in the egg extracts, and decreased the concentration of G6P and NADPH production. Addition of G6P or NADPH inhibited the binding of caspase-2 to its adaptor RAIDD [in a glutathione- S -transferase (GST) pull-down experiment]. Oligomerization of caspase-2 through interactions with adaptors, such as RAIDD, is essential for caspase-2 autocleavage and activation. G6P stimulated the phosphorylation of caspase-2, and pharmacological analysis, in vitro kinase assays, and extract depletion assays indicated that calcium/calmodulin-dependent protein kinase II (CaMKII) was the responsible enzyme. Mutation of one of the two putative CaMKII phosphorylation sites in caspase-2 prevented the G6P- and NADPH-stimulated phosphorylation of caspase-2, and this mutant caspase-2 also promoted cell death of oocytes when its mRNA was injected into the oocytes. Thus, these results define a path from metabolic status through the pentose-phosphate pathway that controls oocyte cell survival in a process that relies on the phosphorylation state of caspase-2. Lipinski and Yuan discuss the implications of this research for understanding aging-related cell death. L. K. Nutt, S. S. Margolis, M. Jensen, C. E. Herman, W. G. Dunphy, J. C. Rathmell, S. Kornbluth, Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2. Cell 123 , 89-103 (2005). [PubMed] M. M. Lipinski, J. Yuan, A cellular response to an internal energy crisis. Cell 123 , 3-5 (2005). [PubMed]

Heterogeneity of Glucose Transport in Rat Liver Microsomal Vesicles

Glucose transport across the membrane of rat liver microsomal vesicles was studied by a rapid filtration method in three different experimental systems: (i) inward transport in the presence of extravesicular glucose, (ii) efflux from passively preloaded vesicles, and (iii) efflux of glucose generated intravesicularly by glucose-6-phosphatase upon addition of glucose 6-phosphate were investigated. The apparent intravesicular glucose space estimated with the rapid filtration method was lower than the total microsomal glucose accessible space both the in the steady-state phase of uptake and at the starting point of efflux: 0.5 versus 2.3 μl/mg protein. The initial rate of influx/efflux was dependent on the extravesicular/intravesicular glucose concentration and was much lower than the rate of influx estimated previously by the light-scattering technique. Both influx and efflux could be inhibited byN-ethylmaleimide and possibly became saturable at high (>100 mM) glucose concentration. Known inhibitors of GLUT transporters (genistein, cytochalasin B, phloretin, and hexoses) did not affect glucose influx. The time course of glucose efflux from vesicles preincubated in the presence of glucose 6-phosphate was similar to that from glucose-loaded vesicles. These data together with that obtained previously (by a light-scattering technique; Marcolongo, P., Fulceri, R., Giunti, R., Burchell, A., and Benedetti, A. (1996)Biochem. Biophys. Res. Commun.219, 916–922) indicate that microsomal vesicles are heterogeneous regarding their glucose-transporting properties and that glucose transport is bidirectional and its feature meets the requirements of a facilitative transport.

Identification of an endogenous NADPH-regenerating system coupled to nitrate reduction in vitro in plant and fungal crude extracts

A low molecular weight factor obtained from the crude extract of nit-1 mutant of Neurospora crassa (NEF) was capable of stimulating the activity of reconstituted NADPH specific nitrate reductase (NR, EC 1.6.6.3; Z.Z. Alikulov, N.A. Savidov, H.S. Lips, A low molecular weight regulator of nitrate reductase in higher plants. 4th International Symposium on Inorganic Nitrogen Assimilation and 1st Fohs Biostress Symposium. Seeheim/Darmstadt, Germany, 1995, p. 9). This factor eluted from a G15 column as glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P). Like the hexose-phosphates, NEF allowed rapid regeneration of NADPH from the NADP +, resulting from nitrate reduction. Sequential treatment of the NEF-containing fraction with commercial preparations of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.43) and phosphoglucose isomerase (PGI, EC 5.3.1.9) resulted in the oxidation of NEF and the reduction of NADP +. NEF, therefore, was a substrate of G6PDH and 6PGDH in a reaction which reduced NADP + to NADPH. It was concluded that the low molecular weight factor enhancing NR activity isolated from fungal and plant crude extracts (Z.Z. Alikulov, N.A. Savidov, H.S. Lips, A low molecular weight regulator of nitrate reductase in higher plants. 4th International Symposium on Inorganic Nitrogen Assimilation and 1st Fohs Biostress Symposium. Seeheim/Darmstadt, Germany, 1995, p. 9) may be a mixture of hexose phosphates. G6PDH, 6PGDH and PGI were active in crude extracts of plants and fungi used for NR assays. Oxidation of one mole of G6P led to the production of approximately two moles of NADPH. The activities of PGI, G6PDH and 6PGDH exceeded that of NR in both fungal and plant crude extract. Fungal crude extracts exhibited a considerably higher capacity to regenerate NADPH than plant extracts. The addition of G6P during the in vitro NR assay increased the stability of the reaction allowing detection of very low NR activity levels. The stimulation of NR in vitro by NADPH generating systems points out the physiological importance of these systems in nitrate assimilation and in the nitrate reduction assays in situ and in vivo.

Permeability of rat liver microsomal membrane to glucose 6-phosphate.

Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer revealed the following. (1) The increase in extravesicular osmolality by addition of glucose 6-phosphate or mannose 6-phosphate (25 mM each) caused a rapid shrinking of microsomal vesicles. After shrinkage, a rapid swelling phase (t1/2 approx. 22 s) was present with glucose 6-phosphate but absent with mannose 6-phosphate, indicating that the former had entered microsomal vesicles, but the latter had not. (2) Almost identical results were obtained in the absence of any glucose 6-phosphate hydrolysis, i.e. with microsomes pre-treated with 100 microM-vanadate. (3) The anion-channel blocker 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid (DIDS) suppressed the glucose 6-phosphate-induced swelling phase. (4) The swelling phase was more prolonged as the glucose 6-phosphate concentration increased (t1/2 = 16 +/- 3, 22 +/- 3 and 35 +/- 4 s with 25 mM, 37.5 mM- and 50 mM-glucose 6-phosphate respectively). The behaviour of glucose-6-phosphatase activity of intact and disrupted microsomes measured in the presence of high concentrations (less than 30 mM) of substrate also indicated the saturation of the glucose 6-phosphate permeation system by extravesicular concentrations of glucose 6-phosphate higher than 20-30 mM. Additional experiments showed that vanadate-treated microsomes pre-equilibrated with 0.1 mM- and 1.0 mM-glucose 6-phosphate (and [1-14C]glucose 6-phosphate as a tracer) rapidly (t1/2 less than 20 s) released [1-14C]glucose 6-phosphate when diluted in a glucose 6-phosphate-free medium. The efflux of [1-14C]glucose 6-phosphate was largely prevented by DIDS, allowing an evaluation of the intravesicular space of glucose 6-phosphate of approx. 1.0 microliter/mg of microsomal protein.

Iron-catalyzed oxidative modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Structural and functional changes.

As a variety of eukaryotic cells age, the specific activity of glucose-6-phosphate dehydrogenase (Glu-6-PDH) declines as much as 50%. Because of the central role of this enzyme in metabolism, it is important to define factors responsible for this loss in enzyme activity. We report that Glu-6-PDH from Leuconostoc mesenteroides is rapidly inactivated by micromolar concentrations of Fe2+ and H2O2. Inactivation correlated with the formation of one carbonyl functionality/enzyme subunit, indicating that inactivation is the result of site-specific oxidative modification. Our results suggest that Fe2+ binds to the glucose 6-phosphate binding site and that interaction of the enzyme-bound Fe2+ with H2O2 leads to the oxidative modification of amino acids essential for enzyme activity. Partially inactivated enzyme remained predominantly in the dimeric form, and no change in the apparent affinity of the remaining active subunits for substrate was observed. Partial inactivation did, however, lead to a decrease in the thermal stability of the remaining activity. This decrease in thermal stability could be largely overcome by the addition of glucose 6-phosphate. Thus, although exposure to H2O2 and Fe2+ results in the irreversible inactivation of Glu-6-PDH, the resulting modification is selective, leads to the formation of heterodimers of both active and inactive subunits, and does not appear to cause large scale structural changes. Our results demonstrate the inherent susceptibility of Glu-6-PDH from L. mesenteroides to modification by an oxidation system known to exist in vivo. An assessment of the physiological significance of Fe(2+)-catalyzed oxidation of Glu-6-PDH awaits extension of these studies to mammalian sources known to accumulate less active or inactive forms of the enzyme as a function of age.

Ca2+ Ionophore and Phorbol Ester Stimulate Diacylglycerol Formation and Phosphatidylcholine Hydrolysis in Rat Parotid Acinar Cells

We investigated the effects of A23187 and phorbol 12,13-dibutyrate (PDBu) on sn-1,2-diacylglycerol (DAG) accumulation and phosphatidylcholine (PC) hydrolysis in rat parotid acinar cells. Both A23187 and PDBu, in concentration ranges of 0.001–0.1 μM, stimulated DAG accumulation and PC hydrolysis in a time- and concentration-dependent manner. Treatment with A23187 and PDBu stimulated the release of [3H]choline and [3H]phosphocholine into the medium, indicating [3H]PC hydrolysis is due to the activation of phospholipases C and D; however, [3H]phosphatidylethanolamine hydrolysis was not indicated. These releases were unaffected by the addition of glucose 6-phosphate, a phosphatase inhibitor. Staurosporine, a protein kinase C inhibitor, significantly inhibited the DAG accumulation and the PC hydrolysis stimulated by these agents. Combinations of A23187 and PDBu potentiated the stimulatory effect which each of these agents alone had on DAG accumulation and PC hydrolysis. This mode of action was additive but not synergistic. These results suggest that DAG accumulation induced by A23187 and PDBu is related to the PC hydrolysis mediated via the activation of phospholipases C and D, and that it is not related to phosphatidylethanolamine hydrolysis.

Toxic heavy metal ions activate the heme-regulated eukaryotic initiation factor-2 alpha kinase by inhibiting the capacity of hemin-supplemented reticulocyte lysates to reduce disulfide bonds

Addition of toxic heavy metal ions (Cd2+, Hg2+, and Pb2+) to hemin-supplemented rabbit reticulocyte lysate brings about the activation of the heme-regulated eukaryotic initiation factor 2 alpha kinase (HRI) and the inhibition of protein chain initiation. In this report we examined the effects of monothiol and dithiol compounds, metal ion-chelating agents, and metallothioneins (MT) on metal ion-induced inhibition of protein synthesis. The dithiol compounds dithiothreitol and 2,3-dimercaptopropane sulfonic acid prevented and relieved the inhibition of protein synthesis caused by Cd2+ and Hg2+ in hemin-supplemented lysates, but the monothiol compounds 2-mercaptoethanol, cysteamine, D-(-)penicillamine, and glutathione had no effect. The inhibition of protein synthesis caused by Cd2+ was reversed by the addition of excess EDTA but not by the addition of excess nitrilotriacetic acid. Toxic heavy metal ions inhibited the capacity of hemin-supplemented lysate to reduce disulfide bonds. Addition of excess EDTA to Cd(2+)-inhibited lysates restored the capacity of the lysate to reduce disulfide bonds and inhibited the phosphorylation of eukaryotic initiation factor eIF-2. MTs and their apoproteins (apoMTs) inhibited the activation of HRI and protected protein synthesis from inhibition by Cd2+, Hg2+, and Pb2+. Addition of apoMTs to heavy metal ion-inhibited lysates restored the capacity of lysates to reduce disulfide bonds. The restoration of the lysate's thioredoxin/thioredoxin reductase activity was accompanied by the inactivation of HRI and the resumption of protein synthesis, indicating that apoMTs can "detoxify" metal ions already bound to proteins. Several observations presented in this report suggest that the binding of metal ions to the alpha-domain of MT is responsible for the ability of MT to sequester bound metal in a non-toxic form. Addition of glucose 6-phosphate or NADPH had no effect on protein synthesis in metal ion-inhibited lysates, and NADPH concentrations in Cd(2+)-inhibited and hemin-supplemented control lysates were equivalent. The data suggest that the metal ions cause the inhibition of protein synthesis by binding to vicinal sulfhydryl groups present in some critical protein(s), possibly the dithiols present in the active site of thioredoxin and (or) thioredoxin reductase, which leads to the activation of HRI.

Inhibition of rabbit reticulocyte lysate protein synthesis by heavy metal ions involves the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2.

The effect of heavy metal ions (in particular Cd2+, Hg2+, and Pb2+) on protein synthesis in hemin-supplemented reticulocyte lysates was investigated. Heavy metal ions were found to inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. The shut off of protein synthesis occurred in conjunction with the phosphorylation of the alpha-subunit of the eukaryotic initiation factor (eIF) 2, the loss of reversing factor (RF) activity, and the disaggregation of polyribosomes. Addition of eIF-2 or RF to heavy metal ion-inhibited lysates restored protein synthesis to levels observed in hemin-supplemented controls. The stimulation of protein synthesis observed upon the addition of cAMP to heavy metal ion-inhibited lysates correlated with the inhibition of eIF-2 alpha phosphorylation and the restoration of RF activity. The partial restoration of protein synthesis observed upon the addition of MgGTP to heavy metal ion-inhibited lysates correlated with a partial inhibition of eIF-2 alpha phosphorylation. Addition of glucose 6-phosphate was found to have no effect on protein synthesis of eIF-2 alpha phosphorylation under these conditions. Antiserum raised to the reticulocyte heme-regulated eIF-2 alpha kinase inhibited the phosphorylation of eIF-2 alpha catalyzed by Hg2+-inhibited lysate. The inhibition of protein synthesis observed in the presence of heavy metal ions correlated with the relative biological toxicity of the ions. Highly toxic ions (AsO-2, Cd2+, Hg2+, Pb2+) inhibited protein synthesis by 50% at concentrations of 2.5-10 microM. Cu2+, Fe3+, and Zn2+, which are moderately to slightly toxic ions, inhibited protein synthesis by 50% at concentrations of 40, 250, and 300 microM, respectively. The data presented here indicate that heavy metal ions inhibit protein chain initiation in hemin-supplemented lysates by stimulating the phosphorylation of eIF-2 alpha apparently through the activation of the heme-regulated eIF-2 alpha kinase rather than through inhibition of the rate of eIF-2 alpha dephosphorylation.

Insulin and glucose 6-phosphate stimulation of Ca2+ uptake by skinned muscle fibers.

Calcium uptake by skinned muscle fibers is stimulated by physiological concentrations of insulin. These fibers, which lack a functional plasma membrane, are permeable to macromolecules but retain extensive portions of their sarcolemma in the form of transverse tubules intercalated between the myofibrils. They have an active sarcoplasmic reticulum that removes 45Ca2+ from solution at concentrations below the threshold that initiates contraction (less than 1 microM). The Ca2+ uptake activity is stimulated by insulin, presumably in response to its binding to those receptors located in the transverse tubules. Addition of glucose 6-phosphate, whose intracellular concentration increases in response to insulin, also stimulates Ca2+ uptake, a unique property of this preparation. These data indicate that insulin and glucose 6-phosphate act in concert to stimulate the sarcoplasmic reticulum. The resulting decrease in myoplasmic Ca2+ and the increase in glucose 6-phosphate would serve to mediate some of the anabolic effects of the hormone.

Glucose 6-phosphate dehydrogenase from brewers' yeast (Zwischenferment): Further observations on the ligand-lnduced macromolecular association phenomenon; kinetic properties of the two-chain protein species; and studies on the enzyme-substrate interactions

In the presence of EDTA 3−, the two-subunit species of apoenzyme ( M r 102,000 g/mole) ( Yue, Noltmann, and Kuby (1967) Biochemistry 6, 1174) maintains its integrity without further dissociation into component subunits over a broad range of pH values to 9.8 and in very dilute solutions, as demonstrated by sedimentation equilibrium studies. In the absence of EDTA 3−, studies on the ligand-induced macromolecular association to the four-subunit species ( Yue, Noltmann, and Kuby (1969) J. Biol. Chem. 244, 1353–1364) have been further extended for NADP + to include ultracentrifugal titrations at pH 7.8 and 3 °C. A significant concentration of EDTA 3−, as before, will inhibit these macromolecular association reactions and stabilize the apoenzyme species in solution. NADPH is similar to NADP + in that it will induce the formation of the four-subunit species; but by way of contrast, glucose 6-phosphate 2−will not. In fact, the addition of glucose 6-phosphate 2− to NADPH solutions may actually inhibit or reverse the association reaction induced by NADPH, and in this respect, glucose 6-phosphate 2− acts in a fashion not unlike the EDTA − ion. Thus, the ratio of ( NADP) 0 ( Glc-6-P) 0 as well as their absolute concentrations will affect the extent of dimerization of the enzyme in the absence of any other modifiers. The use of EDTA 3− has now permitted direct steady-state kinetic measurements on the two-chain species alone, which has proved to be active even in the presence of high concentrations of EDTA 3−. The steady-state kinetic mechanism of the “monomeric” enzyme species appears to have reduced to a relatively simple case, viz., to that of a random quasi-equilibrium type with a rate-limiting step at the interconversion of the ternary complexes and with independent binding of the substrates; values for the kinetic parameters have been derived for this mechanism. The product inhibition pattern of NADPH (competitive with respect to both substrates) would point, in addition, to the absence of any kinetically significant concentrations of dead-end complexes with the two-chain species. To confirm the assigned kinetic mechanism for the two-chain enzyme, measurements of the equilibrium binding of the substrates were undertaken by several techniques. By gel filtration through Sephadex G-25, in the presence of EDTA 3−, two equilavent binding sites for NADP + per mole of apoenzyme (or one per subunit)- is observed, with a value for the intrinsic dissociation constant approximating the kinetically determined value. On the other hand, in the absence of EDTA 3−, the nonlinear Scatchard plot obtained is likely a reflection of the superimposed association-dissociation reactions involving the protein. Similar quantitative studies for the ligand NADP +, in the presence and absence of EDTA 3−, were conducted by equilibrium dialysis, by a uv difference-spectral technique, and by measurement of the protection afforded by either substrate against inactivation by 5,5′-dithiobis(2-nitrobenzoic acid), which apparently reacts with only a single exposed sulfhydryl group per subunit. Coincident with these studies, a total amino acid composition for this protein is reported. In the case of glucose 6-phosphate, by the technique of equilibrium dialysis, there appears to be two major equivalent binding sites (or one per subunit) with an intrinsic dissociation constant approximating that measured kinetically, as well as two additional but very weak sites (or one additional site per subunit). Moreover, EDTA 3− exerts no effect on the equilibrium binding behavior of Glc-6- P, in agreement with the observation that Glc6- P does not induce the formation of the tetra-chain species. Glucose 6-phosphate will also protect the enzyme against inactivation by 5,5′dithiobis(2-nitrobenzoic acid), pointing to a close proximity of both binding sites for Glc-6- P and NADP + to the single exposed sulfhydryl group per subunit. Finally, fluorometric observations have qualitatively confirmed the existence of both NADPH- and NADP +- enzyme binary compounds.

Regulation of the pentose phosphate cycle.

1. A search was made for mechanisms which may exert a ;fine' control of the glucose 6-phosphate dehydrogenase reaction in rat liver, the rate-limiting step of the oxidative pentose phosphate cycle. 2. The glucose 6-phosphate dehydrogenase reaction is expected to go virtually to completion because the primary product (6-phosphogluconate lactone) is rapidly hydrolysed and the equilibrium of the joint dehydrogenase and lactonase reactions is in favour of virtually complete formation of phosphogluconate. However, the reaction does not go to completion, because glucose 6-phosphate dehydrogenase is inhibited by NADPH (Neglein & Haas, 1935). 3. Measurements of the inhibition (which is competitive with NADP(+)) show that at physiological concentrations of free NADP(+) and free NADPH the enzyme is almost completely inhibited. This indicates that the regulation of the enzyme activity is a matter of de-inhibition. 4. Among over 100 cell constituents tested only GSSG and AMP counteracted the inhibition by NADPH; only GSSG was highly effective at concentrations that may be taken to occur physiologically. 5. The effect of GSSG was not due to the GSSG reductase activity of liver extracts, because under the test conditions the activity of this enzyme was very weak, and complete inhibition of the reductase by Zn(2+) did not abolish the GSSG effect. 6. Preincubation of the enzyme preparation with GSSG in the presence of Mg(2+) and NADP(+) before the addition of glucose 6-phosphate and NADPH much increased the GSSG effect. 7. Dialysis of liver extracts and purification of glucose 6-phosphate dehydrogenase abolished the GSSG effect, indicating the participation of a cofactor in the action of GSSG. 8. The cofactor removed by dialysis or purification is very unstable. The cofactor could be separated from glucose 6-phosphate dehydrogenase by ultrafiltration of liver homogenates. Some properties of the cofactor are described. 9. The hypothesis that GSSG exerts a fine control of the pentose phosphate cycle by counteracting the NADPH inhibition of glucose 6-phosphate dehydrogenase is discussed.

Association of epididymal adipose tissue hexokinase with subcellular structure

1. 1. When differential centrifugations were carried out with sucrose homogenates of rat epididymal adipose tissue, it was observed that hexokinase activity was associated with subcellular fractions. 2. 2. Electron microscopy of the precipitates resulting from the differential centrifugations revealed that hexokinase (ATP: d-hexo 6-phosphotransferase, EC 2.7.1.1) activity was associated with both mitochondria and microsomes. Mitochondria were specifically identified by treatment with calcium phosphate and gradient centrifugation. 3. 3. Bound hexokinase activity was observed with both inner and outer mitochondrial membranes separated after hypotonic breakage of the mitochondria. 4. 4. The bound hexokinase activity was slowly released in the course of 50 min incubation at 4° when MgCl 2, KCl or (NH 4) 2SO 4 were added to suspensions of either mitochondria or microsomes. When glucose 6-phosphate was added, hexokinase activity was rapidly released, resulting in a certain partition between soluble and particle-bound enzyme activity which was dependent on the concentration of glucose 6-phosphate. 5. 5. The effect of glucose 6-phosphate was antagonized by inorganic phosphate. This allowed the demonstration of reversible release of bound hexokinase activity. Thus, when enzyme activity was first eluted from the particles by addition of glucose 6-phosphate, a considerable part of the activity was brought back to the particle-bound state by the subsequent addition of inorganic phosphate.

Glycogen Synthetase of Bee Larvae: UTILIZATION OF NATIVE PRIMER AND THE EFFECTS OF GLUCOSE 6-PHOSPHATE

During the larval stage of the honeybee there is an abrupt and very large elevation of glycogen content. Homogenates from larvae of high glycogen content have an active glycogen synthetase that is associated with the particulate glycogen fraction. Although the enzyme resembles mammalian glycogen synthetase in many of its properties there is at least one important difference. The enzyme bound with particulate glycogen of bee larvae, but not of mammals, is an active enzyme-substrate complex, since high rates of glucose incorporation are observed in the absence of added soluble primer. The main factor limiting incorporation of glucose into glycogen under the conditions of these experiments seems to be the length of outer branch chains in the primer. When this limit is reached, incorporation stops. Addition of glucose 6-phosphate causes incorporation to resume until a higher limit is attained. Two alternative interpretations of this effect are possible. Glucose 6-phosphate either changes the specificity of the enzyme for primer outer chains or activates a glucose 6-phosphate-dependent form of the enzyme that can utilize longer outer chains as effective priming units.

The effect of B 12 on enzyme activity in E. coli mutant 113-3

1. 1. It is shown that the stimulation of enzyme activity in E. coli mutant 113-3 by vitamin B 12 is markedly enhanced by the presence of glutathione. The further addition of glucose 6-phosphate and DPN is required for maximum stimulation of many enzymes. 2. 2. The fact that B 12 is not required for the synthesis or activity of individual enzymes in freshly harvested cells suggests that it is not a cofactor in these reactions. 3. 3. The reactivation of enzymes by B 12 after inactivation by aging suggests that B 12 in conjunction with glutathione affects a reversible structural change common to these enzyme proteins.