Glucose-6-phosphate dehydrogenase in lichen planus skin

The apparent Michaelis constant (Km) for glucose-6-phosphate of the enzyme glucose-6-phosphate dehydrogenase has been measured in extracts prepared from biopsies of normal human skin and from both affected and apparently normal skin of patients with lichen planus. No differences of Km were found and starch gel electrophoresis of extracts from lichen planus lesions and normal controls showed similar patterns when stained for glucose-6-phosphate dehydrogenase activity. These results do not support the view that lichen planus is an inborn error of metabolism in which the structure of glucose-6-phosphate dehydrogenase of skin is affected.     

Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions

Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP. Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42 degreesC. Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions.     

Detection of specific glucose-3-phosphatase activity in rat liver

Sugar-3-phosphates are related to aspects of diabetes which depend on protein glycosylation events. Sorbitol-3-phosphate and fructose-3-phosphate occur in normal and diabetic individuals, and glucose-3-phosphate is a potential intermediate in their biosynthesis. Almost nothing is known about enzyme pathways for their metabolic turnover. We have found that part of the phosphohydrolytic activity on glucose-3-phosphate in rat liver supernatants corresponds to a specific, Mg(2+)-dependent, glucose-3-phosphatase much less or not active on other phosphate esters, including glucose-1-phosphate, glucose-6-phosphate, fructose-1-phosphate, fructose-6-phosphate and p-nitrophenyl-phosphate. This finding opens a route to a better understanding of the metabolism and role of sugar-3-phosphates.     

Polycythemia vera: stem-cell and probable clonal origin of the disease

Two women with polycythemia vera and heterozygosity (GdB/GdA) at the X-chromosome-linked locus for glucose-6-phosphate dehydrogenase were studied to determine the nature of the cellular origin of their polycythemia. In contrast to unaffected tissue, such as skin fibroblasts, which consisted of both B and A types, the glucose-6-phosphate dehydrogenase of the patients' erythrocytes, granulocytes and platelets was only of Type A. These results provide direct evidence for the stem-cell nature of polycythemia vera and strongly imply a clonal origin for this disease. The fact that no descendants of the presumed normal stem cells were found in circulation suggests that bone-marrow proliferation in this disorder is influenced by local (intramarrow) regulatory factors.     

The condensing effects of egg lecithin and cholesterol on triolein monolayers are inhibited by substitution of one saturated acyl chain in the triacylglycerol

1. u.v. radiations and copper acetate, as free radical generating systems, determine a significant diminishing of glucose-6-phosphate dehydrogenase activity in the homogenates of Saccharomyces cerevisiae. 2. The inactivation is proportional to the concentration of the formed free radicals, existing a direct dependence on the action time of the free radicals generating systems and on the irradiation dose. The decrease of the enzyme catalytic activity is correlated with the increase of the malondialdehyde concentration. 3. The affinity for the substrate of the enzyme under the action of free radicals does not change significantly compared to the native enzyme: the Km value for NADP is halved, whilst that for glucose-6-phosphate remains unchanged. 4. The electrophoretic study shows evidence of five electrophoretic bands with enzymatic activity in the native extract and the disappearance of one molecular form under the free radical action.     

The gene for glycogen-storage disease type 1b maps to chromosome 11q23

Glycogen-storage disease type 1 (GSD-1), also known as "von Gierke disease," is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase) activity. There are four distinct subgroups of this autosomal recessive disorder: 1a, 1b, 1c, and 1d. All share the same clinical manifestations, which are caused by abnormalities in the metabolism of glucose-6-phosphate (G6P). However, only GSD-1b patients suffer infectious complications, which are due to both the heritable neutropenia and the functional deficiencies of neutrophils and monocytes. Whereas G6Pase deficiency in GSD-1a patients arises from mutations in the G6Pase gene, this gene is normal in GSD-1b patients, indicating a separate locus for the disorder in the 1b subgroup. We now report the linkage of the GSD-1b locus to genetic markers spanning a 3-cM region on chromosome 11q23. Eventual molecular characterization of this disease will provide new insights into the genetic bases of G6P metabolism and neutrophil-monocyte dysfunction.     

Purification, characterization, and high performance liquid chromatography assay of Salmonella glucose-1-phosphate cytidylyltransferase from the cloned rfbF gene

We report the purification and characterization of glucose-1-phosphate cytidylyltransferase, the first of five enzymes committed to biosynthesis of CDP-D-abequose from Salmonella enterica strain LT2. The purification was greatly facilitated by using a cloned rfbF gene encoding this enzyme. Pure enzyme was obtained by 64-fold enrichment in three chromatography steps. The NH2-terminal sequence of the purified enzyme was in agreement with the sequence predicted from the nucleotide sequence of the rfbF gene. The SDS-polyacrylamide gel electrophoresis estimated subunit M(r) of 31,000 agrees well with the M(r) of 29,035 calculated from the amino acid composition deduced from the nucleotide sequence of the rfbF gene. The glucose-1-phosphate cytidylyltransferase catalyzes a reversible bimolecular group transfer reaction and steady-state kinetic measurements, including product inhibition patterns, indicate that this reaction proceeds by a "ping-pong" type of mechanism. The Km values for CTP, alpha-D-glucose 1-phosphate, CDP-D-glucose, and pyrophosphate are 0.28, 0.64, 0.11, and 1.89 mM, respectively.     

Saccharomyces cerevisiae GNA1, an essential gene encoding a novel acetyltransferase involved in UDP-N-acetylglucosamine synthesis

The Saccharomyces cerevisiae gene, YFL017C, for a putative acetyltransferase was characterized. Disruption of YFL017C was lethal, leading to a morphology similar to those caused by the depletion of AGM1 or UAP1, the genes encoding phospho-N-acetylglucosamine mutase and UDP-N-acetylglucosamine pyrophosphorylase, respectively. This implies the involvement of YFL017C in UDP-N-acetylglucosamine synthesis. The recombinant protein for YFL017C displayed phosphoglucosamine acetyltransferase activities in vitro and utilized glucosamine 6-phosphate as the substrate. When incubated with Agm1p and Uap1p, the Yfl017c protein produced UDP-N-acetylglucosamine from glucosamine 6-phosphate. These results indicate that YFL017C specifies glucosamine-6-phosphate acetyltransferase; therefore, the gene was designated GNA1 (glucosamine-6-phosphate acetyltransferase). In addition, whereas bacterial phosphoglucosamine acetyltransferase and UDP-N-acetylglucosamine pyrophosphorylase activities are intrinsic in a single polypeptide, they are encoded by distinct essential genes in yeast. When the sequence of ScGna1p was compared with those of other acetyltransferases, Ile97, Glu98, Val102, Gly112, Leu115, Ile116, Phe142, Tyr143, and Gly147 were found to be highly conserved. When alanine was substituted for these amino acids, the enzyme activity for the substituted Phe142 or Tyr143 enzymes was severely diminished. Although the activity of Y143A was too low to perform kinetics, F142A displayed a significantly increased Km value for acetyl-CoA, suggesting that the Phe142 and Tyr143 residues are essential for the catalysis.     

Red blood cell glucose metabolism in trisomy 10p: possible role of hexokinase in the erythrocyte

Red blood cell glucose metabolism was investigated in a male patient with de novo trisomy 10p. According to previous evidence, when assigning hexokinase gene locus in the 10p11 leads to pter region, a triplex dosage effect of hexokinase activity (HK) was found, while all the other erythrocyte glycolytic enzymes were in the normal values range. Red blood cell glucose utilization was 2.87 mumole/hr/ml RBC as compared to 1.43 in normal controls; the rate of glucose metabolized through the hexose monophosphate shunt (HMPS) was unchanged. Glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, and dihydroxyacetone phosphate increased with respect to normal controls, while normal levels of 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and ATP were found. The HK activity increased in all the red blood cell fractions obtained by density gradient ultracentrifugation. However, a small difference in the distribution of cells through the gradient was evident. The experiments reported in this article show that in the red blood cells of patients with trisomy 10p, an increased level of HK leads to higher concentrations of glucose-6-phosphate and to a faster glucose utilization in the Embden-Meyerhof pathway, while the HMPS rate is unchanged.     

Frequency analysis of the contralateral suppression of evoked otoacoustic emissions by narrow-band noise

Yeast cells defective in the GGS1 (FDP1/BYP1) gene are unable to adapt to fermentative metabolism. When glucose is added to derepressed ggs1 cells, growth is arrested due to an overloading of glycolysis with sugar phosphates which eventually leads to a depletion of phosphate in the cytosol. Ggs1 mutants lack all glucose-induced regulatory effects investigated so far. We reduced hexokinase activity in ggs1 strains by deleting the gene HXK2 encoding hexokinase PII. The double mutant ggs1 delta, hxk2 delta grew on glucose. This is in agreement with the idea that an inability of the ggs1 mutants to regulate the initiation of glycolysis causes the growth deficiency. However, the ggs1 delta, hxk2 delta double mutant still displayed a high level of glucose-6-phosphate as well as the rapid appearance of free intracellular glucose. This is consistent with our previous model suggesting an involvement of GGS1 in transport-associated sugar phosphorylation. Glucose induction of pyruvate decarboxylase, glucose-induced cAMP-signalling, glucose-induced inactivation of fructose-1,6-bisphosphatase, and glucose-induced activation of the potassium transport system, all deficient in ggs1 mutants, were restored by the deletion of HXK2. However, both the ggs1 delta and the ggs1 delta, hk2 delta mutant lack detectable trehalose and trehalose-6-phosphate synthase activity. Trehalose is undetectable even in ggs1 delta strains with strongly reduced activity of protein kinase A which normally causes a very high trehalose content. These data fit with the recent cloning of GGS1 as a subunit of the trehalose-6-phosphate synthase/phosphatase complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of reductive activation of potato tuber ADP-glucose pyrophosphorylase

The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase activity is activated by a incubation with ADP-glucose and dithiothreitol or by ATP, glucose- 1-phosphate, Ca2+, and dithiothreitol. The activation was accompanied by the appearance of new sulfhydryl groups as determined with 5, 5'-dithiobis(2-nitrobenzoic acid). By analyzing the activated and nonactivated enzymes on SDS-polyacrylamide gel electrophoresis under nonreducing conditions, it was found that an intermolecular disulfide bridge between the small subunits of the potato tuber enzyme was reduced during the activation. Further experiments showed that the activation was mediated via a slow reduction and subsequent rapid conformational change induced by ADP-glucose. The activation process could be reversed by oxidation with 5, 5'-dithiobis(2-nitrobenzoic acid). Incubation with ADP-glucose and dithiothreitol could reactivate the oxidized enzyme. Chemical modification experiments with [14C]iodoacetic acid and 4-vinylpyridine determined that the intermolecular disulfide bridge was located between Cys12 of the small subunits of the potato tuber enzyme. Mutation of Cys12 in the small subunit into either Ala or Ser eliminated the requirement of DTT on the activation and prevented the formation of the intermolecular disulfide of the potato tuber enzyme. The mutants had instantaneous activation rates as the wild-type in the reduced state. A two-step activation model is proposed.     

Synthesis and loss of activity of glucose-6-phosphate dehydrogenase in dividing plant cells

1. Using the technique of density-labelling with deuterium oxide, evidence has been obtained for the de novo synthesis of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADPH+ 1-oxidoreductase, EC 1.1.1.49), during the culture of synchronously growing plant cells. 2. The entire increase in enzyme activity during the early cell cycles in this material can be accounted for by the appearance of an enzyme species with increased buoyand density. 3. A method is described for resolving overlapping distribution profiles after density centrifugation, which allows estimation of the amount of each species present at different times, and calculation of the loss of activity of the light species present from the start of culture. 4. Loss of activity of glucose-6-phosphate dehydrogenase in normal growing conditions in the presence of 2,4-dichlorophenoxyacetic acid is very much faster than in conditions which do not lead to cell division: in the absence of 2,4-dichlorophenoxyacetic acid, or in the presence of the inhibitor of RNA synthesis, 6-methylpurine.     

Respiratory abnormalities and ventilatory capacity in a Papua New Guinea Island community

Several molecular forms of human glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) corresponding to different stages of post-synthetic modifications have been purified from human leukocytes. The various enzyme forms were different in their specific activity, their kinetic properties and their isoelectrofusing pattern. The molecular weight of the subunits of the different forms was not modified. The changes in the electrofocusing pattern were not due to modifications of the N-terminal ends, the oxidation of thiol groups or the non-covalent fixation of an acid molecule upon the enzyme. Carboxypeptidase B cleaved a C-terminal lysine from the different enzyme forms and shifted the isoelectric point of the different enzyme active bands towards the acid pH. The different enzyme forms studied here seemed to result from the action upon 'native glucose-6-phosphate dehydrogenase' of 'modifying factors' especially abundant in some leukemic granulocytes. The modifying factors did not seem to be consumed during the 'modification' of glucose-6-phosphate dehydrogenase. Moreover, the storage for one year of unmodified enzyme resulted in changes in its electrofocusing pattern similar to those quickly induced by the 'modifying factors'. Consequently it appears that the modifying factors are catalysts of the modification of special residues of glucose-6-phosphate dehydrogenase. The hypothesis that this modification involves the deamination of asparagine or glutamine residues is put forward.     

The in vivo regulation of liver and kidney glucose-6-phosphatase by dexamethasone

The microsomal glucose-6-phosphatase enzyme is situated with its active site inside the lumen of the endoplasmic reticulum and for normal enzyme activity in vivo, transport systems are needed for the substrates and products of the enzyme. Most studies of glucose-6-phosphatase have been carried out on the liver enzyme and relatively little is known about the regulation of the kidney glucose-6-phosphatase enzyme system. Here we demonstrate that the liver and kidney glucose-6-phosphatase systems are regulated differently by dexamethasone and that dexamethasone acts on both the glucose-6-phosphatase enzyme and T1 its associated glucose-6-phosphate transport protein.     

A 17-amino acid insert changes UDP-N-acetylhexosamine pyrophosphorylase specificity from UDP-GalNAc to UDP-GlcNAc

We previously reported the purification of a UDP-N-acetylhexosamine (UDP-HexNAc) pyrophosphorylase from pig liver that catalyzed the synthesis of both UDP-GlcNAc and UDP-GalNAc from UTP and the appropriate HexNAc-1-P (Szumilo, T., Zeng, Y., Pastuszak, I., Drake, R., Szumilo, H., and Elbein, A. D. (1996) J. Biol. Chem. 271, 13147-13154). Both sugar nucleotides were synthesized at nearly the same rate, although the Km for GalNAc-1-P was about 3 times higher than for GlcNAc-1-P. Based on native gels and SDS-polyacrylamide gel electrophoresis, the enzyme appeared to be a dimer of 120 kDa composed of two subunits of about 57 and 64 kDa. Three peptides sequenced from the 64-kDa protein and two from the 57-kDa protein showed 100% identity to AGX1, a 57-kDa protein of unknown function from human sperm. An isoform called AGX2 is identical in sequence to AGX1 except that it has a 17-amino acid insert near the carboxyl terminus. We expressed the AGX1 and AGX2 genes in Escherichia coli. The protein isolated from the AGX1 clone comigrated on SDS gels with the liver 57-kDa pyrophosphorylase subunit and was 2-3 times more active with GalNAc-1-P than with GlcNAc-1-P. On the other hand, the protein from the AGX2 clone migrated with the liver 64-kDa pyrophosphorylase subunit and had 8-fold better activity with GlcNAc-1-P than with GalNAc-1-P. These results indicate that insertion of the 17-amino acid peptide modifies the specificity of the pyrophosphorylase from synthesis of UDP-GalNAc to synthesis of UDP-GlcNAc.     

Contribution of peroxidation products to oxidative inactivation of rat liver microsomal glucose-6-phosphatase

Exposure of rat liver microsomes to ascorbic acid/Fe(2+) caused decreases in the membrane-bound glucose-6-phosphate (G-6-Pase) activity and the protein thiols after a short lag period (4 min). Under the same conditions, the production of thiobarbituric acid-reactive substances and fluorescent products was also initiated from 4 min after the start of the treatment, although conjugated diene was formed immediately on incubation of the microsomes with ascorbic acid/Fe(2+). After centrifugation of the treated microsomes, the fluorescent products and the enzyme activity remained in the membrane fraction. The results of kinetic studies of the enzyme activity indicated that ascorbic acid/Fe(2+)-induced inhibition of the enzyme activity is mainly due to an increased Km value for the substrate. A decreased activity of the microsomal G-6-Pase was also observed when the microsomes were incubated with aldehydes such as malondialdehyde, n-heptaldehyde, acetaldehyde, and trans-2-nonenal. However, loss of protein thiols was detected only upon treatment of the microsomes with trans-2-nonenal. Glucose-6-phosphate (G-6-P)effectively prevented ascorbic acid/Fe(2+)- or trans-2-nonenal-induced inhibition of the enzyme activity, but the substrate failed to protect the protein thiols in both systems. The results of fluorescence anisotropy measurements of diphenylhexatriene-labeled microsomes suggested that changes in the lipid dynamics are not directly related to peroxidation- mediated inhibition of the enzyme activity. Based on these results, a possible reason for the inhibition of the microsomal G-6-Pase activity associated with ascorbic acid/Fe(2+) treatment is discussed.     

Regulation of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Role of the NH2-terminal region

The role of the NH2-terminal region of the liver and skeletal muscle 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatases was investigated, as well that of a mutant of the liver isoform lacking the first 22 amino acids, by the overexpression of these enzymes in Escherichia coli and the comparison of their kinetic properties. The muscle isoform and the deletion mutant had Km values for fructose 6-phosphate which were 50- and 20-fold higher, respectively, than that of the liver isoform, and the bisphosphatase maximal velocity of the liver deletion mutant was 4-fold higher than that of the native liver isoform. Phosphorylation of the liver isoform increased bisphosphatase activity by 2-3-fold and the Km for fructose 6-phosphate of the 6-phosphofructo-2-kinase by 10-15-fold, but these kinetic effects were greatly diminished for the deletion mutant despite equivalent phosphorylation by cAMP-dependent protein kinase. Arg-173 of the skeletal muscle isoform was found to be functionally equivalent to the residue corresponding to the essential fructose 6-phosphate binding residue of the liver kinase domain, Arg-195. The results suggest that 1) the NH2-terminal regions of the liver and skeletal muscle isoforms are important determinants of fructose 6-phosphate affinity, and 2) the initial 22 amino acids of the liver isoform exert an inhibitory influence on the bisphosphatase and mediate, at least in part, the response of both activities of the enzyme to cAMP-dependent phosphorylation.     

Mannosidosis: clinical, morphologic, immunologic, and biochemical studies

The primary metabolic defect in mannosidosis is the deficiency of the acidic alpha-mannosidase A and B activites which results in the lysosomal accumulation of mannose-rich substrates. Out studies demonstrate that the enzymatic diagnosis of suspect homozygotes can be made reliably using plasma, isolated leukocytes, or cultured skin fibroblasts assayed carefully at the appropriate acidic pH. Immunologic studies of a mannosidosis homozygote revealed significant abnormalities of neutrophil function; these included a depressed chemotactic responsiveness and impaired phagocytosis of bacteria. Lymphocyte transformation studies showed a 20% of normal response to purified phytohemagglutinin and a 25% of normal response to concanavalin A. Three major components of alpha-mannosidase activity in normal human liver were resolved by ion exchange chromatography on DEAE-cellulose and electrophoresis on cellulose acetate gels. Electrophoresis of the liver extract from homozygote I with mannosidosis revealed only one band of activity which coelectrophoresed with the alpha-mannosidase C isozyme partially purified from normal liver. However, ion exchange chromatography revealed the presence of residual hepatic acidic activities; the residual A isozyme was eluted in a position corresponding to that of normal alpha-mannosidase A whereas the residual B activity was eluted at a slightly more electronegative position than that of normal B isozyme. The apparent Km values for alpha-mannosidase activity as determined from Linweaver-burk plots were 1.1 mM for normal liver and 0.9 mM for normal leukocytes. In contrast, the residual activity in these sources from homozygote 1 could not be saturated within the solubility range of the substrate; the apparent Km value was estimated at 15.4 mM in liver extracts. Zinc significantly lowered the apparent Km value of the acidic activity in normal liver (from 1.2 to 0.24 mM), whereas this metallic ion had little effect on the values for mannosidosis hepatic activity (from 15.4 to 12.3 mM). Unlike zinc, cobalt had its major effect on the acidic activity in the mannosidosis liver extract, lowering the apparent Km from 15.4 to 3.9 mM, whereas the apparent Km for the normal activity was increased from 1.2 to 1.9 mM. The residual acidic activities were markedly stimulated by zinc in both leukocytes (approximately 300%) and plasma ( approximately 400%) from the homozygotes and to a lesser extent in those sources from normal individuals. In contrast, cobalt enhanced the residual acidic activities in leukocytes (approximately 500%) and plasma (approximately 200%) from the homozygotes while inhibiting these acidic activities (78.9% and 47.7%, respectively) in normal individuals.     

Participation of cytochrome P450-2B and -2D isozymes in the demethylenation of methylenedioxymethamphetamine enantiomers by rats

The cytochrome P450 isozymes in rat liver microsomes that catalyze the demethylenation of methylenedioxymethamphetamine enantiomers to the corresponding dihydroxymethamphetamine were characterized. Dihydroxymethamphetamine formation in liver microsomes from male Sprague-Dawley rats exhibited multienzyme kinetics, with Km values in the micromolar/millimolar range. The stereoselectivity [(+)-isomer versus (-)-isomer] varied from 0.78 to 1.94 after pretreatment of the rats with phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, or pyrazole, suggesting that different isozymes participate in the reaction. The low-Km demethylenation was not induced by these compounds and was not inhibited by antibodies raised against CYP2C11. Liver microsomes from female Dark-Agouti rats, a strain genetically deficient in CYP2D1, exhibited demethylenation activities that were 9% of those in microsomes from male Sprague-Dawley rats. The low-Km demethylenation was also inhibited by CYP2D substrates such as sparteine, bufuralol, or desipramine and was almost completely inhibited by antibodies against P450 BTL, which belongs to the CYP2D family. The higg-Km demethylation activity was induced by phenobarbital and pregnenolone-16 alpha-carbonitrile and the activity in both untreated and phenobarbital-induced microsomes was suppressed by anti-CYP2B1 IgG. Experiments with IgG raised against cytochrome b5 suggested that the hemoprotein contributed to the low-Km activity but not the high-Km activity. These results indicate that cytochrome P450 isozymes belonging to the CYP2D subfamily catalyze demethylenation with low Km values and that the reaction occurring with high Km values is likely to be mediated by members of the CYP2B family, but with the possible participation of other phenobarbital-inducible isoforms.     

Glucose-6-P control of glycogen synthase phosphorylation in yeast

The SNF1 gene encodes a protein kinase necessary for expression of glucose-repressible genes and for the synthesis of the storage polysaccharide glycogen. From a genetic screen, we have found that mutation of the PFK2 gene, which encodes the beta-subunit of 6-phosphofructo-1-kinase, restores glycogen accumulation in snf1 cells. Loss of PFK2 causes elevated levels of metabolites such as glucose-6-P, hyperaccumulation of glycogen, and activation of glycogen synthase, whereas glucose-6-P is reduced in snf1 cells. Other mutations that increase glucose-6-P, deletion of PFK1, which codes for the alpha-subunit of 6-phosphofructo-1-kinase, or of PGI1, the phosphoglucoisomerase gene, had similar effects on glycogen metabolism as did pfk2 mutants. We propose that elevated glucose-6-P mediates the effects of these mutations on glycogen storage. Glycogen synthase kinase activity was reduced in extracts from pfk2 cells but was restored to that of wild type if the extract was gel-filtered to remove small molecules. Also, added glucose-6-P inhibited the glycogen synthase kinase activity in extracts from wild-type cells, half-maximally at approximately 2 mM. We suggest that glucose-6-P controls glycogen synthase activity by two separate mechanisms. First, glucose-6-P is a direct activator of glycogen synthase, and second, it controls the phosphorylation state of glycogen synthase by inhibiting a glycogen synthase kinase.