Variations in the Localization of Acetyl-Coenzyme A Synthetase in Aerobic Yeast Cells

In cells of Saccharomyces cerevisiae growing aerobically for 24 hr, acetyl-coenzyme A synthetase [acetate: CoA ligase (AMP), EC 6.2.1.1] was localized principally in the microsomal fraction. On density gradients, the enzyme in such cells behaved as a low-density particle, readily separable from the soluble proteins. After 48 hr of incubation, the cells showed a bimodal distribution of enzyme, with most of the activity now sedimenting with the mitochondrial fraction and only a smaller amount with the microsomal fraction. By using density gradients, two forms of synthetase were obtained from these cells: one band denser and the other band less dense than the intact mitochondria. In all preparations containing synthetase activity, appreciable levels of phospholipids were also detected.     

ACR1, a gene encoding a protein related to mitochondrial carriers,is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae

The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds.     

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (−10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).     

ACR1, a gene encoding a protein related to mitochondrial carriers, is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae

The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds.     

Origin of acetate in spinach leaf cell

Mitochondria were isolated from spinach (Spinacia oleracea L.) leaves using a Percoll gradient step. The high purity of the organelle fraction is demonstrated by electron microscopy and biochemical parameters. In the matrix space of these mitochondria, a short-chain acyl-coenzyme A hydrolase is present that converts acetyl-coenzyme A to acetate and coenzyme A with reasonable rates (K_m, 150 micromolar; V_max, 140 nanomoles acetate formed milligram¹ protein hour⁻¹). The enzyme is product inhibited by coenzyme A-sulfhydryl, other thiols are ineffective; however, the disulfides 5,5′-dithio-bis-(2-nitrobenzoate) and cystamine stimulate the hydrolysis. The possible role of this mitochondrial enzyme as a means of generating free acetate from pyruvate via acetyl-coenzyme A in the mitochondria of mature spinach leaves is discussed. It is suggested that free acetate moves rapidly from the mitochondrion to the chloroplast where acetyl-coenzyme A synthetase, solely localized in this organelle, converts the metabolically inert free acetate to the highly active acetyl-coenzyme A.     

Biosynthesis of Glycogen and Starch in Cryptococcus laurentii

Cells of Cryptococcus laurentii, when grown in liquid culture on 2% glucose close to neutral pH, showed glycogen granules throughout the cytoplasm. Glycogen levels of C. laurentii cells reached maximal levels just before onset of stationary phase. Concomitantly, a sharp rise in total and specific activity of glycogen synthetase was observed. Conversely, glycogen phosphorylase reached its highest specific activity approximately 3 hr after the glycogen peaked and remained high until most of the endogenous glycogen was utilized. Uridine diphosphoglucose pyrophosphorylase activity was always an order of magnitude higher than glycogen synthetase during log phase, but fell off rapidly after the cells reached stationary growth. Kinetic properties of the glycogen synthetase showed that the enzyme is always activated by glucose-6-phosphate, although the degree of activation by glucose-6-phosphate was found to be somewhat variable. The accelerated uptake of glucose commencing with the onset of stationary phase is explained by the rapid formation of extracellular acidic polysaccharide, which continues as long as there is glucose in the medium. In cells grown at pH 3.4, where no detectable extracellular acidic polysaccharide was formed, glucose uptake drastically declined when the cells reached stationary phase. These cells also contained glycogen-like granules in the cytoplasm. The evidence presented indicates that these granules are in fact glycogen, and that its structure does not resemble that of the starch excreted by cells grown at acidic pH.     

Mechanisms of acetate formation and acetate activation in halophilic archaea

The halophilic archaea Halococcus (Hc.) saccharolyticus, Haloferax (Hf.) volcanii, and Halorubrum (Hr.) saccharovorum were found to generate acetate during growth on glucose and to utilize acetate as a growth substrate. The mechanisms of acetate formation from acetyl-CoA and of acetate activation to acetyl-CoA were studied. Hc. saccharolyticus, exponentially growing on complex medium with glucose, formed acetate and contained ADP-forming acetyl-CoA synthetase (ADP-ACS) rather than acetate kinase and phosphate acetyltransferase or AMP-forming acetyl-CoA synthetase. In the stationary phase, the excreted acetate was completely consumed, and cells contained AMP-forming acetyl-CoA synthetase (AMP-ACS) and a significantly reduced ADP-ACS activity. Hc. saccharolyticus, grown on acetate as carbon and energy source, contained only AMP-ACS rather than ADP-ACS or acetate kinase. Cell suspensions of Hc. saccharolyticus metabolized acetate only when they contained AMP-ACS activity, i.e., when they were obtained after growth on acetate or from the stationary phase after growth on glucose. Suspensions of exponential glucose-grown cells, containing only ADP-ACS but not AMP-ACS, did not consume acetate. Similar results were obtained for the phylogenetic distantly related halophilic archaea Hf. volcanii and Hf. saccharovorum. We conclude that, in halophilic archaea, the formation of acetate from acetyl-CoA is catalyzed by ADP-ACS, whereas the activation of acetate to acetyl-CoA is mediated by an inducible AMP-ACS.Abbreviations. Hc. Halococcus - Hf. Haloferax - Hr. Halorubrum - Hb. Halobacterium An erratum to this article can be found at     

THE ISOLATION OF LYSOSOMES FROM EHRLICH ASCITES TUMOR CELLS FOLLOWING PRETREATMENT OF MICE WITH TRITON WR-1339

A method is described for obtaining highly purified lysosomes from Ehrlich ascites tumo cells grown in mice injected with Triton WR-1339. The isolated particles show a high specific activity for aryl sulfatase, representing an 80–90-fold purification over the homogenate, and a 15–18% yield of the total enzyme activity. Mitochondrial and microsomal marker enzymes are present in negligible amounts (0.2% of the activity of the homogenate). The biochemical evidence for a rather high degree of homogeneity of the fraction is supported by the electron microscopic examination of the purified lysosomes. The intracellular localizations of N-acetyl-β-glucosaminidase, NADH-cytochrome c reductase and NADPH-cytochrome c reductase in Ehrlich ascites cells are also reported, the first two being present in highest concentration in the combined mitochondrial-lysosomal fraction and the third in the microsomal fraction.     

Subcellular distribution of aminoacyl-transfer RNA synthetases in Chinese hamster ovary cell culture

Chinese hamster ovary cells grown in cell culture were broken and fractionated by differential centrifugation. Four principal fractions: nuclear and membrane, microsomal, postribosomal, and supernatant were obtained. The distribution of aminoacyl-tRNA synthetases in these four fractions was determined for all twenty amino acids.It was shown that there is a differential distribution of synthetases. Activities specific for eight amino acids: Ala, Ser, Gly, Cys, His, Arg, Thr and Pro were found mainly in the supernatant fraction. Activities specific for eleven amino acids: Asp, Asn, Glu, Gln, Ile, Leu, Lys, Met, Phe, Tyr and Val were found mainly in the postribosomal fraction. Four activities were found at significant levels in the microsomal fraction: Asp, Phe, Lys and Pro. The nuclear and membrane fraction contained activity for Lys, His, Asp and Thr.Changes in aminoacyl-tRNA synthetase activities in various fractions from preparations made by breaking cells with a membrane-dissociating detergent showed that some of the aminoacyl-tRNA synthetase activities may be membrane bound.     

Characterization and distribution of a phospholipase A2 activity from adult rabbit lung

A phospholipase A2 activity was characterized in adult rabbit lung. This activity was calcium- and deoxycholate-dependent and displayed an alkaline pH optimum. Km and Vmax were 0.176 mM and 256.8 pmoles/min./mg protein respectively. The microsomal fraction displayed the highest enzymatic specific activity; the lowest activity was present in the cytosol. Yet this latter fraction accounted for the majority of the total activity. Although the specific activity was high within the lamellar body fraction this compartment contained only approximately 2% of the total activity. Phospholipase A2 activity was inhibited by bromophenacyl bromide, chlorpromazine and mepacrine in decreasing order of effectiveness. Treatment of the microsomes with increasing concentrations of NaC1 indicated that the lung phospholipase A2 activity was relatively loosely bound to the microsomal membranes and was maximally removed with salt at a concentration only slightly higher than physiological. Addition of calmodulin to the enzyme assay did not significantly alter hydrolysis of labelled phosphatidylcholine.     

In Salmonella enterica, the sirtuin-dependent protein acylation/deacylation system (SDPADS) maintains energy homeostasis during growth on low concentrations of acetate

Acetyl-coenzyme A synthetase (Acs) activates acetate into acetyl-coenzyme A (Ac-CoA) in most cells. In Salmonella enterica, acs expression and Acs activity are controlled. It is unclear why the sirtuin-dependent protein acylation/deacylation system (SDPADS) controls the activity of Acs. Here we show that, during growth on 10 mM acetate, acs(+) induction in a S. enterica strain that cannot acetylate (i.e. inactivate) Acs leads to growth arrest, a condition that correlates with a drop in energy charge (0.17) in the acetylation-deficient strain, relative to the energy charge in the acetylation-proficient strain (0.71). Growth arrest was caused by elevated Acs activity, a conclusion supported by the isolation of a single-amino-acid variant (Acs(G266S)), whose overproduction did not arrest growth. Acs-dependent depletion of ATP, coupled with the rise in AMP levels, prevented the synthesis of ADP needed to replenish the pool of ATP. Consistent with this idea, overproduction of ADP-forming Ac-CoA-synthesizing systems did not affect the growth behaviour of acetylation-deficient or acetylation-proficient strains. The Acs(G266S) variant was >2 orders of magnitude less efficient than the Acs(WT) enzyme, but still supported growth on 10 mM acetate. This work provides the first evidence that SDPADS function helps cells maintain energy homeostasis during growth on acetate.     

The reversal of experimental porphyria and the prevention of induction of hepatic mixed-function oxidase by acetate

The administration of acetate or sulfanilamide depressed the porphyric response of rats to 3,5-dicarbethoxy-1,4-dihydrocollidine. The induction of δ-aminolevulinate synthetase (EC 2.3.1.37) in porphyric rats was decreased by acetate administration and δ-aminolevulinate synthetase activity in hepatic homogenates was inhibited by acetate. Succinate reversed the inhibition by acetate in vitro. Since an alteration of heme biosynthesis by acetate was observed, the effect of acetate on the induction of hepatic microsomal cytochrome P-450 and microsomal mixed-function oxidase by phenobarbital was examined. Acetate prevented the induction of hepatic mixed-function oxidase and cytochrome P-450 by phenobarbital. Unlike the action of other inhibitors of hepatic heme biosynthesis, acetate also prevented the induction by phenobarbital of NADPH-cytochrome c reductase (EC 1.6.99.3). These findings suggest that acetate may be inhibiting heme biosynthesis by effects on δ-aminolevulinate synthetase, the rate-limiting step in heme biosynthesis, by alteration of the induction of this enzyme and by a direct effect on the enzymic reaction itself. It is suggested that acetate may be involved in the glucose effect related to the inhibition of the induction of δ-aminolevulinate synthetase.     

Biosynthesis of paf-acether. IX. Role for a phosphorylation-dependent activation of acetyltransferase in antigen-stimulated mouse mast cells

Mouse bone marrow-derived mast cells passively sensitized with monoclonal IgE released paf-acether (platelet-activating factor) and beta-hexosaminidase when challenged with the specific antigen. The formation and the release of paf-acether followed an early increase in the activity of the acetyltransferase, the main enzyme in paf-acether biosynthesis. The antigen-induced activation of the acetyltransferase was dependent on physiologic temperature and on the presence of Ca2+. By using microsomal fractions from unchallenged and challenged mast cells, the Vmax values were 3.5 and 12.0 nmol/min/mg of protein, respectively, whereas in both cases a Km value for acetyl-coenzyme A of 172 microM was measured. The stimulation of acetyltransferase could be mimicked in vitro under experimental conditions which favor phosphorylation, i.e. adding ATP and Mg2+ to lysates from unchallenged mast cells. In contrast, ATP and Mg2+ were uneffective on lysates from challenged cells that exhibited high level of acetyltransferase activity, suggesting that phosphorylation of the enzyme already took place at the time of cell stimulation. Moreover, addition of alkaline phosphatase to microsomal fraction obtained from either antigen-challenged mouse bone marrow-derived mast cells or unchallenged cells, resulted in 52% and 43% loss of acetyltransferase activity, respectively. Phorbol myristate acetate treatment of cells doubled the enzyme activity supporting the phosphorylation hypothesis. Thus, we report on the immunologic activation of a key enzyme for paf-acether synthesis and on the mechanism of this activation in a pure mast cell population. A link between bridging of IgE receptors and the activation of an enzyme critical to the formation of a lipid mediator is thereby evidenced.     

Reversible inactivation by noradrenaline of long-chain fatty acyl-CoA synthetase in rat adipocytes.

Incubation of rat adipocytes with the same range of noradrenaline concentrations that stimulate lipolysis caused a rapid and stable decrease in the activity of fatty acyl-CoA synthetase. Corticotropin, glucagon and dibutyryl cyclic AMP also decreased the activity of the enzyme. The effect of noradrenaline was apparent over a wide range of concentrations for the three substrates of the enzyme. A novel fluorescence assay of fatty acyl-CoA synthetase using (1,N6-etheno)-CoA is described. The effect of noradrenaline was not abolished by inclusion of albumin in homogenization buffers, persisted through subcellular fractionation and isolation of microsomes (microsomal fractions) and even survived treatment of microsomes with Triton X-100. The effect of noradrenaline was rapidly reversed within cells by the subsequent addition of insulin or propranolol. The inclusion of fluoride in homogenization buffers did not alter the observed effect of noradrenaline. Additions of cyclic AMP-dependent protein kinase to adipocyte microsomes caused considerable phosphorylation of microsomal protein by [gamma-32P]ATP, but did not affect the activity of fatty acyl-CoA synthetase.     

Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae.

A method is shown to be effective over a wide range of enzyme ratios for the simultaneous detection of the two isoenzymes of acetyl coenzyme A synthetase [acetate:coenzyme A ligase (AMP-forming); EC 6.2.1.1] in homogenates and cellular fractions of Saccharomyces cerevisiae. When this method was used, it was found that cells grown under anaerobic conditions contained only one variety of this enzyme, designated the nonaerobic synthetase, whereas cells grown with vigorous aeration contained principally the other, aerobic, synthetase. In cells grown as standing cultures (i.e., semi-aerobically), both enzymes were present and were found mainly in the extramitochondrial material of homogenates. When anaerobic cultures were aerated, the amount of aerobic enzyme increased steadily over a 24-h period, so that at the end of this time, aerated cells contained predominantly aerobic enzyme. During this same period, the amount of nonaerobic enzyme decreased. The percentage of aerobic enzyme that sedimented with the mitochondria increased steadily during this period of aeration, so that, at the end of 24 h of aeration, essentially all of the aerobic enzyme sedimented with the mitochondria. The nonaerobic enzyme was never found in this cellular compartment.     

Roles of acetate and pyruvate in the metabolism of Streptococcus diacetilactis

Streptococcus diacetilactis required acetate, contained acetate kinase and phosphotransacetylase, and incorporated both radioactive exogenous acetate and acetate from citrate into cell lipids. dl-alpha-Lipoic acid replaced acetate and was required for the oxidation of pyruvate. Stimulation of S. diacetilactis by citrate was found to depend on pyruvate oxidation. Resting cells of the organism produced acetate from 73% of the pyruvate they utilized. However, molar growth yields from glucose were not greater under aerobic compared to anaerobic conditions or when lipoic acid or citrate plus lipoic acid was used in the medium in place of acetate. Data indicate that the growth of S. diacetilactis is limited by the rate of acetyl-coenzyme A synthesis, that the rate of synthesis from pyruvate is higher than the rate from acetate, and that lack of acetyl-coenzyme A not required for growth limits the production of diacetyl and precludes the formation of adenosine triphosphate from acetyl-coenzyme A.     

An association between glutamine synthetase activity and adipocyte differentiation in cultured 3T3-L1 cells

Confluent 3T3-L1 Swiss mouse fibroblasts acquired morphological and biochemical characteristics of adipocytes when maintained in medium containing 10% calf serum and added insulin. Identical cultures maintained in the absence of added insulin did not differentiate into adipocytes. Incubation of confluent cultures for 48 h with 0.25 μm dexamethasone and 0.5 mm 1-methyl-3-isobutylxanthine yielded subsequent adipocyte differentiation when the culture medium contained 10% fetal calf serum. In contrast, differentiation did not occur when similarly treated cultures were maintained in medium containing 10% calf serum. The increase in glutamine synthetase which occurred during adipocyte differentiation was closely associated with an increased rate of triglyceride synthesis from acetate, with increased protein, and with increases in the activities of glycerol-3-P dehydrogenase and glucose-6-P dehydrogenase. Glutamine synthetase activity remained undetectable in insulin-treated confluent 3T3-C2 cells maintained under conditions which yielded high glutamine synthetase activity in 3T3-L1 cells. (3T3-C2 cells did not differentiate into adipocytes.) Glutamine accumulated in the culture medium of 3T3-L1 adipocytes, but it did not accumulate in the medium from identically treated 3T3-C2 cells. A half-maximal increase in glutamine synthetase specific activity occurred at a culture medium insulin concentration of 10 ng/ml. Neither adipocyte differentiation nor the rise in glutamine synthetase activity were substantially altered by maintaining confluent cultures in medium lacking added glutamine. Incubation of confluent 3T3-L1 cultures with 3 mml-methionine sulfone, a reversible inhibitor of glutamine synthetase, increased by two-fold both the activity and the cellular content of glutamine synthetase. Incubation of confluent 3T3-L1 cultures with 4 mml-glutamine and l-methionine-dl-sulfoximine, an irreversible inhibitor of glutamine synthetase activity, decreased glutamine synthetase activity to less than 5% of the activity in control cultures; however, neither cellular content of the enzyme nor synthesis rate of the enzyme were substantially altered. In the presence of added glutamine, neither methionine sulfone nor methionine sulfoximine had a significant effect on phenotypic adipocyte conversion. By contrast, when confluent cultures were incubated with methionine sulfoximine and no added glutamine, glutamine synthetase remained absent and there was no evidence of adipocyte conversion. Our data indicate (1) that added insulin is required for adipocyte differentiation of 3T3-L1 cells maintained in medium containing calf serum, (2) that glutamine synthetase activity increases during adipocyte conversion regardless of the culture conditions employed to achieve differentiation, and (3) that glutamine synthetase activity may be required for adipocyte differentiation when cultures are maintained in medium lacking added glutamine.     

Changes in Viability, Cell Composition, and Enzyme Levels During Starvation of Continuously Cultured (Ammonia-Limited) Selenomonas ruminantium

Under nitrogen (ammonia)-limited continuous culture conditions, the ruminal anaerobe Selenomonas ruminantium was grown at various dilution rates (D). The proportion of the population that was viable increased with D, being 91% at D = 0.5 h⁻¹. Washed cell suspensions were subjected to long-term nutrient starvation at 39°C. All populations exhibited logarithmic linear declines in viability that were related to the growth rate. Cells grown at D = 0.05, 0.20, and 0.50 lost about 50% viability after 8.1, 4.6, and 3.6 h, respectively. The linear rates of decline in total cell numbers were dramatically less and constant regardless of dilution rate. All major cell constituents declined during starvation, with the rates of decline being greatest with RNA, followed by DNA, carbohydrate, cell dry weight, and protein. The rates of RNA loss increased with cells grown at higher D values, whereas the opposite was observed for rates of carbohydrate losses. The majority of the degraded RNA was not catabolized but was excreted into the suspending buffer. At all D values, S. ruminantium produced mainly lactate and lesser amounts of acetate, propionate, and succinate during growth. With starvation, only small amounts of acetate were produced. Addition of glucose, vitamins, or both to the suspending buffer or starvation in the spent culture medium resulted in greater losses of viability than in buffer alone. Examination of extracts made from starving cells indicated that fructose diphosphate aldolase and lactate dehydrogenase activities remained relatively constant. Both urease and glutamate dehydrogenase activities declined gradually during starvation, whereas glutamine synthetase activity increased slightly. The data indicate that nitrogen (ammonia)-limited S. ruminantium cells have limited survival capacity, but this capacity is greater than that found previously with energy (glucose)-limited cells. Apparently no one cellular constituent serves as a catabolic substrate for endogenous metabolism. Relative to losses in viability, cellular enzymes are stable, indicating that nonviable cells maintain potential metabolic activity and that generalized, nonspecific enzyme degradation is not a major factor contributing to viability loss.     

Purification of Phosphatidylinositol Synthetase from Rat Brain by CDP-Diacylglycerol Affinity Chromatography and Properties of the Purified Enzyme

A purification procedure for rat brain phosphatidylinositol synthetase (PI synthetase; CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase; EC 2.7.8.11) is described. The enzyme was purified 200-250-fold from the homogenate by solubilization with Triton X-100 from microsomal membranes and affinity chromatography on CDP-diacylglycerol-Sepharose. Elution of enzyme activity required the presence of Triton X-100, CDP-diacylglycerol, and either phosphatidylcholine or asolectin. The product that was obtained in 5-10% yield from whole brain and in 70% yield from the microsomal fraction contained three protein bands as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final preparation contained levels of CDP-diacylglycerol hydrolase and CDP-diacylglycerol: sn-glycero-3-phosphate 3-phosphatidyltransferase activities that were less than 1% of PI synthetase activity. The purified enzyme displayed a pH optimum of 8.5-9.0, required either Mg2+ or Mn2+ and exhibited a Km of 4.6 mM for myo-inositol.     

UDP-glucose: ceramide glucosyltransferase of rat brain: an association with smooth microsomes and requirement of an intact membrane for enzyme activity

Subcellular distribution of rat brain UDP-glucose:ceramide glucosyltransferase, the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The activity of the enzyme was highest in the fraction rich in microsomes. Subfractionation of crude microsomal fractions resulted in a further enrichment of the enzyme activity in the fraction which contained smooth microsomes, thus suggesting that the enzyme is associated with microsomal membranes. The enzyme does not appear to be associated with synaptosomes or myelin. Treatment of the microsomal fraction with phospholipase A and C or detergents resulted in the loss of enzyme activity. Preincubation of the microsomal fraction at 37 °C also resulted in a loss of enzyme activity. These results suggest the requirement of specific membrane structure for the activity of the enzyme UDP-glucose:ceramide glucosyltransferase of rat brain. The amount of the enzyme activity lost during preincubation was dependent on the composition of the incubation medium and the age of the rats from which microsomal fractions were obtained.