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At least 19 recordsLinked to original sources
PubMed1975

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Phenotypic suppression of sn-glycerol-3-phosphate acyltransferase Km mutants by loss of feedback inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Revertants of Escherichia coli mutants defective in the first enzyme of membrane phospholipid synthesis, sn-glycerol-3-phosphate (glycerol-P) acyltransferase, were investigated. These glycerol-P acyltransferase mutants, selected as glycerol-P auxotrophs, contained membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than the parental activity. The glycerol-P acyltransferase activity was also more thermolabile in vitro than the parental activity. Most revertants no longer requiring glycerol-P for growth regained glycerol-P acyltransferase activity of normal thermolability and apparent Km for glycerol-P. However, two novel revertants were isolated which retained an abnormal glycerol-P acyltransferase activity. The glycerol-P dehydrogenase activities of these novel revertants were about 20-fold less sensitive to feedback inhibition by glycerol-P. The feedback-resistant glycerol-P dehydrogenase co-transduced with gpsA, the structural gene for the glycerol-P dehydrogenase. Further transduction experiments demonstrated that the feedback resistant glycerol-P dehydrogenase phenotypically suppressed the glycerol-P acyltransferase Km lesion. The existence of the class of glycerol-P auxotrophs which owe their phenotype to the glycerol-P acyltransferase Km lesion therefore depends on the feedback regulation of glycerol-P synthesis in E. coli.

Acyltransferases↗
PubMed1978

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Kinetic characterization of wild type and feedback-resistant forms of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous wild type and feedback-resistant forms of the biosynthetic sn-glycerol 3-phosphate (glycerol-P) dehydrogenase of Escherichia coli (EC1.1.1.8) were subjected to two-substrate kinetic analysis. The kinetics of the NADPH-dependent reduction of dihydroxyacetone phosphate (dihydroxyacetone-P) and of the NADP-dependent oxidation of glycerol-P indicate that these reactions proceed by a sequential mechanism. Glycerol-P was a competitive inhibitor with respect to dihydroxyacetone-P for both enzymes. The wild type and feedback-resistant glycerol-P dehydrogenases had Ki values for glycerol-P of 4.4 micrometer and 43 micrometer, respectively. Therefore, the sensitivity of the wild type activity and reduced sensitivity of the feedback-resistant activity, both noted previously in crude extracts, were inherent properties of the enzymes. The patterns of product inhibition for both enzymes were identical, and the difference in the inhibition constants for glycerol-P occurred without significant alteration of any other kinetic constant determined. Kinetic mechanisms consistent with the patterns of product inhibition violated Haldane relationships and other kinetic relationships. These discrepancies suggest that glycerol-P inhibition occurs at a site distinct from the active site. The pH dependencies of the Km for dihydroxyacetone-P and the Ki for glycerol-P were markedly different suggesting the existence of an allosteric site. The addition of glycerol-P in the presence of NADPH stabilized both enzymes against thermal inactivation. Half-maximal stabilization was provided by 5 micrometer and 50 micrometer glycerol-P for the wild type and feedback-resistant enzymes, respectively. These kinetic data, considered in conjunction with previous physiologic and genetic data, indicate that the synthesis of glycerol-P is regulated in vivo by glycerol-P inhibition of the glycerol-P dehydrogenase. The data suggest that glycerol-P inhibition occurs at an allosteric, regulatory site.

Dihydroxyacetone Phosphate↗
PubMed1975

Alpha-Glycerol phosphatase and glycerol kinase activities in tissues of the silkmoth Hyalophora cecropia during the larval-pupal transformation.

Magnesium metabolism has been studied in Hyalophora cecropia during the larval-pupal transformation (LPT) (Jungreis, A. M., Am. J. Physiol. 224: 27--30, 1973). Throughout this period, it accumulated in midgut (0.5 M at ecdysis), presumably as osmotically inactive Mg3(PO4)2. Glycerol accumulation in hemolymph was also first noted during this period. Since total alpha-glycerol phosphate present in hemolymph declined between the larval and pupal stages of development, the relationship between magnesium and alpha-glycerol phosphate metabolism was studied. Specific and total alpha-glycerol phosphatase (degradative) and glycerol kinase (synthetic) enzyme activities were measured in fat-body and midgut tissue throughout the LPT. At both feeding larval and diapause pupal stages in development in both tissues, total glycerol kinase activity is greater than that of alpha-glycerol phosphatase with degradative/synthetic activity ratios of 0.2--0.5. In fat body, ratios remained constant or shifted in the direction of synthesis during the LPT, whereas those measured in midgut tissue increased in the direction of degradation with a maximum ratio of 5.8 noted following spinning. The increase in degradative/synthetic activity ratios in midgut tissue is attributed to a greater rate of loss of glycerol kinase than alpha-glycerol phosphatase enzyme activity. Orthophosphate, presumably released from alpha-glycerol phosphate within the cells of the pharate pupal midgut tissue, combines with magnesium to form osmotically inactive Mg3(PO4)2 crystals.

Animals↗
PubMed1975

Proportional activities of glycerol kinase and glycerol 3-phosphate dehydrogenase in rat hepatomas.

The activities of glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), glycerol kinase (EC 2.7.1.30), lactate dehydrogenase (EC 1.1.1.27), "malic' enzyme (L-malate-NADP+ oxidoreductase; EC 1.1.1.40) and the beta-oxoacyl-(acyl-carrier protein) reductase component of the fatty acid synthetase complex were measured in nine hepatoma lines (8 in rats, 1 in mouse) and in the livers of host animals. With the single exception of Morris hepatoma 16, which had unusually high glycerol 3-phosphate dehydrogenase activity, the activities of glycerol 3-phosphate dehydrogenase and glycerol kinase were highly correlated in normal livers and hepatomas (r = 0.97; P less than 0.01). The activities of these two enzymes were not strongly correlated with the activities of any of the other three enzymes. The primary function of hepatic glycerol 3-phosphate dehydrogenase appears to be in gluconeogenesis from glycerol.

Animals↗
PubMed1979

Conversion of (U-14C)-glycerol, (2-3H)-glycerol and (1-14C)-palmitate into circulating lipoproteins in the rat.

The in vivo formation of labelled very low density lipoproteins (VLDL) from (U-14C)-glycerol, (2-3H)-glycerol and (1-14C)-palmitate was studied in fed female rats. The rate of disappearance of radioactivity from plasma after the i.v. injection with these tracers was similar for (U-14C)-glycerol and (1-14C)-palmitate. With (2-3H)-glycerol, plasma radioactivity at 10 min was lower than with the other substrates although it did not change thereafter. A certain proportion of radioactivity administered as glycerol appeared in plasma lipids, mainly in the VLDL glyceride glycerol fraction, although when (U-14C)-glycerol was the substrate, a considerable portion also appeared in the esterified fatty acids of these lipoproteins. When using (1-14C)-palmitate, practically all the circulating labelled esterified fatty acids appeared in the VLDL fraction, while the labelled free fatty acids appeared in lipoprotein of higher density, presumable free fatty acid-albumin complexes. This data is discussed in terms of the role of the liver in the rapid, continuous cycling of these substrates to yield VLDL-glycerides for their extrahepatic utilization.

Animals↗
PubMed1975

[Effects of parenteral infusion of glycerol on glycerol kinase and adenosintriphosphate in rat kidneys (author's transl)].

Effects of intravenous glycerol infusions on glycerol kinase and adenosintriphosphate in rat kidneys. Intravenous infusions of glycerol with rates near and above the average maximal turnover capacity (0,74 g.kg-1.h-1) cause alterations of the kidneys such as weight increase, decrease of protein content and activity of glycerol kinase with excretion of this enzyme in the urine. At infusion rates of 0,6 g.kg-1.h-1 and more a decrease of the ATP-content of the kidneys is observed. Effects on the liver are not consistent and only demonstrable at infusion rates above the maximal turnover rate of glycerol. The results are discussed with regard to osmotic nephrosis. The appearance of glycerol kinase in the urine is discussed as an early symptom of kidney damage.

Adenine Nucleotides↗
PubMed1979

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.

Acyl Coenzyme A↗
PubMed1977

Regulation of selectivity of CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn-glycerols.

The suitability of monoenoic, dienoic, tetraenoic, and hexaenoic molecular species of 1,2-diacyl-sn-glycerols as substrates for the CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied in rat liver microsomes. No statistically significant difference in the rates of phosphatidylcholine synthesis with the various diacylglycerols was found at 0.40 mM, although a moderate discrimination against hexaenoic species relative to monoenoic and dienoic species was observed at 0.25 mM. The addition of palmitoyl-CoA (7.5 micron) significantly enhanced cholinephosphotransferase activity when tetraenoic diacylglycerols were added at 0.25 or 0.40 mM. CDPethanolamine at 24.4 micron was found to inhibit the rates of phophatidylcholine biosynthesis by 54 and 39% with hexaenoic and monoenoic 1,2-diacyl-sn-glycerols, respectively, whereas no significant effects were observed in the case of dienoic and tetraenoic species. These latter findings may partially explain why 1-saturated 2-docosahexaenoyl diacylglycerols are used to a greater extent for phosphatidylethanolamine than for phosphatidylcholine synthesis in rat liver in vivo. The present results also suggest that the selectivity of the cholinephosphotransferase for certain molecular species of 1,2-diacyl-sn-glycerols is a function of diacylglycerol concentration and may be mediated under physiological conditions by substrates for enzymes which compete for common diacylglycerol precursors.

Animals↗
PubMed1978

Comparative study of the clinical effects of vincamine + glycerol versus glycerol + placebo in the acute phase of stroke.

The treatment of the acute phase of stroke creates a difficult problem to the clinician. The presently used drugs lead to controversial results. The progress in knowledge of the pathogenesis of cerebral damage underlines the determinant role of the metabolic deficits in the ischemic areas. The effect on the clinical symptomatology of patients in the acute phase of stroke was studied during a double-blind comparative clinical trial with an alcaloid of Vinca minor (Pervincamine) which acts favorably against disturbances of oxydative glucose metabolism and of cerebral microcirculation. 20 patients divided into two homogeneous groups received during 5 days either Pervincamine (4 ampoules of 3 ml = 60 mg vincamine p.d.) + glycerol, or glycerol + placebo administered by i.v. infusion. Clinically the results indicate a greater improvement of the neurological status (objectivated by a neurological scale) with vincamine treatment than with placebo. Statistically the analysis confirms the highly significant effect of vincamine on motility of lower and upper limbs (p less than 0.02) and the significant effect on cranial nerves (homonymous hemianopsia and conjugated deviation of eyes) (p less than 0.05) and on the sphincter control level (p less than 0.05).

Adult↗
PubMed1975

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Properties of wild type and Km defective sn-glycerol-3-phosphate acyltransferase activities.

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase, the first enzyme of membrane phospholipid synthesis in Escherichia coli, was investigated in a wild type and a mutant strain defective in this activity. The mutant strain, selected as a glycerol-P auxotroph, was previously shown to contain a glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of its parent or revertants. The membranous mutant glycerol-P acyltransferase but did not appear to be thermolabile in vivo. Revertants no longer requiring glycerol-P for growth, showed glycerol-P acyltransferase activity with thermolability properties similar to the wild type. The second phospholipid biosynthetic enzyme, 1-acylglycerol-P acyltransferase, was not thermolabile in membranes containing a thermolabile glycerol-P acyltransferase activity. The pH optimum for the mutant acyltransferase was over 1 pH unit higher than that of the parental activity. Further, the mutant and wild type glycerol-P acyltransferase differed in their response to magnesium chloride and potassium chloride. The palmitoyl-CoA dependence of the wild type and mutant glycerol-P acyltransferase activities were different. The mutant glycerol-P acyltransferase activity was inhibited greater than 90% by Triton X-100 under conditions where the wild type activity was not affected. These experiments provide novel information about the wild type glycerol-P acyltransferase activity of E. coli and provide six additional lines of evidence for the mutant character of the glycerol-P acyltransferase in the mutant strains.

Acyltransferases↗
PubMed1975

Kinetics of glycerol uptake by the perfused rat liver. Membrane transport, phosphorylation and effect on NAD redox level.

The kinetics of glycerol uptake by the perfused rat liver were determined according to a model which includes membrane transport, intracellular phosphorylation and competitive inhibition of glycerol phosphorylation by L-glycerol 3-phosphate. The membrane transport obeys first-order kinetics at concentrations below 10 mM in the affluent medium. The K-m of the glycerol phosphorylation was 10 muM and the K-i of the L-glycerol 3-phosphate inhibition was 50 muM. The maximum activity (V) was 3.70 mumoles/min per g liver wet wt. These results are similar to in vitro kinetics of the glycerol kinase, except that K-i was found to be somewhat lower in the intact organ. At low glycerol concentrations, a steep concentration gradient exists across the liver cell membrane. The increase in the lactate to pyruvate concentration ratio during glycerol metabolism is related to the actual concentration of L-glycerol 3-phosphate, not to the rate of glycerol uptake.

Animals↗
PubMed1978

Stopped-flow spectrophotometric assay of glycerol permeation in Escherichia coli: applicability and limitations.

The passive permeation and facilitated diffusion of glycerol in various strains of Escherichia coli have been studied by stopped-flow spectrophotometry. Contrary to the prediction for glycerol entry by simple diffusion, the reciprocal relaxation time (1/tau, s-1) for the passive permeation of glycerol in cells grown in the presence of glucose was not constant but decreased as the glycerol concentration increased above 100 mM. This anomaly was not due to refractive index differences or to the presence of residual levels of the glycerol facilitator protein in non-induced cells. Although reciprocal relaxation times for glycerol-induced E. coli exhibited the expected elevation relative to non-induced cells, a similar anomalous decrease 1/tar (s-1) with increasing glycerol concentration was observed. In addition, at early times after suspension in dilute buffer, the 1/tau (s-1) values obtained for induced or non-induced E. coli swelling in glycerol were considerably greater than for organisms incubated in dilute buffer for longer times. We concluded that either this spectrophotometric technique was not monitoring solely the permeation of glycerol into E. coli, or concentrations of glycerol above 100 mM significantly perturbed the structure of the E. coli cell envelope.

Cell Count↗
PubMed1978

Relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives by Pseudomonas aeruginosa.

The relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives in Pseudomonas aeruginosa was studied by comparing the growth on glycerol and enzymatic constitution of strain PAO with these characteristics of glucose-catabolic mutants and revertants. Growth of strain PAO on glycerol induced a catabolic oxidized nicotinamide adenine dinucleotide-linked glyceraldehyde-phosphate dehydrogenase and seven glucose-catabolic enzymes. The results indicated that these enzymes were induced by a six-carbon metabolite of glucose. All strains possessed a constitutive anabolic Embden-Meyerhof-Parnas pathway allowing limited conversion of glycerol-derived triosephosphate to hexosephosphate derivatives, which was consistent with induction of these enzymes by glycerol. Phosphogluconate dehydratase-deficient mutants grew on glycerol. However, mutants lacking both phosphogluconate dehydrogenase and phosphogluconate dehydratase were unable to grow on glycerol, although these strains possessed all of the enzymes needed for degradation of glycerol. These mutants apparently were inhibited by hexosephosphate derivatives, which originated from glycerol-derived triosephosphate and could not be dissimilated. This conclusion was supported by the fact that revertants regaining only a limited capacity to degrade 6-phosphogluconate were glycerol positive but remained glucose negative.

Gluconates↗
PubMed1976

Triacylglycerol synthesis in isolated fat cells. Evidence that the sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities are dual catalytic functions of a single microsomal enzyme.

The acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (glycerol-P acyltransferase) and acyl-CoA:dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) (DHAP acyltransferase) activities were investigated in vitro in order to evaluate the quantitative contribution of the glycerol-P and DHAP pathways for the synthesis of triacylglycerols in isolated fat cells and to test the hypothesis that these two activities may be dual catalytic functions of a single enzyme. More than 85% of both acyltransferase activities was associated with the microsomal subcellular fraction. The microsomal glycerol-P acyltransferase activity showed an apparent Km of 8 muM for glycerol-P with a Vmax of 15.6 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent Km of 40 muM for DHAP with a Vmax of 9.7 nmol/min/mg. Glycerol-P was a competitive inhibitor (Ki = 7.2 muM) of the DHAP acyltransferase, and DHAP was a competitive inhibitor (Ki = 92 muM) of the glycerol-P acyltransferase. The two acyltransferase activities showed virtual identity in their pH dependence, acyl-CoA chain length dependence, thermolability, and inactivation by N-ethylmaleimide. Trypsin, detergents, collagenase, phospholipases, and various salts and organic solvents also had similar effects on both activities. Taken as a whole, the data strongly suggest that the microsomal glycerol-P and DHAP acyltransferase activities actually represent dual functions of a single enzyme. Calculations based on the above kinetic constants and previously reported glycerol-P and DHAP pools in adipocytes suggest that the in vivo ratio of glycerol-P to DHAP acylation should be greater than 24:1.

Acyltransferases↗