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Biomedical subjects

M Benziman

Publications and source records attributed to M Benziman.

At least 37 records · Page 2Linked to original sources
PubMed1982

Achievement of high rates of in vitro synthesis of 1,4-beta-D-glucan: activation by cooperative interaction of the Acetobacter xylinum enzyme system with GTP, polyethylene glycol, and a protein factor.

Regulatory properties of a cellulose synthase (UDP-forming)(UDPglucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase, EC 2.4.1.12) have been demonstrated by using enzyme preparations derived from cells of Acetobacter xylinum. Preparation of a particulate fraction in the presence of 20% (wt/vol) polyethylene glycol-4000 (PEG-4000) yields enzyme with activity 3- to 10-fold higher than that previously reported. The enzyme prepared in this fashion also shows a further marked, specific activation by GTP. The Ka for GTP is 34 microM. Guanosine 5'-[gamma-thio]triphosphate, an analog of GTP, is even more effective than GTP (Ka for guanosine 5'-[gamma-thio]triphosphate = 17 microM). A large number of other nucleotides and nucleotide derivatives were tested with no effect. Enzyme prepared in the absence of PEG-4000 does not respond to GTP because it lacks a protein factor necessary for GTP activation. PEG-4000 promotes the interaction of the protein factor with the enzyme. The factor itself has no synthase activity nor does it stimulate activity of the enzyme in the absence of GTP. In the presence of GTP, protein factor, and PEG-4000, initial rates of enzyme activity 200 times greater than those previously reported can be achieved. Such rates exceed 40% of the in vivo rate of cellulose synthesis from glucose.

Bacterial Proteins↗
PubMed1980

Calcofluor white ST Alters the in vivo assembly of cellulose microfibrils.

The fluorescent brightener, Calcofluor White ST, prevents the in vivo assembly of crystalline cellulose microfibrils and ribbons by Acetobacter xylinum. In the presence of more than 0.01 percent Calcofluor, Acetobacter continues to synthesize high-molecular-weight beta-1,4 glucans. X-ray crystallography shows that the altered product exhibits no detectable crystallinity in the wet state, but upon drying it changes into crystalline cellulose I. Calcofluor alters cellulose crystallization by hydrogen bonding with glucan chains. Synthesis of this altered product is reversible and can be monitored with fluorescence and electron microscopy. Use of Calcofluor has made it possible to separate the processes of polymerization and crystallization leading to the biogenesis of cellulose microfibrils, and has suggested that crystallization occurs by a cell-directed. self-assembly process in Acetobacter xylinum.

Acetobacter↗
PubMed1980

Cellulose biogenesis: Polymerization and crystallization are coupled processes in Acetobacter xylinum.

Calcofluor White ST, stilbene derivative used commerically as an optical brightener for cellulose, increased the rate of glucose polymerization into cellulose by resting cells of the gram-negative bacterium Acetobacter xylinum. This bacterium normally produces a ribbon of cellulose that is a composite of crystalline microfibrils. In concentrations above 0.1 mM, Calcofluor disrupts the assembly of crystalline cellulose I microfibrils and their integration into a composite ribbon by stoichiometric binding to glucose residues of newly polymerized glucan chains. Under these conditions, the rate of glucose polymerization increases up to 4 times the control rate, whereas oxygen uptake increases only 10-15%. These observed effects are readily reversible. If free Calcofluor is washed away or depleted below the threshold value by binding to cellulose as polymerization continues, ribbon production and the normal rate of polymerization resume. It is concluded that polymerization and crystallization are cell-directed, coupled processes and that the rate of crystallization determines the rate of polymerization. It is suggested that coupling must be maintained for biogenesis of crystalline cellulose I.

Journal Article↗
PubMed1978

Purification and regulatory properties of the oxaloacetate decarboxylase of Acetobacter xylinum.

The oxaloacetate (OAA) decarboxylase (EC 4.1.1.3) activity of Acetobacter xylinum cells grown on glucose or glycerol is the same as that of cells grown on intermediates of the citrate cycle. The enzyme was purified 92-fold from extracts, and its molecular weight was determined to be 100,000 by gel filtration. Initial velocity studies revealed marked positive cooperativity for OAA (Hill coefficient [n(H)] = 1.8; S(0.5) = 21 mM). The affinity of the enzyme for OAA was markedly increased upon addition of nicotinamide adenine dinucleotide (NAD), NAD phosphate (NADP), and some other pyridine nucleotides. S(0.5(OAA)) decreased to 1 mM but n(H) and V(max) were unchanged. Saturation kinetics for the pyridine nucleotides were hyperbolic, and a half-maximal effect was obtained with 8 muM NAD and 30 muM NADP. The enzyme also catalyzed the exchange of (14)CO(2) into OAA but not the net carboxylation of pyruvate. Exchange activity, too, exhibited sigmoidal kinetics for OAA and was strongly stimulated by NAD at low substrate concentrations. The enzyme was inhibited by acetate competitively with respect to OAA. The K(I) for acetate (12 mM) was well within the physiological range of this compound inside the cell. The regulatory properties of the decarboxylase with respect to OAA cooperativity, NAD activation, and acetate inhibition were retained in situ within permeabilized cells. These properties seem to provide for a control mechanism which could insure the maintenance of OAA and the citrate cycle during growth of cells on glucose and, conversely, the required supply of pyruvate during growth on intermediates of the citrate cycle.

Carbon Dioxide↗
PubMed1977

Alpha-ketoglutarate dehydrogenase complex of Acetobacter xylinum. Purification and regulatory properties.

The alpha-ketoglutarate dehydrogenase complex of Acetobacter xylinum was purified to homogeneity. It consists of three main polypeptide chains with a total molecular weight of about 2.4 X 10(6). It catalyzes the overall Mg2+ and thiamin pyrophosphate-dependent, NAD+- and CoA-linked oxidative decarboxylation of alpha-ketoglutarate, as well as the partial reactions characteristic of the three enzyme components described for the complex from other sources. Initial velocity studies revealed marked positive cooperativity for the substrate alpha-ketoglutarate (Hill coefficient (nH) = 2.0; concentration of ligand at half-maximum effect (S0.5) = 8 mM). The sigmoidal [alpha-ketoglutarate]-velocity relationship became hyperbolic upon addition of AMP or 3-acetylpyridine adenine dinucleotide (AcPyAD) or in the presence of high concentrations of NAD. S0.5 (alpha-ketoglutarate) decreased to 1 mM, but Vmax was unchanged. Saturation curves for NAD and AMP are sigmoidal (nH = 2) at low alpha-ketoglutarate concentrations and become hyperbolic at high alpha-ketoglutarate concentrations. As judged by S0.5, the relative efficiency of the allosteric effectors is AcPyAD greater than AMP greater than alpha-ketoglutarate- greater than NAD+. Half-maximal changes in nH, S0.5, and activation by AMP occur at a pH significantly different from that of half-maximal activity. A model for the allosteric behavior of the complex is proposed in which the first enzyme component of the complex (E1) is the site for the allosteric interactions and AMP is the primary positive modifier, whereas NAD and AcPyAD act as AMP analogues. The overall reaction is competitively inhibited by NADH with respect to NAD (K1 = 20 micronM) and by succinyl-CoA with respect of CoA (K1 = 3 micronM). The properties of the alpha-ketoglutarate dehydrogenase complex of A. xylinum appear to provide for appropriate partitioning of alpha-ketoglutarate carbon between competing pathways in response to the energy state of the cells.

Adenosine Monophosphate↗
PubMed1976

Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.

Acetyl Coenzyme A↗
PubMed1976

Activities of citrate synthase and other enzymes of Acetobacter xylinum in situ and in vitro.

The activities of a number of enzymes, extracted from Acetobacter xylinum, that are involved in carbohydrate metabolism may be accounted for in situ in permeabilized cells. The kinetic properties of citrate synthase and glycerokinase observed in vitro are also retained in situ. So is the regulatory sensitivity of these enzymes. Both in vitro and in situ, (a) citrate synthase, in contrast with the enzyme for other Gram-negative bacteria, is inhibited by ATP and is insensitive to NADH, and (b) glycerokinase is inhibited by fructose diphosphate and the ratio of its activities towards glycerol and dihydroxyacetone is the same.

Cell Membrane Permeability↗
PubMed1976

Phosphorylation of glycerol and dihydroxyacetone in Acetobacter xylinum and its possible regulatory role.

Extracts of Acetobacter xylinum catalyze the phosphorylation of glycerol and dihydroxyacetone (DHA) by adenosine 5'-triphosphate (ATP) to form, respectively, L-alpha-glycerophosphate and DHA phosphate. The ability to promote phosphorylation of glycerol and DHA was higher in glycerol-grown cells than in glucose- or succinate-grown cells. The activity of glycerol kinase in extracts is compatible with the overall rate of glycerol oxidation in vivo. The glycerol-DHA kinase has been purified 210-fold from extracts, and its molecular weight was determined to be 50,000 by gel filtration. The glycerol kinase to DHA kinase activity ratio remained essentially constant at 1.6 at all stages of purification. The optimal pH for both reactions was 8.4 to 9.2. Reaction rates with the purified enzyme were hyperbolic functions of glycerol, DHA, and ATP. The Km for glycerol is 0.5 mM and that for DHA is 5 mM; both are independent of the ATP concentration. The Km for ATP in both kinase reactions is 0.5 mM and is independent of glycerol and DHA concentrations. Glycerol and DHA are competitive substrates with Ki values equal to their respective Km values as substrates. D-Glyceraldehyde and l-Glyceraldehyde were not phosphorylated and did not inhibit the enzyme. Among the nucleotide triphosphates tested, only ATP was active as the phosphoryl group donor. Fructose diphosphate (FDP) inhibited both kinase activities competitively with respect to ATP (Ki= 0.02 mM) and noncompetitively with respect to glycerol and DHA. Adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) inhibited both enzymic activities competitively with respect to ATP (Ki (ADP) = 0.4 mM; Ki (AMP) =0.25 mM). A. xylinum cells with a high FDP content did not grow on glycerol. Depletion of cellular FDP by starvation enabled rapid growth on glycerol. It is concluded that a single enzyme from A. xylinum is responsible for the phosphorylation of both glycerol and DHA. This as well as the sensitivity of the enzyme to inhibition by FDP and AMP suggest that it has a regulatory role in glycerol metabolism.

Adenosine Diphosphate↗
PubMed1974

Regulation of hexose phosphate metabolism in Acetobacter xylinum.

The metabolism of glucose and fructose was studied in resting succinate-grown cells of Acetobacter xylinum. From fructose only cellulose and CO(2) were formed by the cells, whereas from glucose, gluconate was formed much more rapidly than these two products. The molar ratio of sugar converted into cellulose to sugar converted into CO(2) was significantly greater than unity for both hexoses. The pattern of label retention in the cellulose formed by the cells from specifically (14)C-labelled glucose, fructose or gluconate corresponded to that of hexose phosphate in a pentose cycle. On the other hand, the isotopic configuration of cellulose arising from variously singly (14)C-labelled pyruvate did not agree with the operation of a pentose cycle on gluconeogenic hexose phosphate. Readily oxidizable tricarboxylic acid-cycle intermediates such as acetate, pyruvate or succinate promoted cellulose synthesis from fructose and gluconate although retarding their oxidation to CO(2). The incorporation into cellulose of C-1 of fructose was greatly increased in the presence of these non-sugar substrates, although its oxidation to CO(2) was greatly diminished. It is suggested that the flow of hexose phosphate carbon towards cellulose or through the pentose cycle in A. xylinum is regulated by an energy-linked control mechanism.

Acetates↗
PubMed1972

Factors affecting hexose phosphorylation in Acetobacter xylinum.

Fructose was oxidized and converted to cellulose by cells of Acetobacter xylinum grown on fructose or succinate, but not by cells grown on glucose. In resting fructose-grown cells, glucose strongly suppressed fructose utilization. Extracts obtained from fructose- or succinate-grown cells catalyzed the adenosine triphosphate (ATP)-dependent formation of the 6-phosphate esters of glucose and fructose, whereas glucose-grown cell extracts phosphorylated glucose but not fructose. Fructokinase and glucokinase activities were separated and partially purified from cells grown on glucose, fructose, or succinate. Whereas fructokinase phosphorylated fructose only, glucokinase was active towards glucose and less active towards mannose and glucosamine. The optimal pH for the fructokinase was 7.4 and for the glucokinase was 8.5. The K(m) values for the fructokinase were: fructose, 6.2 mm; and ATP, 0.83 mm. The K(m) values for the glucokinase were: glucose, 0.22 mm; and ATP, 4.2 mm. Fructokinase was inhibited by glucose, glucosamine, mannose, and deoxyglucose in a manner competitive with respect to fructose, with K(i) values of 0.1, 0.14, 0.5, and 7.5 mm, respectively. Adenosine diphosphate (ADP) and adenosine monophosphate (AMP) inhibited both kinases noncompetitively with respect to ATP. The K(i) values were: 1.8 mm (ADP) and 2.1 mm (AMP) for fructokinase, and 2.2 mm (ADP) and 9.6 mm (AMP) for glucokinase. Fructose metabolism in A. xylinum appears to be regulated by the synthesis and activity of fructokinase.

Adenosine Triphosphate↗