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Biomedical subjects

K Bloch

Publications and source records attributed to K Bloch.

At least 73 records · Page 4Linked to original sources
PubMed1979

Effects of a supernatant protein activator on microsomal squalene-2,3-oxide-lanosterol cyclase.

A soluble protein termed "supernatant protein factor" (SPF) that stimulates microsomal squalene epoxidase has been isolated in this laboratory (Ferguson, J.B., and Bloch, K. (1977) J. Biol. Chem. 252, 5381-5385). We now show that the purified protein also stimulates microsomal squalene-2,3-oxide leads to lanosterol cyclase but has no effect on the subsequent conversion of lanosterol to cholesterol. Phospholipid, specifically phosphatidylglycerol or phosphatidylethanolamine, is required for maximal stimulation of the cyclase by purified SPF. The response of microsomal squalene epoxide-lanosterol cyclase to SPF was abolished by pretreatment of the membranes with phospholipase A2 or by low concentrations of deoxycholate, indicating that an intact membrane system is required. Digestion of intact microsomes with trypsin had no effect on the SPF-stimulated cyclase activity. However, in the presence of 0.4% deoxycholate, trypsin completely inhibited microsomal squalene epoxide-lanosterol cyclase. We conclude that the cyclase is located on the luminal side of the microsomal membrane. SPF also significantly enhances the formation of lanosterol from squalene-2,3-oxide already bound to microsomes. This finding is constant with the proposal that SPF influences intramembrane events.

Animals↗
PubMed1979

On the role of the sterol hydroxyl group in membranes.

The adequacy of sterol derivatives containing a blocked 3-hydroxyl group for sustaining the growth of two sterol auxotrophs has been investigated. Mycoplasma capricolum, a cholesterol-requiring bacterium, grows nearly as well on media supplemented with cholesteryl methyl ether or cholesteryl acetate as on free cholesterol. The two derivatives are recovered unchanged from the bacterial cells. Similarly, cholesteryl methyl ether or ergosteryl methyl ether replace cholesterol or ergosterol as sterol sources for a yeast mutant, strain GL7, defective in 2,3-oxidosqualene-lanosterol cyclization. During aerobic or semianaerobic growth, yeast cells demethylate some of the cholesteryl methyl ether to free cholesterol. However, cells growing on cholesterol methyl ether under strict anaerobic conditions do not produce free sterol. The bearing of these results on the postulated requirement of a free sterol hydroxyl group for membrane function is discussed. Sterol esterification does not appear to be essential for the two microbial systems.

Cell Membrane↗
PubMed1979

Purification of hog liver isomerase. Mechanism of isomerization of 3-alkenyl and 3-alkynyl thioesters.

A hog liver enzyme that catalyzes the reversible conversion of 3-acetylenic fatty acyl thioester to (+)-2,3-dienoyl fatty acyl thioester has been purified to homogeneity. The enzyme is not inhibited by the allenic product that it generates. The same homogenous enzyme catalyzes the conversions of 3-cis- or 3-trans-acyl Coenzyme A derivatives to 2-trans-acyl-CoA derivatives. Four forms of the isomerase differing in charge (pI = 6.57, 6.83, 7.01, and 7.27) have been separated by isoelectric focusing. Ultracentrifugation and sodium dodecyl sulfate-gel electrophoresis indicate that each of these enzyme forms is dimeric and composed of two 45,000-dalton subunits. With 3-acetylenic substrates, all enzyme forms exhibit broad specificity for chain length (C6 to C12) and for the thioester moiety (N-acetylcysteamine (NAC), pantetheine, or CoA). The 3-cis and 3-trans olefinic substrates are active only in the form of their coenzyme A derivatives; their NAC thioesters inhibit competitively. Mechanistic studies favor an isomerization pathway by way of carbanion intermediates. The acetylene-allene isomerase described here and the reported crotonase-catalyzed hydration of allenic thioesters (Branchini, B.R., Miesowicz, F.M., and Bloch, K. (1977) Bioorg. Chem. 6, 49-52) may be responsible for the degradation of naturally occurring acetylenic and allenic acids.

Acyl Coenzyme A↗
PubMed1978

Subunit structure of Mycobacterium smegmatis fatty acid synthetase. Evidence for identical multifunctional polypeptide chains.

Fatty acid synthetase from Mycobacterium smegmatis has been purified to near homogeneity as judged by a variety of electrophoretic criteria under both native and dissociating conditions. A single protein band was obtained on gel electrophoresis in sodium dodecyl sulfate or 8 M urea at various pH values and on isoelectric focusing in 8 M urea. A subunit molecular weight of about 290,000 was found by polyacrylamide gel electrophoresis in sodium dodecyl sulfate or by sedimentation equilibrium ultracentrifugation in 6 M guanidine HCl. Quantitative Quantitative determination of pantetheine, of flavin, and of the number of fatty acids synthesized during a single enzyme turnover all yield values corresponding to a stoichiometry of about 1 mol per mol of subunit, providing strong evidence that M. smegmatis fatty acid synthetase is an oligomer of identical, multifunctional polypeptide chains.

Acyl Carrier Protein↗
PubMed1978

Sterol requirement of Mycoplasma capricolum.

Mycoplasmas require an external source of sterol for growth. For Mycoplasma capricolum this requirement is met not only by cholesterol but also by the methylcholestane derivatives lanosterol, cycloartenol, 4,4-dimethylcholesterol, and 4beta-methylcholestanol. Cholesteryl methyl ether and 3alpha-methylcholestanol serve equally well as sterol supplements. None of the growth-supporting sterol derivatives tested was metabolically modified. The unusual acceptance of diverse cholestane derivatives by a mycoplasma species contrasts with the structural attributes thought to be necessary for sterol function in eukaryotic membranes.

Cholestanes↗
PubMed1977

Mycobacterium smegmatis fatty acid synthetase. Long chain transacylase chain length specificity.

Long chain transacylase activity, acyl-CoA + enzyme in equilibrium acyl-enzyme + CoA, catalyzed by the multienzyme complex fatty acid synthetase from Mycobacterium smegmatis was measured by exchange of radioactive coenzyme A into even numbered fatty acyl-CoA substrates 14 to 24 carbon atoms long. This transacylase activity decreases sharply with increasing chain length. It is suggested that C24 transacylation may be rate-limiting in de novo fatty acid synthesis catalyzed by the myocobacterial system. Mycobacterial polysaccharides stimulate the rate of transacylation, and this enhancement becomes more marked as the chain length of the substrate increases. The magnitude of the effect is similar to polysaccharide stimulation of overall synthetase activity. It is therefore proposed that terminal transacylation is the specific and perhaps only partial reaction catalyzed by the M. smegmatis fatty acid synthetase which is facilitated by polysaccharide. The product distribution of the synthetase is distinctly bimodal, with peaks for acyl chains 16 and 24 carbon atoms long. A scheme based on nonoverlapping unimodal chain length specificities for the rates of two activities, elongation and terminal transacylation, is offered to explain this bimodal distribution.

Acyltransferases↗
PubMed1977

Mycobacterium smegmatis fatty acid synthetase. Polysaccharide stimulation of the rate-limiting step.

An initial activity burst lasting 5 to 10 s is observed for both de novo synthesis with acetyl-CoA as primer and for elongation of palmitoyl-CoA catalyzed by the multienzyme complex fatty acid synthetase from Mycobacterium smegmatis. After the initial burst, synthetase activity slows at least 6-fold to the steady state rate. The size of the initial burst is proportional to the amount of synthetase protein and corresponds to the synthesis of a small number C three to five) of C24 or C26 acyl chains per mol of enzyme. During the initial burst, C24, C26 acyl enzyme is formed and can be isolated by ammonium sulfate precipitation. On incubation with CoA, enzyme-bound acyl chains undergo transacylation to form the corresponding CoA derivatives. Diffusion of C24-CoA and C26-CoA from the enzyme is slow and rate-limiting for overall fatty acid synthesis. Mycobacterial polysaccharides markedly accelerate this rate-determining step but bovine serum albumin does not. This facilitation of product diffusion accounts for the large stimulation of de novo synthesis and of elongation of mycobacterial polysaccharide. It is also shown that the high apparent Km for acetyl-CoA (approximately 400 micrometer) in the steady state reflects the substrate concentration required to shift the product pattern in favor of shorter chain fatty acids (C16,C18). These conditions circumvent the slow, rate-limiting diffusion of C24-CoA and C26-CoA.

Acetyl Coenzyme A↗
PubMed1977

Mycobacterium smegmatis fatty acid synthetase. A mechanism based on steady state rates and product distributions.

The initial steady state rate and product distribution of fatty acid synthesis catalyzed by Mycobacterium smegmatis fatty acid synthetase has been investigated as a function of various concentrations of acetyl-CoA, malonyl-CoA, mycobacterial polysaccharide, and bovine serum albumin. Polysaccharide has a large effect on both rate and chain length. The steady state rate stimulation by polysaccharide is not duplicated by other acyl-CoA-binding molecules such as bovine serum albumin. It is concluded that relief of product inhibition does not adequately explain the specific effects of the mycobacterial polysaccharide. A general mechanism is presented which accounts for variations in reaction rate and produce pattern over a wide range of experimental conditions. We propose that the diffusion of long chain acyl-CoA (C14 to C24) from the enzyme is the rate-limiting step in fatty acid synthesis catalyzed by the M. smegmatis synthetase. Polysaccharide facilitates this rate-limiting step by forming a ternary complex with enzyme-bound acyl-CoA causing rapid release of product.

Acetyl Coenzyme A↗
PubMed1977

Fatty acid synthetase activity in Mycobacterium smegmatis. Characterization of the acyl carrier protein-dependent elongating system.

Mycobacterium smegmatis extracts contain two fatty acyl synthetase systems (Brindley, D.N., Matsumura, S. and Bloch, K. (1966) Nature 224, 666-669). One is the extensively studied multienzyme complex, (molecular weight 1.39 - 10(6)) which produces shorter C16 and C18) and longer (C24 and higher) fatty acids in a bimodal pattern. The second synthetase is acyl carrier-protein (ACP) dependent and elongates the CoA derivatives of C12 and longer chains. In contrast to the type I synthetase which also extends long fatty acyl chains, the ACP-dependent system produces homologous fatty acids up to 30 carbon atoms long in approximately equal proportions. Other properties which distinguish the ACP-dependent system from the multienzyme complex include the resistance to high concentrations of palmitoyl-CoA and to low ionic strength and the lack of stimulation by mycobacterial polysaccharides. The possibility that the two fatty acid synthetases are complimentary in their function is discussed.

Adenosine Triphosphate↗
PubMed1977

Purification and properties of a soluble protein activator of rat liver squalene epoxidase.

A soluble rat liver protein, termed "supernatant protein factor" (SPF), that stimulates microsomal squalene epoxidase has been purified approximately 11,000-fold. The most highly purified preparation obtained by isoelectric focusing shows a single coincident peak for activity and protein (the isoelectric point, pI, was 6.74). SPF is about 95% pure, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it migrates to a position corresponding to an apparent molecular weight of 47,000. An amino acid analysis of SPF is presented, and the properties of SPF and of the various soluble protein activators of microsomal sterol biosynthesis described by other laboratories are compared.

Amino Acids↗
PubMed1977

Differential effects of cholesterol and lanosterol on artificial membranes.

The effects of cholesterol, 4,4-dimethylcholesterol, and lanosterol (4,4',14alpha-trimethyl-delta8,24-cholestadiene-3beta-ol) on some properties of lecithin vesicles have been compared. Unlike cholesterol, lanosterol retards the exit of trapped glucose from phospholipid vesicles only slightly. The 13C nuclear magnetic resonance spectrum of cholesterol/lecithin vesicles shows no resonances attributable to the sterol. By contrast, several resonances attributable to quaternary carbon atoms or methyl groups are seen in the 13C nuclear magnetic resonance spectrum of lanosterol/lecithin vesicles, indicating that lanosterol is much less immobilized than cholesterol. Because the membrane behavior of 4,4-dimethylcholesterol is closely similar to that of cholesterol, it is concluded that the axial 14-alpha-methyl group is responsible for the lessened membrane immobilization of lanosterol. The results emphasize the importance of a planar sterol alpha-face for interaction with phospholipid acyl chains.

Cholesterol↗
PubMed1976

Inhibition of glutamate dehydrogenase and malate dehydrogenases by palmitoyl coenzyme A.

In extension of a previous study with yeast glucose-6-P dehydrogenase (Kawaguchi, A., and Bloch, K. (1974) J. Biol. Chem. 249, 5793-5800), the structural changes accompanying the inhibition of glutamate dehydrogenase and several malate dehydrogenases by palmitoyl-CoA and by sodium dodecyl sulfate have been investigated. Palmitoyl-CoA converts liver glutamate dehydrogenase to enzymatically inactive dimeric subunits (Mr = 1.2 X 10(5)) and tightly binds to the dissociated enzyme. Removal of the inhibitor from the palmitoyl-CoA-dimer complex fails to regenerate enzyme activity. The Ki values for palmitoyl-CoA inhibition of malate dehydrogenases (oxalacetate reduction) are, for the enzyme from pig heart mitochondria, 1.8 muM, 500 muM from pig heart supernatant, and 10 muM from chicken heart supernatant. These inhibitions are readily reversible. Palmitoyl-CoA does not alter the quaternary structure of any of the malate dehydrogenases and binds only weakly to these enzymes. Mitochondrial malate dehydrogenase assayed in the direction malate to oxalacetate is much less sensitive to palmitoyl-CoA, with Ki values of 50 muM at pH 10 and greater than 50 muM at pH 7.4. While the differences in palmitoyl-CoA sensitivity in the forward and backward reactions catalyzed by mitochondrial dehydrogenase are unexplained, a physiological rationale for these differential effects is offered. Sodium dodecyl sulfate dissociates the various dehydrogenases to monomeric subunits in contrast to the more selective effects of palmitoyl-CoA.

Animals↗
PubMed1975

Reversible inhibition of the fatty acid synthetase complex from Mycobacterium smegmatis by palmitoyl-coenzyme A.

Palmitoyl-CoA dissociates the fatty acid synthetase complex from Mycobacterium smegmatis into inactive subunits of molecular weight 250,000 as determined by sucrose density gradient centrifugation. Palmitoyl-CoA binds to the subunits but the binding can be prevented and reversed by the mycobacterial 3-O-methylmannose-containing polysaccharide. When the palmitoyl-CoA containing inactive subunits were isolated by gel filtration on Sepharose 6B, and then concentrated and dialyzed against 0.5 M phosphate buffer, pH 7.0, containing 3 mM of the complexing agent heptakis-(2,6-di-O-methyl)-beta-cyclodextrin, activity was regenerated to the level of 40 percent of a control sample. The reversibility of the dissociation and inactivation of the synthetase by palmitoyl-CoA suggests that this end product might play a regulatory role by acting as a feedback inhibitor.

Coenzyme A↗
PubMed1975

Complex formation between mycobacterial polysaccharides or cyclodextrins and palmitoyl coenzyme A.

The mycobacterial polysaccharides MMP (3-O-methyl-mannose-containing polysaccharide), MGLP (lipolysaccharide containing 6-O-methylglucose and glucose), and the cyclodextrins (cyclohexaamylose and cycloheptaamylose) form stoichiometric complexes with palmitoyl-CoA (Machida, Y., Bergeron, R., Flick, P., and Bloch, K. (1973) J. Biol. Chem. 248, 6246-6247). Complex formation is presumed to result from hydrophobic interactions. In order to enhance the hydrophobic character of the cyclodextrins the following derivatives have been synthesized: heptakis (2,di-O-propyl)-, heptakis (2,6-di-O-methyl)-, pentakis (6-O-methyl)-, heptakis (3-O-methyl)-, and permethylated beta-cyclo-dextrin. These compounds stimulate fatty acid synthesis catalyzed by the Mycobacterium smegmatis fatty acid synthetase, the magnitude of the effect decreasing in the order in which the alkylated cyclodextrins are listed above. MMP or MGLP are qualitatively indistinguishable from alkylated cyclodextrins both with respect to palmitoyl-CoA binding and with respect to effects on enzyme systems, suggesting that they form inclusion complexes of the same type. On the basis of model building it is postulated that MMP in solution assumes a helical conformation with a hydrophobic channel about 6 A in diameter and approximately 29 A long, dimensions appropriate for accommodating the paraffinic chain of palmitoyl-CoA in the form of an inclusion complex. Since palmitoyl-CoA binds to polysaccharide much more tightly than free palmitate it is further postulated that ionized groups of the CoA moiety of acyl CoA participate in the binding and do so by hydrogen bonding to the hydrophilic exterior of helical MMP. Palmitoyl-CoA, and to a lesser extent palmitate, affect the optical rotation of MMP and also of the alkylated cyclodextrins indicating that complex formation induces conformational changes in the polysaccharides.

Binding Sites↗
PubMed1975

Solubilization and partial characterization of rat liver squalene epoxidase.

The microsomal enzyme system from rat liver which catalyzes squalene epoxidation requires a supernatant protein and phospholipids (Tai, H., and Bloch, K. (1972) J. Biol. Chem. 247, 3767). It has now been found that these two cytoplasmic components can be replaced by Triton X-100. The same detergent solubilizes the microsomal squalene epoxidase and the resulting supernatant can be separated into two components, A and B, by DEAE-cellulose chromatography. Neither Fraction A nor B alone has significant squalene epoxidase activity but combining the two affords a reconstituted system 5-fold higher in specific epoxidase activity than that of the original microsomes. FAD and Triton X-100 in addition to molecular oxygen and NADPH are required in the reconstituted system. Subjecting Fraction A to a second DEAE-cellulose chromatography does not change its specific activity but lowers NADH-ferricyanide reductase activity and the protoheme content to 1/25 and 1/4, respectively. When Fraction B was chromatographed on Sephadex G-200, the specific epoxidase activity tested in the presence of Fraction A was increased 3-fold. This procedure also raised the specific activity of NADPH-cytochrome c reductase activity in Fraction B 3-fold. The reconstituted epoxidase system is not inhibited by either carbon monoxide, potassium cyanide, or o-phenanthrolien but Tiron at 1 mM was inhibitory (50%). Erythrocuprein has no effect on epoxidation. No evidence has been found for the participation of hemoproteins (P450 or cytochrome b5) in squalene epoxidation. Component B appears to be identical with the flavoprotein NADPH-cytochrome c reductase. Component A may be a flavoprotein with an easily dissociable prosthetic group.

Animals↗