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K Bloch

Publications and source records attributed to K Bloch.

At least 55 records · Page 3Linked to original sources
PubMed1983

Phosphatidylcholine and cholesterol interactions in model membranes.

Various phosphatidylcholines differing either in the stereochemistry around their chiral center or in the position of a cis double bond along the acyl chains were synthesized in order to study critical contact regions in the phospholipid molecule with adjacent cholesterol in model membranes. Microviscosities calculated from fluorescence depolarization of diphenylhexatriene and chain order from spin label studies were measured to monitor physical membrane properties. The enhancing effect of cholesterol on the microviscosity of membranes containing phosphatidylcholines with comparable acyl chain length was largest when the two acyl chains were saturated and smallest when both were unsaturated. Membranes prepared from phosphatidylcholines having a single cis double bond at different positions along the sn-2 acyl chain showed roughly the same changes of microviscosity or chain order upon incorporation of cholesterol. No discrimination was evident in the interaction between cholesterol and enantiomeric phosphatidylcholines or between the enantiomeric phosphatidylcholine molecules themselves. We conclude that the rigidifying effect of cholesterol in membranes does not depend on specific sites of interaction and that with respect to physical membrane properties phosphatidylcholine behaves as an achiral molecule.

Cholesterol↗
PubMed1983

Sterol synergism in yeast.

Sterol synergism as previously observed [Dahl, C.E., Dahl, J.S. & Bloch, K. (1980) Biochemistry 19, 1462-1467] and defined as a greater-than-additive growth response to pairs of sterols by Mycoplasma capricolum [Dahl, J.S., Dahl, C.E. & Bloch, K. (1981) J. Biol. Chem. 256, 87-91] is now demonstrated in the yeast mutant GL7, which is auxotrophic for sterol and unsaturated fatty acid. Mutant cells growing poorly when provided with cholesterol and oleic acid respond to ergosterol supplements (ergosterol-to-cholesterol ratio, 1:3) by a pronounced increase in growth rates and cell yields. Stigmasterol also elicits a significant synergistic effect, and 7-dehydrocholesterol, a smaller one. Evidence for a metabolic role of ergosterol in yeast membranes is presented. Cells raised on a 1:3 mixture of ergosterol to cholesterol up to midlogarithmic phase subsequently incorporate [1-14C]oleic acid at significantly faster rates into phospholipids than do cells grown on cholesterol alone.

Cell Membrane↗
PubMed1983

Intermembrane transfer of squalene promoted by supernatant protein factor.

Supernatant protein factor (SPF), a protein that stimulates squalene epoxidation, mediates the transfer of squalene between two separable microsomal populations (Kojima, Y., E. J. Friedlander, and K. Bloch, 1981. J. Biol Chem. 256: 7235-7239). We now show that SPF also promotes the transfer of squalene associated with mitochondria or with plasma membranes to total microsomes or rough or smooth microsomal subfractions. Both rough and smooth microsomes have squalene epoxidase activity that is stimulated by SPF.

Animals↗
PubMed1981

Intermembrane transfer of 5 alpha-cholest-7-en-3 beta-ol. Facilitation by supernatant protein (SCP).

The dehydrogenation of 5 alpha-cholest-7-en-3 beta-ol (lathosterol) to cholest-5,7-dien-3 beta-ol (7-dehydrocholesterol) was studied in rat liver microsomes with [3H]lathosterol as substrate. Microsomal delta 5-dehydrogenase activity was stimulated by purified "squalene and sterol carrier protein" (SCP) (Dempsey, M. E., McCoy, K. E., Barker, H. N., Vafiadou, A. D., Lorsbach, T., and Haward, J. B. (1981) J. Biol. Chem. 256, 1867-1873). When the integrity of microsomal membranes was perturbed by treatment with detergents such as 0.2% Triton X-100 or 0.05% sodium deoxycholate or by phospholipase A2, the stimulatory effect of SCP was abolished, indicating that an intact membrane system is required for the response to SCP. Dehydrogenase solubilized with 2% Triton X-100 was not stimulated by SCP. Lathosterol was effectively incorporated into microsomes in the presence or absence of SCP. The effect of SCP on the enzymatic dehydrogenation of lathosterol previously incorporated into microsomes was significantly greater than on the conversion of exogenous substrate. Kinetically, the effect of SCP was not on initial velocity except to maintain it for much longer periods of time. Tryptic digestion of previously lathosterol-loaded microsomes inactivated the delta 5-dehydrogenase. When such trypsin-treated microsomes containing [3H]lathosterol (donor microsomes) were incubated with normal, enzymatically active microsomes (acceptor microsomes), formation of 7-dehydrocholesterol could not be detected. However, if SCP was included in this assay system, dehydrogenation occurred rapidly, suggesting SCP-mediated intermembrane translocation of lathosterol. When the membranes of acceptor microsomes were damaged by detergent or phospholipase A2, the SCP effect disappeared. SCP-mediated lathosterol transfer was shown more directly by sucrose density gradient centrifugation to separate trypsinized and normal microsomes. These findings show that cytoplasmic proteins promote not only intermembrane transfer of squalene (Friedlander, E. J., Caras, T. W., Liu, L.-F. H., and Bloch, K. (1980) J. Biol. Chem. 255, 8042-8045) but also of later intermediates in cholesterol biosynthesis.

Animals↗
PubMed1981

Protein-facilitated intermembrane transfer of squalene. Demonstration by density gradient centrifugation.

Squalene-enriched, trypsinized microsomes display no squalene epoxidase activity either as such or when combined with normal microsomes. On addition of microgram quantities of supernatant protein factor to the combined system, squalene epoxidation commences at once and continues at a rapid rate (Friedlander, E. J., Caras, I. W., Lin, L. F., and Bloch, K. (1980) J. Biol. Chem. 255, 8042-8045). When mixtures of trypsin-treated, [3H]squalene-containing microsomes and normal microsomes are subjected to isopycnic density gradient centrifugation, the two microsomal populations separate readily. Essentially all of the radioactive squalene remains associated with the lighter (trypsinized) fraction of microsomes. However, if the mixture of microsomes is initially incubated with supernatant protein factor and then centrifuged, a large fraction of labeled squalene sediments with the denser, normal microsomes. Thus, supernatant protein factor mediates the transfer of squalene from one microsome population to another. This conclusion had previously been reached on the basis of less direct experiments (Friedlander, E. J., Caras, I. W., Lin, L. F., and Bloch, K. (1980) J. Biol. Chem. 255, 8042-8045). Evidence is presented that the process of supernatant protein factor-mediated squalene transfer does not involve membrane fusion and proceeds also in the reverse direction.

Animals↗
PubMed1981

Utilization and metabolism of methyl-sterol derivatives in the yeast mutant strain GL7.

Sterols modified at various positions of the tetracyclic nucleus were tested as growth supplements for Saccharomyces cerevisiae strain GL7 erg12 heme3. Derivatives of 3 beta-cholestanol or delta 7-3 beta-cholestenol bearing either a single alpha-oriented methyl group of a gem-dimethyl group at C-4 supported the growth of the mutant whereas 4 beta-methyl sterols did not. The nutritionally active alkyl derivatives were metabolized to 4-demethyl sterols while 4 beta-methyl derivatives were incorporated unchanged, indicating that the C-4 demethylase of yeast is specific for alpha-oriented methyl groups. It appears that 4-demethyl sterols are obligatory for growth of this organism. C-4 methyl derivatives of cholesterol did not support growth, suggesting that the delta 5 double bond blocks demethylation at the adjacent C-4. In other experiments, 14 alpha-methyl sterols were effective growth supplements, while 3 alpha-methylcholesterol was totally inactive. Removal of the C-19 methyl group of cholesterol (19-noncholesterol) rendered the sterol somewhat less effective as a sterol source. The sterol specificity for yeast appears to be particularly strict with regard to substituents that add bulk to the A ring of the steroid nucleus.

Cholestanols↗
PubMed1981

Effect of cholesterol on macromolecular synthesis and fatty acid uptake by Mycoplasma capricolum.

The rates of protein and lipid synthesis of Mycoplasma capricolum were essentially synchronous during growth and depended on the sterol supplement in the media increasing in the order cholesterol (0.5 microgram/ml) < lanosterol (10 microgram/ml) < lanosterol (10 microgram/ml) + cholesterol (0.5 microgram/ml) < cholesterol (10 microgram/ml). The effect of lanosterol plus low cholesterol on macromolecular synthesis was synergistic. Whereas protein and lipid synthesis were brought virtually to a halt by cholesterol starvation, DNA synthesis continued for about 8 h. Increasing the palmitate and elaidate concentrations 4-fold in the lanosterol-supplemented media raised the growth rate even in the absence of the small amount of cholesterol (0.5 microgram/ml) needed otherwise for the synergistic effect on growth. Studies of the kinetics of fatty acid uptake by resting cells showed that the apparent Km (17 microM) of oleate uptake in lanosterol-grown cells was specifically lowered to 3 microM, a value equal to that seen in cholesterol-grown cells, by the inclusion of a synergistic amount of cholesterol in the growth media. By contrast, the apparent Km for palmitate uptake was the same (2 microM) for all three cell types. The results are consistent with the membrane cholesterol serving in a dual role, one as a bulk component and another more specific function involving the regulation of unsaturated fatty acid uptake and thereby phospholipid biosynthesis.

Bacterial Proteins↗
PubMed1980

Supernatant protein factor facilitates intermembrane transfer of squalene.

Squalene epoxidation of microsome-associated squalene is stimulated by a soluble protein termed "supernatant protein factor" (SPF) (Saat, Y. A., and Bloch, K. E. (1976)J. Biol. Chem. 251, 5155-5160). In the absence of SPF, the initial rate for microsome-bound squalene epoxidation is rapid for 5 to 10 min but falls off sharply thereafter. SPF does not affect the rapid initial epoxidation rate of reaction but maintains it for longer periods. This SPF effect on enzyme kinetics indicates that SPF facilitates the otherwise rate-limiting access of squalene to the epoxidse site. Trypsin treatment of microsomes totally inactivates squalene epoxidase. When such trypsin-treated squalene-containing microsomes are incubated with normal, squalene-free, enzymatically active microsomes, formation of squalene epoxide is not observed. However, if SPF is included in this system, conversion of squalene to 2,3-oxidosqualene occurs rapidly. Lowering the temperature from 37 degrees to 22 degrees C abolishes the SPF effect in assay systems containing either normal or trypsin-treated plus normal microsomes. These findings show that SPF promotes the transfer of squalene from one microsome population to another, i.e. intermembrane transfer of substrate.

Animals↗
PubMed1980

Interactions of supernatant protein factor with components of the microsomal squalene epoxidase system. Binding of supernatant protein factor to anionic phospholipids.

Supernatant Protein Factor (SPF), a protein that enhances the activities of microsomal squalene epoxidase and 2,3-oxidosqualene-lanosterol cyclase, has been labeled either by acylation with N-succinimidyl[2,3-3H]propionate or by reductive methylation with [14C]-formaldehyde and sodium cyanoborohydride. Labeled SPF preparations, containing 1 to 2 modified lysine residues/molecule of protein which retained full biological activity, were found to bind only weakly to microsomes under a variety of experimental conditions as determined by sucrose density gradient centrifugation. No interaction between SPF and either squalene or squalene-2,3-oxide could be demonstrated by gel filtration. On the other hand, SPF was shown to bind tightly to vesicles of anionic phospholipids (phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, and phosphatidic acid) but not to vesicles of phosphatidylcholine or phosphatidylethanolamine. The capacity of the anionic phospholipids to bind to SPF parallels their ability to enhance the stimulatory activity of SPF. These observations are inconsistent with the designation of proteins of this type as "sterol carrier proteins."

Acylation↗
PubMed1980

Effect of alkyl-substituted precursors of cholesterol on artificial and natural membranes and on the viability of Mycoplasma capricolum.

Various alkyl-substituted sterols and stanols representative of the intermediates in cholesterol biosynthesis from lanosterol have been compared with respect to (a) their effect on the physical state of lecithin vesicles, (b) their efficacy as growth factors for the sterol auxotroph Mycoplasma capricolum, and (c) their effect on the physical state of the respective mycoplasma membranes. By all three criteria, sterol effectiveness progresses in the order lanosterol less than 4,4-dimethylcholestanol less than or equal to 4 beta-methylcholestanol less than 4 alpha-methylcholestanol less than cholestanol less than cholesterol. Since the corresponding steps in cholesterol biosynthesis occur in the same order, we conclude that the nuclear modifications of the lanosterol structure by oxidative demethylation serve to improve the membrane function of the sterol molecule.

Cell Membrane↗
PubMed1980

Effect of sterol side chains on growth and membrane fatty acid composition of Saccharomyces cerevisiae.

Saccharomyces cerevisiae GL7 cells require exogenous sterol and unsaturated fatty acid for growth. When grown in the presence of cholesterol or 7-dehydrocholesterol, the cells incorporated less saturated fatty acid into phospholipids than cells grown with ergosterol, stigmasterol, or beta-sitosterol as the sterol source. This lower saturated fatty acid content was most pronounced in phosphatidylethanolamine, slightly less so in phosphatidylcholine, and least evident in phosphatidylserine and phosphatidylinositol. Growing the cells with the various sterols did not affect the ratios of individual phospholipids. The ability of strain GL7 to use 7-dehydrocholesterol as the only sterol supplement for growth was dependent upon the nature of the unsaturated fatty acids added to the growth medium. In the presence of linoleic, linolenic, or a mixture of palmitoleic and oleic acids, excellent growth was observed with either ergosterol, cholesterol, or 7-dehydrocholesterol. However, when the medium was supplemented with either oleic or petroselenic acid, the cells grew more slowly (oleic) or much more poorly (petroselenic) with 7-dehydrocholesterol than with ergosterol. A specific relationship between sterol structure and membrane fatty acid composition in yeast cells is implied.

Cholestanol↗
PubMed1980

Glipizide and hepatic glycogenolysis.

Glipizide, a new sulfonylurea recently introduced for the treatment of diabetes, was studied to check its possible extrapancreatic effects. Rats were given a subcutaneous injection of 1 g/kg glucosamine: this dose caused marked hyperglycemia and a decrease in hepatic glycogen, but does not alter blood insulin levels. Pretreatment with i.v. dose of 37.5 microgram/kg glipizide 1 hour before the glucosamine load, significantly inhibits the hyperglycemia and the decrease of hepatic glycogen. This dose of glipizide does not affect blood sugar levels, although it does induce a transient rise in insulin secretion, which lasts no more than 10 minutes after administration. Since glucosamine was administered 1 hour after the sulfonylurea by which time the interference of insulin was no longer felt, it may be concluded that in the experiment described, glipizide seems to have some other action apart from stimulating insulin secretion.

Adrenalectomy↗
PubMed1980

Effect of glipizide on muscle metabolism in vitro.

This paper contains the results of further investigation on the possible extrapancreatic activity of glipizide. An in vitro method, using isolated rat diaphragm was selected. Muscle uptake of C14 glucose and output of labelled CO2, were measured. Insulin was used as the reference substance. The results of our experiments indicate that there is a qualitative difference in the effects of the two experimental situations. However, from these findings it may be assumed that glipizide facilitates the uptake and metabolism of glucose in muscle, thus demonstrating an extrapancreatic effect, which contributes to the hypoglycemic activity of the drug.

Animals↗