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Biomedical subjects

S B Shohet

Publications and source records attributed to S B Shohet.

At least 127 records · Page 7Linked to original sources
PubMed1975

Determinants of the formation and activity of factor V-phospholipid complexes. II. Molecular properties of the complexes.

The stability, buoyancy, and intrinsic activity for factor V-phospholipid complexes were investigated. The decay of factor V activity in the absence of phospholipid followed first order kinetics; however, in the presence of several phospholipids a biphasic decay curve was observed. The addition of phosphatidylethanolamine to factor V produced only a small loss of activity in the first 2 minutes but decreased the subsequent rate of inactivation fourfold. A PE-factor V complex with a low bouyant density was separated from uncomplexed factor V by sucrose density ultracentrifugation. The association constant for this complex was 5 X 10(6) M-1 with approximately 2 moles of factor V bound per mole of lipid micelle. The isolated complex was capable of increasing prothrombin conversion 10-fold without additional phospholipid. A still lighter complex increased the rate of prothrombin conversion 18-fold. Phosphatidyl serine produced a concentration-dependent loss of up to 95% of the factor V activity in the first 2 minutes. After ultracentrifugation on a sucrose density gradient, a PS-factor V complex of increased density was detected. This complex failed to accelerate prothrombin conversion in the intrinsic two-stage assay. Except at very high concentrations, phosphatidylcholine did not alter the kinetics of inactivation of factor V. A factor V-phosphatidylcholine complex could not be detected after ultracentrifugation. When added to factor V, cardiolipin (200 mug/ml), produced a rapid 50% decline in activity with a subsequent three-fold increase in the rate of inactivation. No activity was recovered after ultracentrifugation of factor V in the presence of cardiolipin. Saturated phosphatidylethanolamine produced a concentration dependent initial loss of activity, but only a minimal increase in the subsequent rate of inactivation. At 200 mug/ml almost no light complex was detected after ultracentrifugation, but at 800 mug/ml a light complex was observed. This behavior corresponds to the ability of saturated phosphatidylethanolamine to accelerate prothrombin conversion only at very high concentrations. Thus, phospholipids combine with factor V to form complexes which differ in their ability to accelerate prothrombin conversion. The most active species are stable lipoprotein complexes of lower buoyant density than factor V.

Animals↗
PubMed1975

Determinants of the formation and activity of factor V-phospholipid complexes. I. Influence of phospholipid structure.

Previous studies showed that factor V consists of multiple oligomeric forms including a minor component (form L) which contained lipid and was eluted in the excluded volume of a Sepharose 4B column. In the present and following study the hypothesis that factor V is a lipid requiring protein rather than a lipoprotein has been tested. Modification of a previous purification procedure resulted in the separation of factor V from lipoprotein. The lipid extracted from this lipoprotein was qualitatively similar to that previously isolated from form L. Removal of this lipoprotein by sucrose density gradient ultracentrifugation resulted in the isolation of factor V of high specific activity which contained no detectable phospholipid and less than 0.2% cholesterol. No component was excluded from Sepharose 4B after gel filtration of this purified preparation. It is concluded that factor V is not a lipoprotein, but rather a protein which requires lipid for its coagulant properties. To define this requirement further the ability of factor V-phospholipid complexes to accelerate the conversion of prothrombin to thrombin by factor Xa in the presence of calcium was tested. The rate of thrombin formation was highly dependent on the phospholipid employed with each active phospholipid exhibiting a characteristic optimal concentration. At 250 muM, the order of activity was phosphatidyl ethanolamine greater than phosphatidyl inositol greater than cardiolipin greater than phosphatidyl choline. Phosphatidyl serine was inert at all concentrations employed. No single fatty acid was consistently present in active lipid preparations, nor was a specific fatty acid absent from those which had no clotting activity. In comparing different lipid classes, a direct relationship between the overall degree of unsaturation and activity was not observed. However, bovine phosphatidyl inositol, which is highly unsaturated, is active, while plant phosphatidyl inositol, which contains only trace amounts of unsaturated fatty acids, were inert. At lipid concentrations below 300 muM, saturated phosphatidyl ethanolamine produced by hydrogenation of bovine phosphatidyl ethanolamine was less active than an equal concentration of the native lipid. In mixtures containing both lipids, artificially saturated phosphatidyl ethanolamine potentiated the activity of the native compound.

Animals↗
PubMed1975

A congenital haemolytic anaemia with thermal sensitivity of the erythrocyte membrane.

Microspherocytes, measuring 2-3 mum in diameter, and cells with blunted projections or triangular in shape characterized the erythrocoyte morphology in three children with congenital haemolytic anaemia. Since the erythrocyte morphology resembled that associated with thermal injury, heat-induced changes in erythrocyte morphology and membrane composition were studied. Erythrocytes developed filaments and spheroid bodies which fragmented, resulting in microspherocyte transformation. Normal cells required exposure to 49 degrees C, whereas the patients' cells fragmented at 45 degrees C. Fragmentation was also observed during incubation of patients' cells at 37 degrees C for 17h. The heat-induced transformation of the patients' cells was associated with an increase in the membrane cholesterol:phospholipid and cholesterol:protein ratios. The phospholipid:protein ratio was unchanged. This suggests that fragmentation produces a selective loss of membrane components. Splenectomy ameliorated the haemolytic process. We propose that the patients' red-cell morphology is the result of in vivo fragmentation, and that the spleen is the major site of microspherocyte and poikilocyte destruction.

Anemia, Hemolytic, Congenital↗
PubMed1975

Hereditary stomatocytosis: membrane and metabolism studies.

A defect in the protein kinase-mediated phosphorylation of erythrocyte membrane proteins, previously unrecognized in stomatocytosis, was discovered in a boy with hereditary stomatocytosis and severe hemolytic anemia. The high-sodium, low-potassium erythrocytes of this patient were remarkably permeable to both sodium and potassium. The rate of ouabain-inhibitable active cation transport was more than ten times normal and was sustained by an increase of similar magnitude in glycolysis. The deformability in vitro of fresh stomatocytes was reduced and deteriorated further after a brief period of incubation with glucose. Ferrokinetic studies showed that these rigid cells were sequestered by the spleen. When stomatocytes were deprived of glucose in vitro, ATP depletion and ATPase cation pump failure rapidly ensued. Because of their permeability defect, such depleted cells rapidly became swollen and lysed. Prolonged entrapment in acidic, hypoglycemic regions of the spleen would recapitulate these unfavorable events in vivo. In this regard, splenectomy was followed by an improvement in erythrocyte survival, although evidence of continuing hemolysis was obtained.

Abnormalities, Multiple↗
PubMed1974

Lipid peroxidation in the killing of phagocytized pneumococci.

To directly examine the role of hydrogen peroxide in the killing of bacteria after ingestion by granulocytes, we have studied some of the events of phagocytosis of a mutant strain of pneumococci which is relatively deficient in peroxide production. The hydrogen peroxide-deficient pneumococci and the otherwise identical wild type were grown with [(14)C]arachidonic and [(3)H]palmitic acid labels to label their lipids with unsaturated and saturated fatty acids, respectively. They were then incubated with both normal and chronic granulomatous disease granulocytes. The rates of ingestion and bacterial killing and the stability of fatty acids in the cell-bacteria complex were followed. Radioactive carbon dioxide released from glucose was also independently followed to measure glucose oxidation. Ingestion was similar for all cell-bacteria combinations. Chronic granulomatous disease cells killed the peroxide-positive wild pneumococci much more effectively (20-fold) than the peroxide-deficient mutant. Normal cells killed both peroxide-positive and -negative strains effectively. A considerable loss of [(14)C]arachidonic acid ( approximately 40%) consistent with lipid peroxidation of this unsaturated fatty acid was observed in all normal cells and in chronic granulomatous disease cells with peroxide-positive pneumococci. However, no loss of [(14)C]arachidonic acid occurred in chronic granulomatous disease cells with the peroxide-deficient pneumococci. No loss tritiated palmitic acid occurred in any cell-bacteria combination. Glucose oxidation was impaired in the chronic granulomatous disease cells in comparison to normal cells at rest and was especially impaired in chronic granulomatous disease cells ingesting the peroxide-deficient mutant pneumococci. This defect was partially corrected after phagocytosis of the peroxide-positive strain. These data directly support the hypothesis that bacterial killing is partially dependent upon an intact peroxide-generating system in the leukocyte-bacteria complex. Moreover, they indicate that bacterial lipid peroxidation is associated with the generation of peroxide during phagocytosis. Finally, they suggest that such peroxidation may contribute to effective phagocytic bacterial killing.

Animals↗
PubMed1974

Remodeling of granulocyte membrane fatty acids during phagocytosis.

During phagocytosis, new phospholipid is synthesized from triglyceride fatty acid and may be utilized to form the membranes of phagocytic vesicles. In addition, hydrogen peroxide, which can peroxidize unsaturated fatty acids, is generated. Since both of these processes could change membrane fatty acid composition during the conversion of cytoplasmic granules and plasma membranes to phagosomes, the lipid compositions of these structures were examined. Phagocytic vesicles were prepared by density gradient centrifugation of polystyrene latex particles after phagocytosis. Granule and plasma membrane fractions were isolated by density gradient and differential centrifugation. Phospholipids and fatty acids were analyzed by thin-layer chromatography and gas-liquid chromatography. While whole cells, granules, plasma membranes, and phagosomes were all similar in phospholipid composition, phagosome fatty acids were significantly more saturated than those of the other fractions. This was primarily due to reduced oleic and arachidonic acids and increased palmitic acid in the phagocytic vesicle lipids. Plasma membrane was also more saturated in comparison to whole cells and granules. However, this difference was not sufficient to explain the marked comparative saturation of the phagosomes. The observed increase in fatty acid saturation in these lipids may have been induced by a combination of either peroxidative destruction of polyunsaturated fatty acids or phospholipase activity, coupled with reacylation mechanisms favoring saturated fatty acids.

Arachidonic Acids↗
PubMed1974

Permeability changes induced by peroxidation in liposomes prepared from human erythrocyte lipids.

The diffusion of [2-(14)C]glucose out of liposomes prepared from extracted human erythrocyte lipids was examined. Increased glucose efflux was observed when the lipids were treated with hydrogen peroxide and CuCl(2) before liposome formation, and this phenomenon required both peroxide and metal. Peroxidation of these lipids also resulted in the destruction of polyunsaturated fatty acids and the generation of conjugated dienes, but neither of these processes appeared to be the sole cause of increased glucose efflux. Thin-layer chromatography and the effects of aqueous washes suggested that surface-active lysophosphatides or other lipid degradation products were responsible for the increased permeability of the treated liposomes. It is suggested that the behavior of this liposome model system may be relevant to the high permeability and fragility of vitamin E-deficient erythrocytes.

Carbon Radioisotopes↗