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S Fleischer

Publications and source records attributed to S Fleischer.

At least 235 records · Page 13Linked to original sources
PubMed1978

The orientation of D-beta-hydroxybutyrate dehydrogenase in the mitochondrial inner membrane.

D-beta-Hydroxybutyrate dehydrogenase of beef heart mitochondria is a lipid-requiring enzyme, bound to the inner membrane. The orientation of this enzyme in the membrane has been studied by comparing the characteristics of the enzyme in mitochondria and 'inside-out' submitochondrial vesicles. We observe that the enzymic activity is (1) latent in intact mitochondria; (2) relatively stable to trypsin digestion in mitochondria but rapidly inactivated in submitochondrial vesicles by this treatment; and (3) released more rapidly from submitochondrial vesicles by phospholipase A2 digestion than from mitochondria. Conclusive evidence that D-beta-hydroxybutyrate dehydrogenase is localized on the matrix face of the mitochondrial inner membrane is provided by the correlation that the enzyme is released from submitochondrial vesicles before the membrane becomes leaky to cytochrome c. The arrangement of D-beta-hydroxybutyrate dehydrogenase in the membrane is discussed within a generalized classification of the orientation of proteins in membranes. The evidence indicates that D-beta-hydroxybutyrate dehydrogenase is an amphipathic molecule and as such is inlaid in the membrane, i.e. the enzyme is partially inserted into the hydrophobic milieu of the membrane, with the polar, functional end extending into the aqueous milieu.

Hydroxybutyrate Dehydrogenase↗
PubMed1978

The calcium accumulation in a microsomal fraction from porcine coronary artery smooth muscle. A study of the heterogeneity of the fraction.

1. Microsomes prepared from the combined media and intima of pig coronary artery, take up Ca in an ATP-dependent way. This uptake is stimulated by oxalate. 2. Conditions have been determined to optimize the preparation of the microsomes in terms of their Ca accumulation activity. Careful homogenization of the tissue mince in 0.25 M sucrose by means of a Potter-Elvehjem homogenizer gives microsomal preparations with the highest specific activity for Ca accumulation. 3. Arguments are presented to support the hypothesis that, even in the absence of oxalate, Ca accumulation occurs into the lumen of the vesicles, and that these vesicles have a low Ca permeability. 4. Density gradient analysis shows that the microsomal fraction prepared from pig coronary artery media and intima is composed of vesicles that are heterogeneous in enzymatic composition. 5. Adenylate cyclase appears to be a predominantly plasma membrane-bound enzyme. Rotenone-insensitive NADH-cytochrome c reductase and choline phosphotransferase, two putative markers for internal membranes, give distinct banding patterns on on isopycnic centrifugation, indicating different intracellular localization. 6. There is a difference between the density gradient distribution pattern of Ca uptake measured in the presence or absence of oxalate. The latter coincides more closely with plasma membrane markers. The former resembles more the distribution of rotenone-insensitive NADH-cytochrome c reductase.

Animals↗
PubMed1978

Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid.

Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.

Animals↗
PubMed1977

Characterization of triton X-100-solubilized prostaglandin E binding protein of rat liver plasma membranes.

Rat liver plasma membranes bind prostaglandins E1 and E2 (PGE) with high affinity and specificity. We have solubilized plasma membranes, prelabeled with radioactive PGE1, in water solutions of Triton X-100. We sedimented this material into sucrose density gradient containing H2O and D2O. From numerical integration of the sedimentation equation, taking explicitly into account the density and viscosity gradients present during the centrifugation, we have determined a value of s20,w = 5.6 to 5.7 X 10(-13) s and a partial specific volume, v = 0.80 to 0.81 cm3/g, for the PGE binding protein-Triton X-100 composed of 60% (w/w) protein and 40% (w/w) detergent. Gel filtration in water solutions of Triton X-100 gives a Stokes radius of 53 A for the complex. These data imply a molecular weight of 105,000 for the detergent-free binding protein and a frictional ratio of 1.3 for the complex. If the detergent is bound to the protein in a monolayer, about 40% of the PGE binding protein's surface would be covered with detergent. The procedures used in the analysis of the sedimentation behavior of the PGE binding protein-detergent complex, when coupled with a gel filtration measurement of the Stokes radius, allow valid determination of the size, shape, and extent of detergent binding of a wide variety of membrane proteins, even when they are present as minor components of complex mixtures.

Animals↗
PubMed1976

Protein purification: adsorption chromatography on controlled pore glass with the use of chaotropic buffers.

Chromatography on controlled pore glass in combination with chaotropic buffers makes possible, in a single step, protein purifications of several hundredfold. The new emphasis is on highly selective controllable adsorption. The method is useful for the purification and concentration of proteins from large volumes of complex media and for the purification of proteins that are poorly soluble or tend to aggregate in aqueous solution D-(-)-Beta-Hydroxybutyrate dehydrogenase, a mitochondrial membrane-bound protein, several soluble proteins, and staphylococcal alpha toxin, which can be purified directly from large volumes of culture medium, are used to illustrate the method.

Adsorption↗
PubMed1975

Preparation of a homogeneous soluble D-beta-hydroxybutyrate apodehydrogenase from mitochondria.

D-beta-Hydroxybutyrate dehydrogenase of bovine heart mitochondria has been purified to apparent homogeneity. The membrane-bound enzyme is first released by phospholipase A digestion of the mitochondria. Lithium bromide, 0.4 M, is used to aid release, and dithiothreitol is required to stabilize the enzyme. The membranous material is removed by centrifugation, and the apoenzyme is recovered in the supernatant and precipitated with ammonium sulfate to 50 percent of saturation. The main purification (100-fold) is achieved by selective adsorption and elution on controlled pore glass beads. The purified enzyme has been purified approximately 250-fold from the mitochondria. The purified enzyme is homogeneous as shown by poly-acrylamide gel electrophoresis in sodium dodecyl sulfate or acid-urea systems; a sharp band is obtained which is equivalent to a subunit molecular weight of 31,500. The apoenzyme is devoid of lipid and is completely inactive as isolated. It can be reactivated by adding aqueous microdispersions of lecithin or phospholipids containing lecithin. The apoenzyme is stable, i.e. it has a half-life of about 450 hours at 0-2 degrees in 0.4 M lithium bromide, containing 5 mM dithiothreitol at pH 7, and is soluble at these conditions, existing mainly as a monomer and dimer in dilute solution. It has a tendency to associate into larger aggregates when the salt concentration is lowered. The enzyme does not have a distinctive amino acid composition as compared with other proteins or soluble dehydrogenases. The purified apodehydrogenase is well suited for study of specific protein-lipid interaction, as well as the molecular basis for the role of phospholipid in this lipid-requiring enzyme.

Amino Acids↗
PubMed1975

Interaction of D-beta-hydroxybutyrate apodehydrogenase with phospholipids.

The interaction of a soluble homogeneous preparation of D-beta-hydroxybutyrate apodehydrogenase with phospholipid was studied in terms of restoration of enzymic activity and complex formation. The purified apoenzyme, which is devoid of lipid, is inactive. It is reactivated specifically by the addition of lecithin or mixtures of phospholipids containing lecithin. Mitochondrial phospholipid, i.e. the mixture of phospholipids in mitochondria, reactivates with the highest specific activity (approximately 100 micromol of DPN reduced/min/mg at 37 degrees and with the greatest efficiency (2.5 to 4 mol of lecithin/mol of enzyme subunit). Each of the lecithins of varying chain length and unsaturation reactivated the enzyme, albeit to differing extents and efficiencies. In general, lecithins containing unsaturated fatty acid moieties reactivated better than those containing the comparable saturated lipid. Optimal reactivation can be obtained for the various lecithins when they are microdispersed together with phosphatidylethanolamine. When the lecithins are added microdispersed together with both phosphatidylethanolamine and cardiolipin, maximal efficiency is obtained. Also, PC6:0 and 8:0 reactivate as soluble molecules, so that a phospholipid bilayer is not necessary to reactivate the enzyme. Complex formation was studied using gel exclusion chromatography. It can be shown that each of the phospholipids which reactivate combines with the apoenzyme. Mitochondrial phospholipid, which reactivates the best, binds most effectively; PC8:0, which reactivates with poor efficiency, can be shown to bind with low affinity, and negligible binding occurs at concentrations which do not reactivate the enzyme. Since the apoenzyme is apparently homogeneous and devoid of phospholipid or detergents, it would appear that reactivation does not involve reversal of inhibition such as by removal of a regulatory subunit or detergent from the catalytic subunit. Rather, we conclude that phospholipid is a necessary and integral portion of this enzyme whose active form is a phospholipid-protein complex. The apoenzyme also forms a complex with phosphatidylethanolamine and/or cardiolipin, which do not reactivate enzymic activity. Salt dissociates such complexes in contrast with the lecithin-apoenzyme complex. Binding of phospholipid is a necessary but not sufficient requisite for enzymic activity. The same energies of activation are obtained from Arrhenius plots for the membrane-bound enzyme and for the purified soluble enzyme reactivated with mitochondrial phospholipid or different lecithins. This observation is compatible with the view that the purified enzyme has not been adversely modified in the isolation. Furthermore, essentially the same energies of activation were obtained for saturated lecithins below their transition temperatures and for unsaturated lecithins above their transition temperatures. Hence, there is no indication that a lipid phase transition occurs to influence the activity of this enzyme.

Animals↗
PubMed1975

Lipid composition of the Golgi apparatus of rat kidney and liver in comparison with other subcellular organelles.

Golgi apparatus isolated from both rat liver and rat kidney have been characterized with respect to their neutral and phospholipid content and their phosphopipid composition and compared with mitochondria, rough endoplasmic reticulum and plasma membranes. In addition, the distribution of sulfatide in the subcellular fractions of rat kidney was determinich are rich in cholesterol esters and ubiquinone. Removal of about 75% of the cisternal contents of rat liver Golgi reduced its content of cholesterol esters but not of ubiquinone. The Golgi complex of liver most closely resembles endoplasmic reticulum in its phospholipid composition except for a higher content of sphingomyelin. Removal of most of the contents of the Golgi cisternae did not appreciably alter the phospholipid composition of the Golgi apparatus of liver. Goligi apparatus from kidney has a phospholipid composition which resembles liver Golgi much more closely than it does any other cell fraction from kidney. The sulfatide content of kidney Golgi, the cell fraction richest in this glycolipid, is about 14% of the total lipid present in this fraction. Sulfatide was present in plasma membranes, mitochondria and rough microsomes, but at about one-third the level found in Golgi. Sulfatide is the main glycosphingolipid present in all the cell fractions from kidney which were studied.

Animals↗