Purification and functional reconstitution of the voltage-sensitive sodium channel from rabbit T-tubular membranes

The voltage-sensitive sodium channel has been purified from rabbit T-tubular membranes and reconstituted into defined phospholipid vesicles. Membranes enriched in T-tubular elements (specific [3H]nitrendipine binding = 41 +/- 9 pmol/mg of protein, n = 7) were isolated from fast skeletal muscle. After solubilization with Nonidet P-40, the sodium channel protein was purified to greater than 95% of theoretical homogeneity based on the specific activity of [3H]saxitoxin binding. Two subunits of Mr approximately 260,000 and 38,000 were found; these bands co-distributed with the peak of [3H]saxitoxin binding on sucrose gradients. The purified protein was reconstituted into egg phosphatidylcholine vesicles and retained the ability to gate specific 22Na+ influx in response to activation by batrachotoxin or veratridine. All activated fluxes were blocked by saxitoxin and tetrodotoxin. On sucrose gradients, the distribution of protein capable of functional channel activity paralleled the distribution of specific [3H]saxitoxin binding and of the Mr 260,000 and 38,000 components. The cation selectivity for the reconstituted, batrachotoxin-activated channel was Na+ greater than K+ greater than Rb+ greater than Cs+, with flux ratios of 1:0.13:0.02:0.008. Nine of 25 monoclonal antibodies raised against the rat sarcolemmal sodium channel cross-reacted with the rabbit T-tubular sodium channel in a solid-phase radioimmunoassay. Six of these antibodies showed specific binding to immunoblot transfers of T-tubular membrane proteins. Each labeled a single band at Mr approximately 260,000 corresponding in mobility to the large subunit of the sodium channel.     

Degradation of transplanted rat liver mitochondrial-outer-membrane proteins in hepatoma cells.

Reductively [3H]methylated 3H mitochondrial-outer-membrane vesicles from rat liver and vesicles where monoamine oxidase has been derivatized irreversibly by [3H]-pargyline have been deliberately miscompartmentalized by heterologous transplantation into hepatoma (HTC) cells by poly(ethylene glycol)-mediated vesicle-cell fusion. Fluorescein-conjugated mitochondrial-outer-membrane vesicles have also been used to show that transplanted material is patched, capped and internalized. Reductively methylated outer-membrane proteins and monoamine oxidase are destroyed at the same rate (t1/2 24 h). Mitochondrial-outer-membrane proteins are not degraded at the same rate as HTC plasma-membrane proteins, endogenous cell protein, or endocytosed protein. Transplanted radiolabelled mitochondrial-outer-membrane proteins accumulate intracellularly in structures that are distinct from plasma membrane and lysosomes. However, when mitochondrial-outer-membrane vesicles derivatized with [14C]sucrose are transplanted, the acid-soluble degradation products accumulate in the lysosomal fraction. [14C]Sucrose-conjugated HTC cell plasma membrane accumulates in intracellular structures that are again distinct from plasma membrane and lysosomes. In contrast with the above observations, homologously transplanted mitochondrial-outer-membrane proteins from rat liver are destroyed in hepatocytes at rates that are remarkably similar (t1/2 60-70 h) to the rates in rat liver in vivo [Evans & Mayer (1982) Biochem. Biophys. Res. Commun. 107, 51-58].     

Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on the membranes.

The outer and inner cytoplasmic membranes of Pseudomonas aeruginosa were separated as small and large membranes, respectively, from the cell envelope of this organism treated with lysozyme in Tris-chloride buffer containing sucrose and MgCl2 by differential centrifugation. The small membrane fraction contained predominantly 2-keto-3-deoxyoctonate (KDO), and little cytochromes or oxidase activities. The small membrane was composed of only 9 polypeptides and showed homogeneous small vesicles electron-microscopically. On the other hand, the large membrane fraction had high cytochrome contents and oxidase activities, and little KDO. The large membrane was composed of a number of polypeptides and showed large fragments or vesicles electron-microscopically. These results indicate that the small and large membranes are the outer and inner cytoplasmic membranes of P. aeruginosa, respectively. The isolated outer membrane showed a symmetrical protein peak with a density of 1.23 on sucrose density gradient centrifugation and the isolated inner membrane showed an unusually high density, probably due to association with ribosomes and extrinsic or loosely bound proteins. EDTA lowered the density of both membranes and caused lethal damage to the outer membrane, causing disintegration with the release of lipopolysaccharide (LPS), proteins and phospholipid.     

Oligomeric C-terminal truncated Bax preferentially releases cytochrome c but not adenylate kinase from mitochondria, outer membrane vesicles and proteoliposomes

The mechanism by which the proapoptotic protein Bax releases cytochrome c from mitochondria is not fully understood. The present work approaches this problem using C-terminal truncated oligomeric Bax (BaxDeltaC). Micromolar concentrations of BaxDeltaC released cytochrome c from isolated rat heart and liver mitochondria, while the release of adenylate kinase was not significantly affected. BaxDeltaC also released cytochrome c but not adenylate kinase from outer membrane vesicles filled with these proteins. However, BaxDeltaC was ineffective in releasing cytochrome c when outer membrane vesicles were obtained in the presence of glycerol, conditions under which the number of contact sites was drastically reduced. BaxDeltaC did not liberate encapsulated cytochrome c and adenylate kinase from pure phospholipid vesicles or vesicles reconstituted with porin. However, when the hexokinase-porin-adenine nucleotide translocase complex from brain mitochondria was reconstituted in vesicles, BaxDeltaC released internal cytochrome c but not adenylate kinase. In all these systems, only a small portion of total cytochrome c present in either mitochondria or vesicles could be liberated by BaxDeltaC. BaxDeltaC also increased the accessibility of external cytochrome c to either oxidation by complex IV or reduction by complex III in intact liver and heart mitochondria. CONCLUSIONS: (1) BaxDeltaC selectively releases cytochrome c and enables a bidirectional movement of cytochrome c across the outer mitochondrial membrane. (2) A multiprotein complex that resembles the mitochondrial contact sites is a prerequisite for BaxDeltaC action. (3) A limited pool of cytochrome c becomes the first target for BaxDeltaC.     

THE SUBMITOCHONDRIAL LOCALIZATION OF MONOAMINE OXIDASE : An Enzymatic Marker for the Outer Membrane of Rat Liver Mitochondria

Controlled osmotic lysis (water-washing) of rat liver mitochondria results in a mixed population of small vesicles derived mainly from the outer mitochondrial membrane and of larger bodies containing a few cristae derived from the inner membrane. These elements have been separated on Ficoll and sucrose gradients. The small vesicles were rich in monoamine oxidase, and the large bodies were rich in cytochrome oxidase. Separation of the inner and outer membranes has also been accomplished by treating mitochondria with digitonin in an isotonic medium and fractionating the treated mitochondria by differential centrifugation. Treatment with low digitonin concentrations released monoamine oxidase activity from low speed mitochondrial pellets, and this release of enzymatic activity was correlated with the loss of the outer membrane as seen in the electron microscope. The low speed mitochondrial pellet contained most of the cytochrome oxidase and malate dehydrogenase activities of the intact mitochondria, while the monoamine oxidase activity could be recovered in the form of small vesicles by high speed centrifugation of the low speed supernatant. The results indicate that monoamine oxidase is found only in the outer mitochondrial membrane and that cytochrome oxidase is found only in the inner membrane. Digitonin treatment released more monoamine oxidase than cytochrome oxidase from sonic particles, thus indicating that digitonin preferentially degrades the outer mitochondrial membrane.     

Multiple forms of the glucocorticoid receptor steroid binding protein identified by affinity labeling and high-resolution two-dimensional electrophoresis

Potential charge heterogeneity within the glucocorticoid binding protein (GBP) of the glucocorticoid receptor was examined by a combination of affinity labeling, immunopurification, and high-resolution two-dimensional (2D) gel electrophoresis. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of [3H]dexamethasone 21-mesylate ([3H]DM) labeled cytosol identified a major, competable, component of Mr approximately equal to 92 000 (92K). This component was recognized by anti-human glucocorticoid receptor antibodies but not by nonimmune serum, indicating that the 92K component was the reduced denatured GBP. Examination of [3H]DM-labeled GBP by conventional 2D electrophoresis utilizing equilibrium isoelectric focusing in the first dimension failed to resolve the 92K GBP into discrete isoelectric components. This behavior was not representative of other, nonspecifically [3H]DM-labeled proteins or proteins in general. Nonequilibrium pH gradient electrophoresis (NEPHGE) was therefore employed to achieve separation in the first dimension. Immunopurified, [3H]DM-labeled GBP subjected to NEPHGE reached isoelectric equilibrium after 6 h of electrophoresis at 400 V. A single, broad peak of radioactivity was identified at pH approximately equal to 6.3. Second-dimension analysis of the NEPHGE-separated GBP by SDS-PAGE resolved this peak into two discrete, 92K, isoforms of apparent pI = 5.7 and 6.0-6.5. The GBP charge heterogeneity was confirmed by NEPHGE 2D analysis of [3H]DM-labeled GBP prepared directly from crude cytosol. Two isoforms indistinguishable from those observed in immunopurified samples were identified. An additional, more acidic, isoform (apparent pI approximately equal to 5.2) was also identified. Thus, there are at least two, and perhaps three, isoforms of the GBP. These data therefore suggest that there is significant charge heterogeneity in the GBP of the glucocorticoid receptor.     

Isolation and characterization of transposon Tn5-induced mutants of Pseudomonas perfectomarina defective in nitrous oxide respiration.

Outer membranes of Haemophilus influenzae type b were fractionated to yield Triton X-100-insoluble material and lipopolysaccharide and phospholipids. Liposomes reconstituted from lipopolysaccharide and phospholipids were impermeable to sucrose (Mr, 342) and to a high-molecular-weight dextran (average Mr, 6,600). When the Triton X-100-insoluble material was introduced into the reconstituted liposomes, the vesicles became permeable to sucrose, raffinose (Mr, 504), and stachyose (Mr, 666) and fully retained dextrans of Mr greater than 1,500. Inulin (average Mr, 1,400) was tested for its efflux from the reconstituted outer membrane vesicles; 62% of the added inulin was trapped. The molecular weight exclusion limit for the outer membrane of H. influenzae type b was therefore estimated at approximately 1,400. A protein responsible for the transmembrane diffusion of solutes was purified from H. influenzae type b by extraction of whole cells with cetyl trimethyl ammonium bromide. When this extract was passed over DEAE-Sepharose, three protein-containing peaks (I, II, and III) were eluted. Peaks I and II contained mixtures of proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; when tested for their pore-forming properties, these proteins were unable to render liposomes of lipopolysaccharide and phospholipid permeable to sucrose. Peak III contained only one molecular species of protein of molecular weight 40,000; this protein acted as a porin in reconstituted vesicles. The molecular weight exclusion limit for 40,000-molecular-weight protein matched the estimate of approximately 1,400 which was determined for outer membranes. A series of homologous saccharides of increasing degree of polymerization was prepared from agarose by hydrolysis with beta-agarase and fractionation on gel filtration chromatography. These oligosaccharides of Mr, 936, 1,242, 1,548, and 1,854 were assayed for retention by the complete vesicles containing 40-kilodalton protein and lipopolysaccharide and phospholipids. All of these oligosaccharides were lost by efflux through the porin. Since the molecular conformation of the largest oligosaccharide is an elongated semirigid helix, it is suggested that the pore formed by the 40-kilodalton protein does not act as a barrier to the diffusion of this compound.     

Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase.

When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed.     

Rat liver canalicular membrane vesicles. Isolation and topological characterization

Canalicular plasma membranes were isolated from rat liver homogenates using nitrogen cavitation and calcium precipitation methods. Compared with homogenates, the membranes were enriched 55- to 56-fold in gamma-glutamyltransferase, aminopeptidase M, and alkaline phosphatase activities and showed very low enrichment in markers of other membranes. By electron microscopy, the membrane preparation contained neither junctional complexes nor contaminating organelles and consisted exclusively of vesicles. The presence of vesicles was also evident from the osmotic sensitivity of D-[6-3H]glucose uptake into the membrane preparation. Antisera obtained from rabbits immunized with highly purified rat kidney gamma-glutamyltransferase inhibited the transferase activity of intact or Triton X-100-solubilized membranes by 45-55%. Treatment of vesicles with anti-gamma-glutamyltransferase antisera and anti-rabbit IgG antisera increased the apparent density of the membranes during sucrose density gradient centrifugation. gamma-Glutamyltransferase and aminopeptidase M activities were selectively removed from the vesicles by limited proteolysis with papain without changing the intravesicular space or alkaline phosphatase activity of the membranes. Specific binding of anti-gamma-glutamyltransferase antibody to the outer surface of isolated hepatocytes was observed as measured by the antisera and 125I-labeled protein A; binding followed saturation kinetics with respect to antibody concentration. These data indicate that the isolated canalicular membrane vesicles are exclusively oriented right-side-out and that gamma-glutamyltransferase and aminopeptidase M are located on the luminal side of rat liver canalicular plasma membranes.     

Relative rates of turnover of subunits of mitochondrial proteins.

1. The double-isotope concept [Arias, Doyle & Schimke (1969) J. Biol. Chem. 244, 3303--3315] for the measurement of protein turnover was used to estimate the turnover rates of protein subunits from rat liver submitochondrial fractions resolved by means of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. NaH14CO3 and [5-3H]arginine were used as first and second precursors respectively. 2. Marked heterogeneity of protein subunit turnover rates is seen for protein subunits from water-soluble, salt-soluble and Tween 20-soluble mitochondrial proteins. 3. Much lower heterogeneity is seen in the turnover of protein subunits in Triton X-100-soluble material not binding to DEAE-cellulose at low ionic strength. The relative rates of turnover of proteins in this fraction are lower than for proteins in any other submitochondrial fraction. This fraction contains the integral membrane proteins. 4. Incorporation of [3H]arginine into subunits of the cytochrome oxidase complex is greatest for subunits with molecular weights in excess of 20000. 5. No correlation is seen between protein subunit size and the rate of turnover of the protein subunits in any of the submitochondrial fractions.     

Purification of a hexokinase-binding protein from the outer mitochondrial membrane.

Brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) binds selectively to the outer membrane of rat liver mitochondria but not to inner mitochondrial or microsomal membranes nor to the plasma membrane of human erythrocytes. A protein having subunit molecular weight of 31,000, determined by sodium dodecyl sulfate-gel electrophoresis, has been highly purified from the outer mitochondrial membrane by repetitive solubilization with octyl-beta-D-glucopyranoside followed by reconstitution into membranous vesicles when the detergent is removed by dialysis. When incorporated into lipid vesicles, the protein confers the ability to bind brain hexokinase in a Glc-6-P-sensitive manner as is seen with the intact outer mitochondrial membrane. Hexokinase binding ability and the 31,000 subunit molecular weight protein co-sediment during sucrose density gradient centrifugation. Both hexokinase binding ability and the 31,000 subunit molecular weight protein are resistant to protease treatment of the intact outer mitochondrial membrane while other membrane proteins are extensively degraded. It is concluded that this protein, designated the hexokinase-binding protein (HBP), is an integral membrane protein responsible for the selective binding of hexokinase by the outer mitochondrial membrane.     

Partial Purification and Characterization of the Vacuolar H+-ATPase of Mammalian Synaptic Vesicles

Several major proteins of synaptic vesicles from rat or cow brain sediment as a large complex on sucrose density gradients when solubilized in nonionic detergents. A vacuolar H(+)-ATPase identified by sensitivity to bafilomycin A1 appears to be associated with this oligomeric protein complex. Two subunits of this complex, synaptic vesicle proteins S and U, correspond to the 57-kDa (B) and 39-kDa accessory (Ac39) subunits, respectively, of bovine chromaffin granule vacuolar H(+)-ATPase as shown by Western immunoblot analysis. The five subunits of the oligomeric complex constitute approximately 20% of the total protein of rat brain synaptic vesicles. Taken together, these results strongly suggest that the abundant, multisubunit complex partially purified from brain synaptic vesicles by density gradient centrifugation is a vacuolar H(+)-ATPase. Bafilomycin A1 completely blocks proton pumping in rat brain synaptic vesicles as measured by [14C]methylamine uptake and also blocks catecholamine accumulation measured by [3H]dopamine uptake. Moreover, ATPase activity, [14C]methylamine uptake, and [3H]dopamine uptake are inhibited by bafilomycin A1 at similar I50 values of approximately 1.7 nmol/mg of protein. These findings indicate that the vacuolar H(+)-ATPase is essential for proton pumping as well as catecholamine uptake by mammalian synaptic vesicles.     

Two apparent molecular weight forms of human and monkey phenylalanine hydroxylase are due to phosphorylation

Two-dimensional polyacrylamide gel analyses of purified human and monkey liver phenylalanine hydroxylase reveal that the enzyme consists of two different apparent molecular weight forms of polypeptide, designated H (Mr = 50,000) and L (Mr = 49,000), each containing three isoelectric forms. The two apparent molecular weight forms, H and L, represent the phosphorylated and dephosphorylated forms of phenylalanine hydroxylase, respectively. After incubation of purified human and monkey liver enzyme with purified cAMP-dependent protein kinase and [gamma-32P]ATP, only the H forms contained 32P. Treatment with alkaline phosphatase converted the phenylalanine hydroxylase H forms to the L forms. The L forms but not the H forms could be phosphorylated on nitrocellulose paper after electrophoretic transfer from two-dimensional gels. Phosphorylation and dephosphorylation of human liver phenylalanine hydroxylase is not accompanied by significant changes in tetrahydrobiopterin-dependent enzyme activity. Peptide mapping and acid hydrolysis confirm that the apparent molecular weight heterogeneity (and charge shift to a more acidic pI) in human and monkey liver enzyme results from phosphorylation of a single serine residue. However, phosphorylation by the catalytic subunit of cAMP-dependent protein kinase does not account for the multiple charge heterogeneity of human and monkey liver phenylalanine hydroxylase.     

Protein degradation in rat liver during post-natal development.

Protein degradation rates for liver subcellular and submitochondrial fractions from neonatal (8-day), weanling (25-day) and adult rats were estimated by the double-isotope method with NaH14CO3 and [3H] arginine as the radiolabelled precursors [Dice, Walker, Byrne & Cardiel (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2093-2097]. Decreased protein degradation rates were found during post-natal development for homogenate, nuclear, mitochondrial, lysosomal and microsomal proteins. A decrease in degradation rates for the immunoisolated subunits of monoamine oxidase and pyruvate dehydrogenase was also observed in neonatal and weanling rats respectively. The results suggest coordinate degradation of the subunits of the multi-subunit enzyme pyruvate dehydrogenase. Pyruvate dehydrogenase has a faster rate of degradation in adult rat liver than does cytochrome oxidase. Data analysis suggests heterogeneity of protein degradation rates in the mitochondrial outer membrane and intermembrane space fractions at each developmental stage but not in the mitochondrial inner membrane or matrix fractions. Results obtained for protein degradation rates in adult rat liver by the method of Burgess, Walker & Mayer [(1978) Biochem. J. 176, 919-926] in general confirmed the results obtained for the adult rat liver by the above method. No evidence of a subunit-size relationship for protein degradation was found for proteins in any subcellular or submitochondrial fraction.     

Two-dimensional gel electrophoretic resolution of the polypeptides of rat liver mitochondria and the outer membrane

The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.     

Soluble and Particulate Forms of Rat Catechol-O-Methyltransferase Distinguished by Gel Electrophoresis and Immune Fixation

Catechol-O-methyltransferase (COMT) was visualized in homogenates and subcellular fractions of rat tissues, including liver and brain, by gel electrophoresis, electrophoretic transfer of proteins to nitrocellulose (Western blotting), and immune fixation with antiserum to highly purified soluble rat liver COMT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of all tissue homogenates examined revealed three major immune-specific proteins with apparent molecular weights 23,000, 26,000, and 66,000 (23K, 26K and 66K). Centrifugation of homogenates at 100,000 X g for 60 min resulted in the enrichment of the 26K species protein in the pellet whereas the 23K and 66K proteins were the predominant forms in the supernatant. The 66K protein appeared in variable amounts depending on the tissue being examined and the length of transfer of protein and is assumed to be an "aggregate" of the smaller form(s). The 26K protein was essentially the only immunoreactive species seen in a purified preparation of rat liver outer mitochondrial membrane. Isoelectric focusing (IEF) under denaturing conditions and two-dimensional gel electrophoresis of brain and liver fractions showed that the 23K protein was resolved into three bands of pI 5.1, 5.2, and 5.3, whereas the 26K protein had a pI of 6.2. Analysis of COMT activity in slices from nondenaturing IEF gels indicated that the pI 5.1-5.3 species are biologically active; the pI 6.2 species could not be detected under these conditions. COMT activity was demonstrated, however, in outer mitochondrial membranes from rat liver, which contain predominantly the 26K, pI 6.2 immunoreactive species. The major form of COMT in all rat tissues examined is "soluble" with an apparent Mr of 23K and a pI of 5.2. The nature of the modifications giving rise to pI 5.1 and 5.3 forms of this enzyme are not clear, nor is the relationship between the 23K and 26K forms. Further studies are needed to elucidate the relationship of immunoreactive forms of COMT to each other, their intracellular location, and their functional significance.     

Isolation and reconstitution of an N-ethylmaleimide-sensitive phosphate transport protein from rat liver mitochondria

An N-ethylmaleimide-sensitive phosphate transport protein has been isolated from rat liver mitochondria, substantially purified, and reconstituted into phospholipid vesicles. Purified inner mitochondrial membrane vesicles depleted of F1-ATPase by urea treatment proved to be the most satisfactory starting material. Treatment of these membrane vesicles with Triton X-100 resulted in solubilization of the phosphate transport protein. Further purification was achieved using hydroxylapatite powder. Polyacrylamide gel electrophoresis of the purified fraction in sodium dodecyl sulfate indicated the presence of two Coomassie blue-staining bands with apparent Mr's of 30,000 and 35,000. Labeling of the 35,000 Mr band by the Pi transport inhibitor diazobenzene sulfonate was reduced markedly by prior treatment of the mitochondria with the inhibitor N-ethylmaleimide. The purified fraction containing both proteins could be reconstituted into liposomes prepared from purified asolectin. Phosphate efflux from these vesicles was inhibited by N-ethylmaleimide, by the impermeant mercurial agent, p-chloromercuribenzoate, and by diazobenzene sulfonate. Treatment of the purified fraction with N-ethylmaleimide prior to incorporation into liposomes resulted in a reconstituted system incapable of catalyzing Pi efflux. These studies summarize the first detailed attempt to purify the Pi/H+ transport system from rat liver mitochondria and emphasize the need to commence the purification with purified inner membrane vesicles depleted of F1-ATPase. In addition, these studies show that the final fraction contains a reconstitutively active transport system which when incorporated into phospholipid vesicles has its essential sulfhydryl groups oriented outward. Finally, it is shown that the purified fraction also contains a 30,000 Mr component.     

The glucose transport system of muscle plasma membranes: characterization by means of [3H]cytochalasin B binding

A membrane-rich preparation was isolated from adult rat skeletal muscle in low salt media and further fractionated in sucrose gradients. Fraction F2, with a relative density of 1.092-1.119, consisted of sealed membrane vesicles which were enriched in plasma membrane markers. These vesicles were capable of stereospecific D-glucose uptake which was sensitive to cytochalasin B (CB). The membranes were also enriched in high affinity [3H]CB binding activity (Kd of 0.28 microM). [3H]CB binding to the glucose carrier of these plasma membranes, estimated as the fraction of binding protectable by D-glucose, ranged between 2.5 and 7.4 pmol/mg protein in several membrane preparations. The amount of [3H]CB binding to muscle membranes from newborn and adult rats was not markedly different. Trypsin, at low concentrations, altered the molecular weight of several membrane components, without affecting [3H]CB binding. Higher concentrations of trypsin abolished [3H]CB binding. Both 2,4-dinitrofluorobenzene (0.1 mM) and N-ethylmaleimide (15 mM) inhibited [3H]CB binding; inhibition by these reagents was prevented by inclusion of micromolar concentrations of CB in the reaction mixture. Several procedures that extracted specific proteins enriched the D-glucose-sensitive [3H]CB binding to the protein-depleted membranes. Antibody raised against the glucose carrier of human red cell membranes cross-reacted with a polypeptide of Mr about 45K of muscle membranes which might represent the glucose carrier.     

Identification and preliminary characterization of Vibrio cholerae outer membrane proteins.

Outer membrane proteins of Vibrio cholerae were purified by sucrose density centrifugation and Triton X-100 extraction at 10 mM Mg2+. The proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. V. cholerae outer membrane proteins presented a unique pattern when compared with the patterns of other gram-negative rods. There were 8 to 10 major bands (Mr 94,000 to 27,000), with most of the protein located in band 5 (Mr approximately 45,000), which thus appears to be the major structural protein of the outer membrane. Lipid and carbohydrate were associated with band 6.     

Isolation of hepatic mitochondrial contact sites: previously unrecognized inner membrane components

An improved, fast, and relatively simple procedure for isolation of hepatic mitochondrial contact sites is described. These contact sites include conventional outer membrane, but the inner membrane component (which we term fusion patches) has a unique biochemical composition characterized by a clustering of three specific inner membrane proteins of 54, 52, and 31 kDa identified by proteomics, respectively, as the alpha and beta subunits of ATP synthase and the liver isoform of adenine nucleotide transferase. The contact site fraction was prepared using a discontinuous sucrose gradient from crude outer membranes derived from swollen/shrunk rat liver mitochondria. The resultant contact sites were analyzed using a continuous sucrose density gradient, revealing an apparent heterogeneity due to varying amounts of retained fusion patches in relation to the unvarying outer membrane component. By electron microscopy, contact sites consist of small vacuoles that contain one or several tiny vesicles, many of which are composed of multiple, closely packed lamellae. The contact site subfraction morphology is consistent with the biochemical variation. Thus, contact sites are not haphazard fusions of outer and inner membrane, but consist in part of regions of inner membrane of novel composition (fusion patches) and of conventional outer membrane.