The amphiphilic membrane glycoproteins of Semliki forest virus are attached to the lipid bilayer by their COOH-terminal ends

The membrane location of the Semliki Forest virus glycoproteins E1, E2 and E3 was studied by protease treatment of (1) virus particles and (2) rough micro somes from cells infected with SF virus2. Protease treatment of virus particles removes all but the membrane-associated segments of the glycoproteins. Analyses of protease-treated SF virus membranes in 15% to 22.5% gradient acrylamide gels demonstrate the presence of three distinct peptide species with apparent molecular weights of 9000, 6000 and 5500. The 9000 and the 5500 molecular weight peptides have been aligned to the COOH-terminal end of E2 and the 6000 molecular weight peptide to the COOH-terminal end of El. The mapping of the peptides was done in a “Dintzis”-type of experiment (Dintzis, 1961) where we labelled the proteins of the virus with a gradient of [³⁵S]methionine increasing towards their COOH-terminal end.Protease treatment of microsomes from cells infected with SF virus removes only those parts of the viral glycoproteins that are transversing the lipid bilayer. Analyses of such treated membranes in sodium dodecyl sulphate-containing gels show that a 3000 molecular weight piece is digested from the COOH-terminal end of p62, the cellular precursor of E2 and E3. The COOH-terminus of p62 is shown to be equivalent to that of E2. These results thus demonstrate that the two amphiphilic membrane proteins of SF virus, E1 and E2 (p62) are attached to the lipid bilayer by their COOH-terminal ends. The COOH-terminal end of p62 (E2) spans the microsomal membrane. The third membrane protein, E3, probably does not interact with membrane lipids but is bound to the virus on E1 and (or) E2.     

Desmutagenic substance in water extract of grass-wrack pondweed (Potamogeton oxyphylus Miquel)

It was found that the water extract of grass-wrack pondweed (Potamogeton oxyphylus Miquel) had inhibitory activity for reverse mutations induced by benzo[a]pyrene, 2-aminoanthracene and 2-nitrofluorene with Salmonella typhimurium TA100 and TA98. The active substance was heat-resistant, and the molecular weight was above 300 000. The substance acted as a desmutagen rather than an antimutagen or inhibitor of metabolic activation.     

Assembly pathway of newly synthesized LamB protein an outer membrane protein of Escherichia coli K-12

The assembly of newly induced LamB protein (phage lambda receptor) was investigated in an operon fusion strain of Escherichia coli, in which the lamB gene is expressed under lac promoter control. The induction kinetics both for total cellular and for cell surface-exposed LamB protein were studied by immunochemical detection methods, using two distinct antisera directed against detergent-solubilized LamB trimers and completely denatured LamB monomers, respectively. Anti-trimer antibodies recognized both monomers and trimers, whereas anti-monomer antibodies only reacted with monomers. Provided appropriate solubilization conditions were used, both antisera were able to immunoprecipitate intracellular mature LamB protein quantitatively. Following induction, the first LamB antigenic determinants were detected after 60 to 80 seconds; detection of the newly synthesized protein by anti-monomer antibodies slightly preceded that by anti-trimer antibodies, a finding that could be partly explained by the observation that anti-monomer antibodies recognized a larger fraction of nascent LamB than did anti-trimer antibodies. Exposure of antigenic determinants at the cell surface was delayed for 30 to 50 seconds with respect to their synthesis. Therefore, either translocation or conformational changes must be rate-limiting in the series of processes that eventually convert the newly synthesized protein into its mature outer membrane state. LamB protein was found to occur in at least three clearly distinguishable states. State I is the LamB monomer, state II corresponds to a metastable trimer that dissociates in sodium dodecyl sulphate above 60 degrees C, and state III is the state LamB trimer that dissociates in sodium dodecyl sulphate only at temperatures above 90 degrees C. The chase kinetics of these states showed that conversion of newly synthesized LamB monomers to stable LamB trimers occurred in two stages: state I monomers were chased into metastable state II trimers rapidly (t 1/2 = 20 s), whereas stabilization of state II trimers to state III trimers was a relatively slow (t 1/2 = 5.7 min) process. Based on our results, a timing sequence in the assembly of outer membrane LamB protein is proposed.     

Structural and functional properties of chromatophores and membrane vesicles from Rhodepseudomonas sphaeroides

Membrane vesicles have been isolated by a modified procedure from Rhodopseudomonas sphaeroides, grown phototrophically under high light intensity. In addition,chromatophores have been isolated from this organism grown phototrophically with low light intensities.Structural, chemical and functional properties of both preparations have been investigated and compared. The orientation of the membrane preparations has been studied by freeze-etch electron microscopy, the localization of cytochrome c₂, and light-driven active transport of amino acids and Ca²⁺. The results demonstrate that the orientation of the vesicle membrane is the same as the cytoplasmic membrane of intact cells; the membranes in chromatophores, however, have an inverted orientation.On a dry weight basis, the membrane vesicles contain less protein, carotenoids and bacteriochlorophyll and more lipids than do chromatophores. Qualitatively, however, the composition of both preparations is similar.It is concluded that the intracytoplasmic structures from which the chromatophores are derived are structurally and functionally similar to (and most likely continuous with) the cytoplasmic membranes from which the vesicles are derived.     

Analysis of the pigment content of an antenna pigment-protein complex from three strains of Rhodopseudomonas sphaeroides

The pigment content of a B800–850 light-harvesting pigment-protein complex isolated from three different stains of Rhodopseudomonas sphaeroides has been determined. In each case the ratio of carotenoid to bacteriochlorophyll present is very nearly 1 : 3 an no specificity with regard to carotenoid type was observed.The fourth derivative of the infra-red absorption bands of the complex was determined and it is concluded that the minimal functional unit of B800–850 complex consists of 1 carotenoid molecule and three bacteriochlorophyll molecules. The data presented here, together with the previous study of Austin, (Austin, L.A. (1976) Ph.D. Thesis, University of California at Berkeley, Lawrence Berkeley Laboratory Report No. LBL 5512) suggest that the 800 nm absorption band represents one of these bacteriochlorophyll molecules while the remaining two bacteriochlorophylls are responsible for the 850 nm band.The absorption spectra and circular dichroism spectra of the complexes suggests that their structure has not been greatly altered during the purification.     

Biochemical and ultrastructural characterization of a high molecular weight soluble Mg2+ -ATPase from human erythrocytes

The isolation and purification of a 600,000 Mr cytosolic Mg2+ -ATPase from human erythrocytes is described. The electrophoretic properties of the native and sodium dodecyl sulphate-dissociated protein are presented and compared with those of the erythrocyte protein cylindrin . The Mg2+-ATPase has a single subunit of Mr 100,000 and it has an isoelectric point of 4.9. From transmission electron microscopy of negatively stained specimens, it is proposed that the Mg2+-ATPase is hexameric, containing two superimposed trimers of the 100,000 Mr subunit, which gives rise to a 13 nm pseudohexagonal particle with a central 3 nm cavity. Varying the orientation of the protein in the negative stain also produces images that are not hexagonal. When orientated on-edge, the protein produces a double-disc image, which is most clearly defined under acidic negative staining conditions with uranyl acetate, when some aggregation of the protein is produced. The ultrastructure of the Mg2+-ATPase is shown to be distinctly different from that of cylindrin . A comparative discussion of the negatively stained transmission electron microscopical images of the Mg2+-ATPase, mitochondrial F1-ATPase and several other oligomeric proteins and enzymes is presented.     

Membrane ATPase of Bacillus subtilis. I. Purification and properties

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step.The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s_20,w of 13,4 and an ámino acid composition very similar to bacterial ATPases already studied.After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (α) and 57 000 (β) with different charges.Kinetic studies showed that Ca²⁺ or Zn²⁺ are required for ATPase activity, although Mg²⁺ was uneffective. At optimal Ca²⁺ concentration, the Mg²⁺ has an inhibitory effect. The K_m for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na⁺ + K⁺)-ATPase are studied.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH₂:cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands.

2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c₁, band 5 the Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c₁ and identified with the so-called oxidation factor, and band 7 a polypeptide associated with cytochrome b.

3. The validity of molecular weight estimates for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively.

4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide bands.     

Subunit composition of the membrane glycoprotein complex of Semliki Forest virus

The quaternary structure of the membrane glycoproteins E1, E2 and E3 of Semliki Forest virus has been determined in intact virus and in the protein complexes obtained after Triton X100 solubilization. Intact and solubilized virus were treated with a cleavable cross-linking reagent and the covalently cross-linked glycoprotein complexes were isolated and characterized using antibodies specific for the E1 and E2 membrane glycoproteins. The isolation and characterization procedure was done in a low sodium dodecyl sulphate concentration which prevented non-covalent association between glycoprotein species, but did not abolish antigen-antibody binding.The major glycoprotein complex seen after cross-linking of either intact or Triton X100 solubilized virus was an approximately 100,000 molecular weight species composed of E1-E2 heterodimers only. These findings show that E1 and E2 form a complex in the virus and that this complex is retained after solubilization with Triton X100. The smallest membrane glycoprotein E3 was not cross-linked to the other proteins and was therefore lost in the isolation procedure. However, the presence of E3 together with E1 and E2 in complexes obtained after Triton X100 solubilization of intact virus suggests that an E1-E2-E3 trimer is present in the virus. It is likely that this trimer forms the spike-like structures seen on the surface of the virus.We have observed that antibody specific for one component of the virus glycoprotein complex can induce rearrangement of uncross-linked complexes in Triton X100 solubilized form. This fact should be considered when using specific antibody for characterization of protein complexes.     

Primary organization of the nucleosome core particles sequential arrangement of histones along DNA

A high-resolution map for the arrangement of histones along DNA in the nucleosome core particles has been determined by a new sequencing procedure. The lysine groups of histones were crosslinked to partly depurinated DNA at neutral pH. One strand of DNA was split at the points of crosslinking, thus leaving the 5′-terminal DNA fragments bound to histones. The lengths of these crosslinked DNA fragments were measured to determine the position of histones on one strand of the core DNA from its 5′ end.The results demonstrate that histones are bound to regularly arranged, discrete DNA segments about six nucleotides long. These segments are separated by histone-free gaps about four nucleotides wide located at a distance of about 10n nucleotides from the 5′ end of DNA. The first 20 nucleotides from the 5′ ends of DNA seem to be free of histones. Histones appear to be arranged symmetrically and in a similar way on both DNA strands. Any one histone, being bound predominantly to discrete segments on one or other of the strands, can oscillate at the same time between the two strands across the major DNA groove. Two symmetrical models for the arrangement of two molecules of each core histone on linearized and folded DNA are proposed.     

Production of both interstitial and basement membrane procollagens by fibroblastic WI-38 cells from human embryonic lung

The synthesis and secretion of type IV procollagen, in addition to that of procollagen types I and III, was detected in cells derived from human embryonic lung (WI-38) by immunofluorescence, metabolic labeling, immunoprecipitation, collagenase digestion and the characteristic polypeptide sizes of both intact procollagen type IV chains and their initial pepsin-resistant fragments as determined by polyacrylamide gel electrophoresis. Locally obtained human embryonic lung cells secreted the same procollagens, but neither embryonic nor adult human skin fibroblasts were found to secrete type IV procollagen in amounts detectable by the same methods.     

Electron spin resonance investigations of membrane proteins in erythrocytes in muscle diseases. Duchenne and myotonic muscular dystrophy and congenital myotonia

Comparison of electron spin resonance spectra of spin labeled erythrocyte membranes from patients with the dystrophic conditions Duchenne and myotonic muscular dystrophy with those of normal controls suggests that alterations in membrane protein conformation and/or organization are present in these disease states. These protein alterations are not apparent in the nondystrophic disease congenital myotonia. The results suggest a correlation between changes in the physical state of protein in membranes with the presence of dystrophy. In addition, the present results from erythrocytes lend support for the concept of a generalized membrane defect in these diseases.     

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A₂ cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B. (1976) Biochim. Biophys. Acta 436, 353–365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys. Acta 323, 178–193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of < 10⁶ dalton.Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents.It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.     

Nitrofurazone-induced DNA damage to tissues of mice

Cytotoxicity and DNA damage by nitrofurans has previously been correlated with metabolic reduction of these drugs in vitro. In the present study, nitrofurazone increased the rate of disappearance of stable [³H]thymidine labelled DNA from tissues of mice fed 0.1% nitrofurazone in the diet. Significant loss of labelled DNA occurred within 25 days after the start of the diet in all tissue observed, and loss was in relation to the rate of metabolic reduction of nitrofurazone. A similar correlation was found when another endpoint for DNA damage was used; nitrofurazone reduced by mouse tissue slices caused DNA single-strand breaks in cultured mouse L cells incubated in vitro with the tissues. Again, the ability of each tissue to produce toxic nitrofurazone metabolites determined the amount of DNA damage to the L cells.