Purification and properties of the latent F1-ATPase of Micrococcus lysodeikticus

The latent coupling factor (F₁)-ATPase of Micrococcus lysodeikticus has been purified to homogeneity as determined by a number of criteria including, non-denaturing polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and analytical ultracentrifugation. By inclusion of 1 mM phenylmethyl sulfonyl fluoride, a serine protease inhibitor, in the shock-wash step of release of F₁ from the membranes, the spontaneous activation of both crude and purified ATPase by endogenous membrane protease(s) can be prevented, thereby yielding a highly latent ATPase preparation. Equilibrium ultracentrifugation of the latent ATPase gave a molecular weight of 400 000. The ATPase contained five different subunits α, β, γ, δ, and ? and their molecular weights determined by SDS-polyacrylamide gel electrophoresis were 60 000, 54 000, 37 000, 27 000 and 9000, respectively. The subunit composition was determined with ¹⁴C-labelled, F₁-ATPase prepared from cells grown on medium containing [U-¹⁴C]-labelled algal protein hydrolysate. Within the limitations of this method the results tentatively suggest a subunit composition of 3 : 3 : 1 : 1 : 3.     

Solubility characteristics of Micrococcus lysodeikticus membrane components in detergents and chaotropic salts analyzed by immunoelectrophoresis.

In order to evalute the effectiveness and selectivity of various reagents in the solubilization of bacterial membranes, membranes of Micrococcus lysodeikticus were treated with detergents and chaotropic agents. The composition of the extracts so obtained was analyzed by rocket and two-dimensional immunoelectrophoretic techniques. Recoveery of succinate-, malate-, and reduced nicotinamide adenine dinucleotide- (NADH) dehydrogenases, ATPase, succinylated lipomannan and cytochromes in the extracts was measured. Treatment with a variety of non-denaturing detergents produced extracts that were generally qualitatively uniform although quantitative differences were observed. The degree of extraction of various components was correlated with the hydrophile-lipophile balance. Several chaotropic agents were also evaluated as reagents for membrane solubilization. These agents were less effective in extraction of bulk protein, but produced extracts enriched in some membrane components.     

Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits

The F₁-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F₁s, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230–240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F₁-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F₁ are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual δ- and ?-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F₁; and (4) all subunit antibodies as well as anti-native F₁ were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F₁ and anti-α- and anti-β-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F₁-ATPases and in general may offer an additional method for the examination of multimeric proteins.     

Dissociation of yeast 80 S ribosomes by chaotropic salts

Exposure of yeast 80 S ribosomes to chaotropic salts such as NaClO₄ or NaSCN at concentrations as low as 0.4 M resulted in complete dissociation and subsequent aggregation of the ribosomal proteins. However, under similar conditions, both NaCl and NaBr did not cause dissociation and aggregation. The protein precipitate obtained by exposing the ribosomes to 0.5 M NaClO₄ was free of any rRNA contamination as judged by ultraviolet-absorption analysis. Comparison of the two-dimensional polyacrylamide gel electrophoretic analysis of the above ribosomal protein precipitate with that ribosomal proteins isolated by the standard acetic acid extraction procedure revealed that the protein precipitate contained all the ribosomal proteins. Based on these results, a simple method for the isolation of total ribosomal proteins and rRNA under mild, nondenaturing conditions is proposed. A possible mechanism for the dissociation of proteins from the ribosome by chaotropic salts is also discussed.     

Two detergent-insoluble proteins of the human lymphocyte membrane are enriched in an isolated membrane fraction

Human lymphocytes isolated from peripheral blood on Ficoll/Paque density gradients were surface-labelled by ¹²⁵I/lactoperoxidase or ³H/reductive alkylation and lysed in buffer solutions containing non-ionic or amphoteric detergents (octylphenylpolyoxyethylenes, octylglucoside, cholylamidopropyldimethylammoniopropane sulfonate) under a variety of conditions. The cell lysate was fractionated by sedimentation or by density gradient centrifugation. The large majority of the labelled proteins is solubilized by the detergents. Two proteins of 45 000 and 30 000 molecular weight are the main detergent-insoluble, surface-labelled components. They can be fractionated from detergent lysates of cells in relatively pure form from the other membrane proteins and from nuclear material on density gradients. The same two proteins are specifically enriched in a membrane fraction isolated from a detergent-free cell homogenate by density gradient centrifugation. Cytoskeletal and other intracellular proteins remain associated with these two proteins when fractionated by either of these two independent methods.     

F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein

It has been claimed (Andreu, J.M., Warth, R. and Muñoz, E. (1978) FEBS Lett. 86, 1–5) that the F₁-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F₁-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F₁ preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F₁-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F₁-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (, β, γ, δ and ε) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F₁-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F₁-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan.     

Analysis of Staphylococcus aureus cytoplasmic membrane proteins by isoelectric focusing

Cytoplasmic membranes were isolated from late-exponential phase Staphylococcus aureus 6538 P and the membrane proteins examined under non-denaturing conditions by thin-layer isoelectric focusing (TLIEF) in a pH 3.5–9.5 gradient. Isolated membrane preparations retained protein integrity as judged by the demostration of membrane bound adenosine triphosphatase (ATPase) activity in addition to four solubilzed membrane enzyme markers. Membranes were effectively solubilized with 2.5% Triton X-100 (final concentration). Examination of Triton X-100 solubilized membrane preparations established the presence of 22 membrane proteins with isoelectric points between 3.7 and 6.0. The focused proteins displayed the following enzymatic activities and isoelectric points by zymogram methods: ATPase (EC 3.6.1.3), 4.20; malate dehydrogenase (EC 1.1.1.37), 3.90; lactate dehydrogenase (EC 1.1.1.27), 3.85; two membrane proteins exhibited multiple bands upon enzymatic staining: NADH dehydrogenase (EC 1.6.99.3), 4.25, 4.35; succinate dehydrogenase (EC 1.3.99.1), 4.85, 5.10, 5.35.     

Molecular organization in bacterial cell membranes II. Reevaluation and identification of some chemical components of Micrococcus lysodeikticus membranes

Standard membranes of Micrococcus lysodeikticus were prepared by protoplastlysis in the presence of 50 mM Mg²⁺ and by repeated washings in the absence of this cation. Different washing procedures with EDTA-30 mM Tris (pH 7.5) and low-ionic-strength Tris buffers (pH 7.5) yielded three distinct depleted membranes. RNA and carbohydrates were reevaluated in all these membranes after extraction and/or partial fractionation of the membrane complexes. Values higher than 10% of membrane dry weight have been found for both components in the standard membranes. Figures between 6–12% for carbohydrates and 0.8–16% for RNA were found in the three depleted membranes. The protein: lipid ratio of depleted membranes is lower than that of the standard membrane.The existence of RNA has been confirmed by polyacrylamide gel electrophoresis and by sensitivity to ribonuclease (EC 2.7.7.16). Different patterns of proteins, RNAs and carbohydrate-containing material were revealed by gel electrophoresis in sodium dodecyl sulphate of each type of M. lysodeikticus membrane. These results confirm and extend previous findings obtained with a depleted membrane of M. lysodeikticus (Estrugo, S. F., Larraga, V., Corrales, M. A.; Duch, C. and Munõz, E. (1972) Biochim. Biophys. Acta 255, 960–973). They suggest the existence of specific interactions between membrane components which are broken down and/or altered by different membrane treatments. They also suggest the likely significance in bacterial membranes of components other than lipids and proteins.     

Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis

The solubilizing power of various nonionic and zwitterionic detergents as membrane protein solubilizers for two-dimensional electrophoresis was investigated. Human red blood cell ghosts and Arabidopsis thaliana leaf membrane proteins were used as model systems. Efficient detergents could be found in each class, i.e. with oligooxyethylene, sugar or sulfobetaine polar heads. Among the commercially available nonionic detergents, dodecyl maltoside and decaethylene glycol mono hexadecyl ether proved most efficient. They complement the more classical sulfobetaine detergents to widen the scope of useful detergents for the solubilization of membrane proteins in proteomics.     

Ethanol-induced changes in the membrane lipid composition of Clostridium thermocellum

When ethanol is added to the growth medium of Clostridium thermocellum ATCC 27405 and C9, a different membrane composition is observed after the period of growth arrest. Changes in fatty acid composition and some unsaturated, branched hydrocarbons have been monitored by GLC-MS. There is a marked increase in normal and anteiso-branched fatty acids at the expense of isobranched fatty acids and an increase in short and unsaturated fatty acids. Thus, an adaptive response to growth in the presence of ethanol induces a membrane containing fatty acids with lower melting points and produces a more ‘fluid’ membrane. The suggestion is made that these membrane changes may be maladaptive to the performance of C. thermocellum.     

A study on the renaturation of membrane proteins after solubilization in SDS or following polyacrylamide gel electrophoresis in SDS,with special reference to a phosphatase from acholeplasma laidlawii

It may be easier to renature SDS-denatured hydrophobic proteins than to renature SDS-denatured water-soluble proteins. This paper presents some support for this hypothesis in the form of literature reports and an experiment of our own with an intrinsic membrane protein (a phosphatase from Acholeplasma laidlawii), that could be completely renatured, to judge from the restored activity, which was equal to (or higher than) that of the untreated enzyme. If this hypothesis is correct it might be possible to devise general methods to reverse the SDS denaturation of hydrophobic membrane proteins. This would be a breakthrough in the purification of at least some membrane proteins, because the high-resolving polyacrylamide gel electrophoresis in SDS could then be used to prepare membrane proteins in a native state. The method used for the renaturation of the SDS-denatured, entirely inactive, phosphatase comprised removal of SDS with the aid of conventional dialysis against a buffer containing the neutral, very efficient and non ultraviolet light-absorbing detergent G3707. For renaturation of the enzyme following an SDS-electrophoresis in polyacrylamide the gel was immersed in the same buffer for several hours; by staining for phosphatase the enzyme could easily be localized in the gel in the form of a yellow band, coinciding with a protein zone.     

Chemical composition of the plasma membrane from epimastigote forms of Trypanosoma cruzi

The chemical composition of two plasma membrane fractions from epimastigote forms of Trypanosoma cruzi is reported. Fraction M, a preparation obtained by conventional methods of cell fractionation is composed of 31% proteins, 34% lipids, 16% carbohydrates and 3% of the lipopeptidophosphoglycan. Phospholipids and sterols account for 7.5 and 9%, respectively, of the total mass. Phosphatidylethanolamine is the major phospholipid in fraction M, representing 45% of the total membrane phospholipids. The other fraction, fraction V (vesicles), was obtained by treatment of the cell with a vesiculating agent. This fraction contains 42% lipids, 20% carbohydrates, 13% proteins and 21% of the lipopeptidophosphoglycan. Phospholipids and sterols make up 17 and 8%, respectively, of the total mass of this fraction. Phosphatidylcholine and phosphatidylethanolamine are the main phospholipids found in fraction V. Phosphonolipids and sialic acid have not been detected in either membrane fraction. Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis show that the glycoproteins ABC and the lipopeptidophosphoglycan are 50- and 10-times more concentrated, respectively, in fractions V and M than in the whole cell homogenate. The high molar sterol/phospholipid ratio found in fraction M suggests that this fraction is less fluid than fraction V, perhaps reflecting a migration of certain membrane components in the presence of the vesiculating agent. Hence, fraction M is, probably, more representative of the epimastigote plasma membrane as a whole than fraction V.     

An analysis of the repair processes in ultraviolet-irradiated Micrococcus luteus using purified ultraviolet-endonuclease

The measurement of the frequency of endonucleolytic incisions in ultraviolet-irradiated DNA serves as the test for the presence of pyrimidine dimers. In accordance with this approach, the lysates of three Micrococcus luteus strains containing radioactively labeled chromosomes were treated with purified M. luteus ultraviolet-endonuclease to trace segregation of dimers amongst parental and newly synthesized DNA and their removal during postreplication and excision DNA repair. A considerable proportion of the dimers in all strains tested proved to be insensitive to the action of exogenous incising enzyme. The use of chloramphenicol as an inhibitor of postirradiation protein synthesis in combination with ultraviolet-endonuclease treatment of DNA allowed to reveal at least two alternative pathways of postreplication repair: constitutively active recombinational pathway and inducible nonrecombinational one.     

Electrophoretic patterns of protein synthesis and turnover in apical plasma membrane and outer bilayer of Schistosoma mansoni

Polypeptide fractions labelled with [¹⁴C]leucine and associated with fractioned inner plasma membrane and outer bilayer (envelope) from the apical double bilayer complex of the surface epithelium of the human blood fluke, Schistosoma mansoni, were analyzed by two-dimensional electrophoresis and fluorography. In contrast to the distribution of alkaline phosphatase, the polypeptide profiles of the two bilayer fractions were similar due to cross contamination between one membrane containing larger amounts of protein (inner) and the second bilayer having more heavily labelled proteins (outer bilayer). Convincing evidence for only two of 35 polypeptides could be provided for localization to the outer bilayer. These results suggest that the marker enzyme used for the inner bilayer, alkaline phosphatase, may not be homogeneously distributed in this membrane. In pulse-chase studies a correction factor for cross-contamination was derived. The rate to turnover of the polypeptide fractions was twice as fast for the outer compared to the inner membrane, this difference being consistent with the view that multilamellar bodies are the precursors of the apical double bilayer complex. Comparing the rates of surface renewal in adult and juvenile schistosomes leads to the suggestion that membrane turnover can be correlated with susceptibility to host immune effector mechanisms.     

Orientation of reaction center and antenna chromophores in the photosynthetic membrane of rhodopseudomonas viridis

Whole cells of Rhodopseudomonas viridis were oriented in a magnetic field. The degree of orientation of the cells was determined by using a photoselection technique. In order to deduce the orientation of the antennae and chromophores of the reaction centers with respect to the membrane plane, we performed linear dichroism measurements of absolute spectra and light induced difference spectra linked to states P⁺I and PI^? on oriented cells. These measurements lead to the following conclusions:The antennae bacteriochlorophyll molecular plane is nearly perpendicular to the membrane. The Q_y and Q_x transitions moments of these molecules make respectively angles of 20 and 70°ith the membrane plane. The antenna carotenoid molecules make an angle of 45°ith the membrane.The primary electron donor possesses two transition moments centered respectively at 970 and 850 nm. The 970 nm transition moment is parallel to the membrane plane, the 850 nm transition is tilted out of the plane. Upon photooxidation of this primary electron donor, a monomer-like absorption band appears at 805 nm. Its transition makes an angle smaller than 25° with the membrane. The photooxidation of the dimer also induces an absorption band shift for the two other bacteriochlorophyll molecules of the reaction center. The absorption band shifts of the two bacteriochlorophyll molecules occur in opposite direction.One bacteriopheophytin molecule is photoreduced in state PI^?. This photoreduction induces an absorption band shift for only one bacteriochlorophyll molecule. Finally, the geometry of the dimeric primary donor seems to be affected by the presence of a negative charge in the reaction center.     

Modifications of membrane lipid composition following the nutritional shift-up of starved cells a comparison with membrane biogenesis in Tetrahymena

Detailed analyses of lipid composition have been made on various membrane fractions isolated at different intervals after 24 h-starved Tetrahymena cells were refed with nutrient-rich medium. During starvation there was a marked alteration in both phospholipid polar headgroup and acyl chain compositions: an increase in 2-aminoethylphospholipid and γ-linolenic acid (18 : 3) with a concurrent decrease in phosphatidylethanolamine and palmitoleic acid (16 : 1). However, following refeeding, such an altered lipid composition was rather rapidly restored to the initial level of the control cell membranes prior to starvation. This membrane lipid modification was found to occur in good accordance with the recovery of cell size and lipid synthesis. The considerable changes in the principal unsaturated fatty acids, 16 : 1 and 18 : 3, which are formed via the palmitate and stearate desaturation pathways, respectively, were suggested to be accounted for by the levels of desaturases activities. The results of the labeling experiments with radioactive precursors have demonstrated that in the refed cells, there was a more rapid and dynamic transfer or exchange between membranes as compared with that in the exponentially growing control cells. Thus, rapid ameliorative modifications of membrane lipid composition are thought to be required for the urgent growth of membrane systems in the refed cell which should be ready to initiate new division.     

Studies on the damage to Escherichia coli cell membrane caused by different rates of freeze-thawing

Freeze-thawing of Escherichia coli cells caused a release of cell membrane components such as protein, phospholipids and lipopolysaccharides. A greater amount of release and a lesser extent of cell survival were seen in slow freeze-thawing than in rapid freeze-thawing. Several dehydrogenases in the cells were also freed. The mode of release was also dependent on the rate of freeze-thawing.The materials released by slow freeze-thawing were found to be mostly composed of outer membrane components, whereas the materials released by rapid freeze-thawing contained cytoplasmic as well as outer membrane components. The chemical composition of these fragments differed significantly from that of the original membranes. The relative content of cytoplasmic membrane-bound enzymes in these fragments also differed from that of the cytoplasmic membrane.The fragmentation was assumed to have resulted mainly from the crystallization of external water. In slow freeze-thawing, it was considered that the phase separation of the membrane phospholipid bilayer increased the possibility of outer membrane fragmentation. Rapid freeze-thawing caused cytoplasmic membrane damage to the cells as well as to the outer membrane. In rapid freeze-thawing, the effect of phase separation appeared to be small because of rapid passage through the transition temperatures.The presence of 10% glycerol completely inhibited the release of cellular materials and enzymes. Cell survival was maintained at a high level in the glycerol-treated samples whether freeze-thawed slowly or rapidly.     

Architecture of the outer membrane of Escherichia coli K12 IV. Relationship between outer membrane particles and aqueous pores

The hypothesis that intramembraneous particles, observed in the outer membrane of Escherichia coli by freeze-fracture electron microscopy, are the morphological representation of aqueous pores, was tested. A mutant which is deficient in five major outer membrane proteins, b, c, d, e and the phage λ receptor protein, contains a largely decreased number of intramembraneous particles and also shows a greatly decreased rate of uptake of several solutes. In derivatives of this strain which contain only one of these proteins in large amounts a strong decrease of the number of intramembraneous particles is observed, which is accompanied by a complete restoration of the rate of uptake of those solutes which use pores in which the protein in question is involved. The results provide strong evidence for the notion that an individual pore contains only one protein species, a property which has been found earlier for individual particles. The observed correlation between particles and aqueous pores strongly supports the hypothesis that the particles are the morphological representation of pores. Implications of this hypothesis for the structure of the particles are discussed.     

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. I. Formation of transmembrane channels in lipid bilayers

The addition of nanomolar amounts of a toxin preparation derived from the sea anemone Stoichactis helianthus to black lipid membranes increases their electrical conductance by one million-fold. In addition, the membranes become permeable predominantly to monovalent cations. The elevated bilayer conductance is voltage-dependent, and the current-voltage curves of these bilayers display rectification as well as a region of negative resistance. The membrane activity of the toxin is proportional to the third power of its concentration, and at very low concentrations the membrane conductance increases in discrete uniform steps. These observations indicate that the mechanism of toxin action involves the formation of transmembrane channels constructed by the aggregation of protein molecules which are inserted in the bilayer. The voltage-dependent membrane conductance arises from two distinct channel characteristics: (1) the unit conductance of individual channels is dependent on the polarity of applied voltage; (2) the number of ion-conducting channels is influenced by the polarity as well as the magnitude of applied potential. It is believed that these effects are due to the influence of an electric field on the insertion of toxin molecules into the bilayer or on their subsequent association with each other to produce channels. Partial chemical characterization of the toxin material has shown that the membrane active factor is a basic protein with a molecular weight of 17 500.