Membranes of Rhodopseudomonas sphaeroides. V. Identification of bacteriochlorophyll alpha-depleted cytoplasmic membrane in phototrophically grown cells.

The separation of membrane fragments was investigated in extracts of phototropically grown Rhodopseudomonas sphaeroides to determine if the plasma membrane contains discrete regions. A highly purified fraction of bacteriochlorophyll alpha-deficient membrane fragments was isolated by differential centrifugation, chromatography on Sepharose 2B, reaggregation, and isopycnic sedimentation on sucrose gradients. Significant levels of b- and c-type cytochromes and succinate dehydrogenase were demonstrated in the isolated membrane fragments and their appearance in electron micrographs, their polypeptide profile in dodecyl sulfate-polyacrylamide gel electrophoresis, and overall chemical composition were essentially identical to a similar fraction isolated from aerobically grown cells. Their polypeptide profiles were distinct from those of the intracytoplasmic chromatophore and outer membranes, and on the basis of bacteriochlorophyll content the phototrophic fraction was contaminated with chromatophores by less than 9%. The membrane fragments contained no diaminopimelic acid or glucosamine. It is condluded that the membrane fragments isolated from phototrophically growing Rp. sphaeroides have arisen from photosynthetic pigment-depleted regions of the plasma membrane structurally and functionally differentiated from the intracytoplasmic chromatophore membrane. These regions represent conserved chemotrophic cytoplasmic membrane whose synthesis continues under photoheterotrophic conditions.     

Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains

A rat liver plasma membrane preparation was isolated and characterized both biochemically and morphologically. The isolation procedure was rapid, simple and effective in producing a membrane fraction with the following biochemical characteristics: approximately 40-fold enrichment in three plasma membrane markers, 5'-nucleotidase, alkaline phosphodiesterase I (both putative bile canalicular membrane enzymes), and the asialo-glycoprotein (ASGP) receptor (a membrane glycoprotein present along the sinusoidal front of hepatocytes); a yield of each of these plasma membrane markers that averaged approximately 16%; and minimal contamination by lysosomes, nuclei, and mitochondria, but persistent contamination by elements of the endoplasmic reticulum. Morphological analysis of the preparation revealed that all three major domains of the hepatocyte plasma membrane (sinusoidal, lateral, and bile canalicular) were present in substantial amounts. The identification of sinusoidal membrane was further confirmed when ASGP binding sites were localized predominantly to this membrane in the isolated PM using electron microscope autoradiography. By morphometry, the sinusoidal front membrane accounted for 47% of the total membrane in the preparation, whereas the lateral surface and bile canalicular membrane accounted for 6.8% and 23% respectively. This is the first report of such a large fraction of sinusoidal membrane in a liver plasma membrane preparation.     

Synthesis and assembly of the membrane proteins in E. coli.

Kinetics of integration of membrane proteins were studied in E. coli to discover how membrane proteins find their final location in the functional membrane. The experiments make use of a simple and convenient method developed for isolating inner and outer membranes from a number of small-scale cultures with high recovery. Among the proteins that constitute the cell surface structures, inner membrane proteins are integrated most rapidly after synthesis, whereas outer membrane proteins delay somewhat, and periplasmic proteins delay further in reaching their destinations. Protein I, a major outer membrane protein with molecular weight of about 37,000 daltons, exhibits significantly slower rates of integration than other outer membrane proteins. The decreased fluidity of membrane lipids by temperature shiftdown of an unsaturated fatty acid auxotroph grown on elaidate results in abnormally slow assembly of the outer membrane proteins and also in an anomalous assembly of the inner membrane proteins, suggesting that the fluid state of the lipids is required for normal operation of these processes. The possible relevance of these findings to the mechanism of membrane formation is discussed.     

Freeze-fracture studies of Cryptosporidium muris.

The attachment site of Cryptosporidium muris to host cells was investigated using the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with the host plasma membrane at the dense band, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was dramatically altered at the above two junctures. The outer parasitophorous membrane showed low IMP-density as compared to the host plasma membrane, although both membranes were continuous. The inner parasitophorous membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP. When the attachment sites of parasites and host cells were fractured, circular-shaped fractured faces were observed on both sites of the parasite and host cell. These exposed faces corresponded to the dense bands and were very similar in size in each parasite.     

Lipid fluidity-dependent biosynthesis and assembly of the outer membrane proteins of E. coli.

The reduction of the membrane lipids of E. coli to a nonfluid state resulted in the accumulation in the cell envelope of a high molecular weight precursor of the protoIG protein, a major outer membrane protein. The protoIG protein was as sensitive to trypsin as the mature toIG protein assembled in the outer membrane. In contrast to the toIG protein, however, the accumulated protoIG protein was easily released from the envelope fraction by both sodium lauryl sarcosinate extraction and sonication. This indicated that the precursor protein was loosely associated with the cell membrane. When a fluid lipid state was restored, the protoIG protein was processed to the mature form which was then correctly assembled in the outer membrane. These results suggest that the protoIG protein produced under nonfluid lipid conditions was properly translocated across the cytoplasmic membrane, but could not be assembled in the outer membrane due either to the reversible inhibition of the processing of the ProtoIG to the toIG protein or to the lack of interaction with a specific outer membrane component(s). Reduced lipid fluidity also caused various alterations in the biosynthesis and assembly of other membrane proteins. In addition to the toIG protein, a large number of new proteins were accumulated in the membrane. Alternatively, the matrix protein as well as the promatrix protein were not detected in the cell envelope. On the other hand, the lipoprotein was normally produced, processed, modified and assembled in the outer membrane. These results indicate that the outer membrane proteins are synthesized and assembled according to several different mechanisms, on which the physical state of the membrane has various effects.     

Lysosomes and membrane recycling. A hypothesis.

A mechanism for intralysosomal membrane recycling is proposed. After invagination of the lysosomal membrane during autophagy, intralysosomal vesicles are formed. It is suggested that membrane can bleb out from these internal vesicles, probably in the form of very small vesicles, and return to the external lysosomal membrane by membrane fusion. This mechanism would conserve lysosomal membrane during autophay, and is analogous to current models of plasma-membrane recycling. Its relationship to turnover of lysosomal-membrane proteins and other proteins is discussed.     

Model for the dynamic responses of taste receptor cells to salty stimuli. I. Function of lipid bilayer membranes.

The dynamic response of the lipid bilayer membrane is studied theoretically using a microscopic model of the membrane. The time courses of membrane potential variations due to monovalent salt stimulation are calculated explicitly under various conditions. A set of equations describing the time evolution of membrane surface potential and diffusion potential is derived and solved numerically. It is shown that a rather simple membrane such as lipid bilayer has functions capable of reproducing the following properties of dynamic response observed in gustatory receptor potential. Initial transient depolarization does not occur under Ringer adaptation but does under water. It appears only for comparatively rapid flows of stimuli, the peak height of transient response is expressed by a power function of the flow rate, and the membrane potential gradually decreases after reaching its peak under long and strong stimulation. The dynamic responses in the present model arise from the differences between the time dependences in the surface potential phi s and the diffusion potential phi d across a membrane. Under salt stimulation phi d cannot immediately follow the variation in phi s because of the delay due to the charging up of membrane capacitance. It is suggested that lipid bilayer in the apical membrane is the most probable agency producing the initial phasic response to the stimulation.     

Characterization of the mycoplasma membrane proteins. V. Release and localization of membrane-bound enzymes in Acholeplasma laidlawii.

The peripheral membrane protein fraction released by washing Acholeplasma laidlawii membranes with low-ionic strength buffers contained about 50% of the total membrane-bound ribonuclease and deoxyribonuclease activities. The ATPase, NADH oxidase and p-nitrophenylphosphatase activities remained bound to the membrane even when EDTA was added to the wash fluids, and thus appear to belong to the integral membrane protein group. Serving as a marker for peripheral membrane proteins, the membrane-bound ribonuclease activity was solubilized by bile salts much more effectively than the integral membrane-bound enzymes. On the other hand, the solubilized ribonuclease showed a much lower capacity to reaggregate with other solubilized membrane components to membranous structures. Yet, most of the ribonuclease molecules which were bound to the reaggregated membranes could not be released by low-ionic strength buffer. The reaggregated membranes differed from the native membranes in the absence of particles on their fracture faces obtained by freeze cleaving, and by their much higher labeling by the [125-I]lactoperoxidase iodination system. These results suggest that most of the proteins are exposed on the reaggregated membrane surfaces, with very little, if any, protein embedded in its lipid bilayer core. Enzyme disposition in the A. laidlawii membrane was studied by comparing the activity of isolated membranes with that of membranes of intact cells after treatment with pronase or with an antiserum to membranes. The data indicate the asymmetrical disposition of these activities, the ATPase and NADH oxidase being localized on the inner membrane surface, while the nucleases are exposed on the external membrane surface.     

Fusion in phospholipid spherical membranes. II. Effect of cholesterol, divalent ions and pH.

Effect of cholesterol, divalent ions and pH on spherical bilayer membrane fusion was studied as a function of increasing temperature. Spherical bilayer membranes were composed of natural [phosphatidylcholine (PC) and phosphatidylserine (PS)] as well as synthetic (dipalmitoyl-PC, dimyristoyl-PC and dioleoyl-PC) phospholipids. Incorporation of cholesterol into the membrane (33% by weight) suppressed the fusion temperature and also greatly reduced the percentage of membrane fusion. The presence of 1 mM divalent ions (Ca++, Mg++ or Mn++) on both sides or one side of the PC membrane did not affect appreciably its fusion characteristic with temperature, but the PS membrane fusion with temperature was greatly enhanced by the presence of divalent ions. The variation of pH of the environmental solution in the range of 5.5 approximately 7.0 did not affect the membrane fusion characteristic. However, at pH 8.5, the fusion with respect to temperature was shifted toward the lower temperature by approximately 3degreesC for PC and PS membranes, and at pH 3.0 the opposite situation was observed as the fusion temperature was increased by 6degreesC for PS membranes and by 4degreesC for PC membranes The results seem to indicate that membrane fluidity and structural instability in the bilayer are important for membrane fusion to occur.     

Carotenoid-containing outer membrane of Synechocystis sp. strain PCC6714.

Outer membranes, free of cytoplasmic or thylakoid membranes and peptidoglycan components, were obtained from Synechocystis sp. strain PCC6714. Electron microscope studies revealed double-track outer membrane vesicles with a smooth-appearing exoplasmic surface, an exoplasmic fracture face covered by closely packed particles and a corresponding plasmic fracture face with regularly distributed holes. Lipopolysaccharide, proteins, lipids, and carotenoids were the constituents of the outer membrane of Synechocystis sp. PCC6714. Twelve polypeptides were found in outer membrane fractions, among them two dominant outer membrane proteins (Mrs, 67,000 and 61,000). Lipopolysaccharide-specific components were GlcN and an unidentified heptose. Outer membrane lipid extracts contained phosphatidylglycerol, sulfolipid, phosphatidylcholine, and unknown lipids. The carotenoids, myxoxanthophyll, related carotenoid-glycosides, zeaxanthin, echinenone, and beta-carotene were found to be true constituents of the outer membrane of Synechocystis sp. PCC6714.     

Physico-chemical studies of taste reception. IV. Response of individual phospholipid membrane to a variety of chemical stimuli.

Variations in the membrane potential across model membranes made of Millipore filter paper and various single phospholipids were measured in response to salt, acid and distilled water. The phospholipids used were phosphatidylcholine (c), spingomyelin (SM), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Results were compared with those obtained with the model membrane made of the total lipids extracted from bovine tongue epithelium, which simulated well the receptor potential observed with intact tast organs. The membrane potential of PE- and PS-membranes increased monotonously with increase of the concentration of 1:1 type salt, while that of PC- and SM-membranes exhibited no appreciable change in 1:1 salt solutions. Application of CaC12 to the membranes brought about a varity of response depending on the species of lipids used. PE- and PS-membranes showed a larger change in the membrane potential than PC- and SM-membranes when pH of the solution was varied. Fe-3+ was strongly absorbed on the surface of PC and SM-membranes, while Fe-3+ bound to PE- and PS-membranes was easily removed by an application of salt solution. A transient increase in the membrane potential was observed when distilled water was applied to the membrane adapted to an appropriate salt solution, which was similar to the water response observed in taste cells. PC- and SM-membranes responded to water when the membrane adapted to either NaC1 or CaC12, but PS-membrane responded only when the membrane was adapted to a solution containing CaC12. PE-membrane did not respond to water in any cases examined. The membrane prepared with a mixture of two species of phospholipids responded neither to salt nor to water, while the membranes prepared with the total lipids or a mixture of three species of lipids in appropriate ratio responded to both. The water response of the total lipids membrane vanished in a high temperature medium, while the water response of PC-membrane retained in all temperature ranges examined, i.e. between 20 degrees and 62 degrees C. The results obtained suggest that a mosaic structure, where each domain has different functions against various chemical stimuli, is formed on the surface of the model membrane made of the total lipids.     

Glycoprotien biosynthesis in liver mitochondria. II. Mitochondrial localization of mannosyltransferase

The preparation of liver mitochondria in the pure state, and submitochondrial fractionation in inner membrane, outer membrane and matrix, show that the mannosyltranferase of the mitochondria is localized in the inner membrane.     

Interactions of C-reactive protein and complement with liposomes. II. Influence of membrane composition.

We found previoulsy that interaction of C-reactive protein (CRP) with liposomal model membranes resulted in complement(C)-dependent membrane damage. In the present study, we investigated the influence of membrane composition on the interactions of CRP and C with liposomes. Adsorption experiments showed that binding of CRP was greatest to strongly positive liposomes. A lesser, but still substantial, extent of CRP binding also was observed with negative liposomes, but negligible amounts of CRP bound to neutral or weakly positive liposomes. CRP-mediated consumption of hemolytic C, and C-dependent glucose release from liposomes both were strongly influenced by liposomal charge, positive being superior to negative. Glucose release and, to a lesser extent, consumption of hemolytic C were inversely related to phospholipid fatty acyl chain length. Phospholipid fatty acyl unsaturation and liposomal cholesterol concentration both had strong influences on C consumption and glucose release. The data suggest that CRP-mediated C consumption and membrane damage require an optimum membrane fluidity. Complement damage in the presence of CRP was enhanced by certain sphingolipids and also by digalactosyl diglyceride, but not by sphingomyelin. Our results thus demonstrate that CRP-mediated C consumption and C-dependent membrane damage both are influenced by the liposomal membrane composition.     

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Arylamidases of rat liver and chemically induced hepatomas. 1. Subcellular distribution of L-leucine. 2. Naphthylamidase-active antigens.

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using L-leucine 2-naphthylamide as substrate. Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase). The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal and cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.     

Properties of membrane stationary states. 2. The isobaric isothermal ensemble

A multi-state membrane transport system serves as the basic model for a comparison of equilibrium and stationary state distributions of ions within an ensemble of membrane channels. Transition probabilities for intramembrane transitions are developed for consistency with an equilibrium Donnan potential and the magnitude of the flux in the stationary state can be controlled with either the bath concentrations or an externally applied potential. The stationary state distribution and operational thermodynamic parameters for the stationary state are expressed as functions of the ratio (j^net/p) where j^net is the observable net membrane flux and p is a characteristic decay constant for the intramolecular transitions. Both parameters are experimentally accessible so that their time independent ratio serves as an experimental measure of the membrane configuration. When operational thermodynamic parameters are expressed as functions of the ratio (j/p) for systems which obey the condition of detailed balance, the free energy varies quadratically with the ratio while the enthalpy, expressed as an average membrane potential, varies linearly with the ratio. As the system is driven from its equilibrium configuration by the net membrane flux the free energy increases relative to the enthalpy of the membrane. The free energy is localized in the membrane region at the expense of dissipation in the bathing solutions.     

The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase.

The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient. The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by (Na+ + K+)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of (Na+ + K+)-ATPase in purified bisolateral plasma membranes was 13-fold. F--activated adenyl cyclase co-purified with (Na+ + K+)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K+-stimulated phosphatase and 5'-nucleotidase also followed (Na+ + K+)-ATPase during fractionation.     

Extensional flow of erythrocyte membrane from cell body to elastic tether. I. Analysis.

This is the first of two papers on an analytical and experimental study of the flow of the erythrocyte membrane. In the experiment to be discussed in detail in the second paper, preswollen human erythrocytes are sphered by aspirating a portion of the cell membrane into a small micropipette; and long, thin, membrane filaments or "tethers" are steadily withdrawn from the cell at a point diametrically opposite to the point of aspiration. The aspirated portion of the membrane furnished a "reservoir" of material that replaces the membrane as it flows as a liquid from the nearly spherical cell body to the cylindrical tether. In this paper we show that an application of the principle of conservation of mass permits the tether radius (approximately 200 A or less) to be measured with the light microscope as the tether is formed and extended at a constant rate. A static analysis of the axisymmetric cell deformation and tether formation process reveals that the tether radius is uniquely determined by the isotropic tension in the membrane and the elastic constitutive (material) behavior of the tether itself. A dynamic analysis of the extensional flow process reveals that the tether radius must decrease as the velocity of the tether is increased and that the decrease depends on both the viscosity of the membrane and the elasticity of the tether. The analysis also shows that these two factors (membrane viscosity and tether elasticity) are readily decomposed and determined separately when flow experiments are performed at different isotropic tensions.     

Protein kinases associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates.

When highly purified myelin from rat sciatic nerve was incubated with [gamma-32P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and 32P from [gamma-32P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg2+-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3',5'-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing. From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.