Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.     

Membrane reorganization during chilling: implications for long-term stabilization of platelets

This essay is a review of the various biophysical and biochemical events that make up the factors responsible for platelet cold-induced activation. It describes the formation of large membrane domains composed of raft aggregates that occur during chilling and storage. It also presents strong evidence that platelet membranes undergo lateral phase separation during prolonged storage in the cold and suggests that raft aggregation and lateral phase separation are key events which must be obviated to stabilize platelets and store them either in the frozen or in the dry state.     

Characterization of the ATPase activities of myosins isolated from the membrane and the cytoplasmic fractions of human platelets

Myosin was purified from the membrane fraction and the cytoplasm of human platelets, and the K+(EDTA)- and Ca2+-dependent ATPase activities were studied under various experimental conditions. The ATPase activity of the myosin from the membrane fraction was slightly lower than that of its cytoplasmic counterpart, regardless of the different assay conditions (pH, ionic strength, and temperature). Both myosins showed the same pH optima and a similar ionic strength dependence for the two ATPase activities measured. In addition, they exhibited the same substrate specificity using ATP, CTP, and GTP as substrates. The activation energy of the Ca2+-dependent ATPase activity was essentially the same for the two myosins, while the activation energy of the K+(EDTA)-dependent ATPase activity of the membrane myosin was higher than that of the cytoplasmic myosin. The ATPase activity of the membrane myosin was found to be more sensitive to freezing and thawing than the cytoplasmic myosin. The alkylation of the thiol groups by N-ethylmaleimide or N-iodoacetyl-N-(5-sulfo-1-naphtyl)ethylenediamine, and the trinitrophenylation of the lysyl residues by 2,4,6-trinitrobenzenesulfonate caused a significant decrease in the K+(EDTA)-dependent ATPase activity of the two myosins. However, the membrane myosin was much less affected than the cytoplasmic myosin. Actin induced inhibition of the K+ (EDTA) ATPase of both myosins, and much smaller quantities of actin were needed to inhibit the cytoplasmic myosin ATPase compared to quantities needed to inhibit the myosin ATPase from the membrane fraction. This indicates that the membrane myosin has a lower affinity toward actin. The observed variations in the ATPase activity of the myosins isolated from the membrane and the cytoplasm fractions of human platelets may reflect differences in their respective physiological functions.     

Transbilayer incorporation of 1-pyrenebutyltrimethylammonium by blood platelets and its application for analyzing changes in physico-chemical properties of the membrane lipid bilayer induced by platelet activation.

The binding of cationic butyltrimethylammonium derivative of pyrene to bovine platelets was initially rapid and then increased gradually, unlike the bindings of other anionic and neutral derivatives of pyrene tested. The rate of increase in binding of the cationic probe depended on temperature and was due to its incorporation into the cytoplasmic side of the platelet membranes, as shown quantitatively by monitoring decrease in its extractability with albumin. The penetration into the inner membrane compartment did not reach equilibrium even after 4 h at 37 degrees C. Slow penetration of a fluorescent probe such as this is useful in studies on the physico-chemical properties of the outer layer and cytoplasmic side of the platelet membranes and their changes. Initial rapid binding of the cationic probe to platelets, representing the binding of the probe to the outer layer of the plasma membrane, was increased by ionomycin-induced platelet activation. Fluorescence spectra in the presence of a relatively high concentration of the cationic probe showed increase of the excimer of the cationic probe accompanied with the incorporation of the probe to the cytoplasmic side. On ionomycin-induced activation, the excimer-to-monomer intensity ratio of the probe in the cytoplasmic side of the platelet membranes decreased, possibly due to decrease in fluidity of the lipid layer near the probe or change in distribution of the probe.     

Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease

Caveolae are 50-100-nm membrane microdomains that represent a subcompartment of the plasma membrane. Previous morphological studies have implicated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial cells, (b) the uptake of small molecules via a process termed potocytosis involving GPI-linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartmentalization of certain signaling molecules, including G- protein coupled receptors. Caveolin, a 22-kD integral membrane protein, is an important structural component of caveolae that was first identified as a major v-Src substrate in Rous sarcoma virus transformed cells. This finding initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation. We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured cells. To facilitate biochemical manipulations, we have applied this procedure to lung tissue--an endothelial and caveolin-rich source-allowing large scale preparation of these complexes. These membrane domains retain approximately 85% of caveolin and approximately 55% of a GPI-linked marker protein, while they exclude > or = 98% of integral plasma membrane protein markers and > or = 99.6% of other organelle-specific membrane markers tested. Characterization of these complexes by micro-sequencing and immuno- blotting reveals known receptors for modified forms of LDL (scavenger receptors: CD 36 and RAGE), multiple GPI-linked proteins, an anion transporter (plasma membrane porin), cytoskeletal elements, and cytoplasmic signaling molecules--including Src-like kinases, hetero- trimeric G-proteins, and three members of the Rap family of small GTPases (Rap 1--the Ras tumor suppressor protein, Rap 2, and TC21). At least a fraction of the actin in these complexes appeared monomeric (G- actin), suggesting that these domains could represent membrane bound sites for microfilament nucleation/assembly during signaling. Given that the majority of these proteins are known molecules, our current studies provide a systematic basis for evaluating these interactions in vivo.     

Loss of phospholipid asymmetry in dilauroylphosphatidylcholine induced plasma membrane vesicles from human platelets

Incubation of human platelets with unilamellar vesicles composed of dilauroylphosphatidylcholine (DLPC) induces shedding of small vesicular structures from the platelet plasma membrane. No significant cell lysis is observed during the process of shedding. Isolated spicules contain the major membrane glycoproteins, Ib, IIb, and IIIa, which are used to define the sidedness of the spicule membrane. These glycoproteins are completely susceptible to chymotrypsin treatment, whereas cytoskeletal proteins are inaccessible towards this enzyme. This demonstrates that the spicule membranes have a right-side-out orientation in as far as membrane proteins are concerned. Isolated spicules were 30-fold more active than platelets in stimulating prothrombin conversion to thrombin by the prothrombinase complex (factors Xa, Va and Ca2+). The increased prothrombinase activity reflects an increased amount of phosphatidylserine in the outer leaflet of the spicule membrane. Protein analysis of platelet spicules and native platelets reveals a number of differences, the most conspicuous of which is the virtual absence of myosin in the spicule preparations. It is proposed that a lack of myosin produces a different cytoskeletal organization in the spicules. This enables phosphatidylserine to become exposed at the outer surface of the spicule membrane.     

Phospholipase activities in subcellular fractions of human platelets.

A homogenate of human platelets was fractionated by zonal ultracentrifugation into membranes, various granules and mitochondria. The membrane fraction was composed of two populations. The first, which represented 75% of the proteins, was rich in plasma membranes; the second, which represented the remaining 25%, was rich in microsomal membranes. Lysophospholipase was essentially localised in the cytosol. Phospholipase A1 which was only weakly bound to membranes, was mostly found in the soluble fraction (75%); the remainder was located in the plasma membranes and the mitochondria. Two-thirds of the phospholipase A2 was found in the particulate fractions.     

Proteomic analysis of isolated membrane fractions from superinvasive cancer cells

The superinvasive phenotype exhibited by paclitaxel-selected variants of an in vitro invasive clonal population of the human cancer cell line, MDA-MB-435S were examined using DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry. Isolation of membrane proteins from the MDA-MB-435S-F/Taxol-10p4p and parental populations was performed by temperature-dependent phase partitioning using the detergent Triton X-114. Subsequent DIGE-generated data analysed using Decyder software showed many differentially-expressed proteins in the membrane fraction. 16 proteins showing statistically significant upregulation in the superinvasive cells were identified by MALDI-ToF. Proteins upregulated in the superinvasive population include Galectin-3, Cofilin, ATP synthase beta subunit, voltage-dependent anion channel 1, voltage dependent anion channel 2, ER-60 protein, MHC class II antigen DR52, Beta actin, TOMM40 protein, Enolase 1, Prohibitin, Guanine nucleotide-binding protein, Annexin II, Heat shock 70 kDa protein, Stomatin-like protein 2 and Chaperonin. Many of these proteins are associated with inhibition of apoptosis, the progression of cancer, tumourigenicity, metastasis, actin remodelling at the leading edge of cells, polarized cell growth, endocytosis, phagocytosis, cellular activation, cytokinesis, and pathogen intracellular motility. These results suggest a correlation between the increased abundance of these proteins with the superinvasive phenotype of the paclitaxel-selected MDA-MB-435S-F/Taxol-10p4p population.     

Solubilization of prostacyclin membrane receptors from human platelets

Prostacyclin (PGI2) receptors have been identified on platelets and other tissues but their physicochemical properties remain unknown due to difficulties in obtaining active solubilized receptors. We evaluated the ability of several detergents to release the receptors from platelet membrane preparations. In contrast to the results of Dutta-Roy and Sinha (Dutta-Roy, A. K., and Sinha, A. K. (1987) J. Biol. Chem. 262, 12685-12691) which revealed selective solubilization of PGE1/PGI2 receptors by 0.05% Triton X-100, we found that CHAPS (3-[(3-chlamidopropyl)dimethylammonio]-1-propanesulfonic acid) (10 mM) was far superior in releasing the PGI2 receptors. In fact, Triton X-100 failed to release detectable PGI2 binding activity into the supernatant. The CHAPS-solubilized receptor degraded rapidly unless 30% glycerol was added which greatly enhanced its stability. By employing an improved binding assay using [3H]iloprost as the ligand and selective membrane filters (AP-15 or GF/B) pretreated with polyethyleneimine for achieving a higher trapping efficiency, we showed by equilibrium binding measurements that the solubilized receptors exhibited a single class of binding sites with a KD of 18.5 nM and Bmax 0.5 pmol/mg. These values were similar to those of the membrane receptors, i.e. KD of 16.6 nM and Bmax 1.0 pmol/mg. Kinetic binding measurements of the solubilized receptors revealed an association rate constant of 0.51 x 10(6) M-1 s-1 and dissociation rate constant of 0.0041 s-1 yielding a calculated KD of 8.0 nM. Displacement of [3H]iloprost (Ki values) from the solubilized and the membrane receptors by diversified eicosanoids was parallel. Our data demonstrate for the first time a successful solubilization of platelet PGI2 receptors. The solubilized receptors retained almost identical binding characteristics as the native membrane receptors.     

Immunological properties of isolated protein fractions from Artemia ribosomes

Acid-soluble ribosomal proteins from cysts of Artemia salina were separated by high-resolution polyacrylamide gel electrophoresis at pH 4.3. Three distinct protein bands, occurring in different parts of the electrophoretic pattern, were used for immunization in rabbits, and the γ-globulin fractions of the antisera were prepared. These preparations produced precipitation lines in agarose gel with protein extracted from whole 80S ribosomes and from 60S and 40S ribosomal subunits. With γ-globulin preparations from non-immune or anti-ovalbumin sera no reactions were obtained.     

Insulin induced phosphorylation and activation of the cGMP-inhibited cAMP phosphodiesterase in human platelets.

Insulin induced phosphorylation and activation of the cGMP inhibited cAMP phosphodiesterase (cGI-PDE) in human platelets were demonstrated after isolation of the enzyme with specific polyclonal cGI-PDE antibodies. The demonstration of this insulin effect required suppression of basal cGI-PDE phosphorylation, through the use of the protein kinase inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine). The human platelet insulin receptor beta-subunit, previously identified as a 97 kDa polypeptide, was detected with the use of wheat germ agglutinin chromatography and anti-phosphotyrosine antibodies. These results suggest that insulin, through phosphorylation/activation of cGI-PDE, could decrease cAMP/cAMP dependent protein kinase (cAMP-PK) activity and thereby make the platelets more sensitive towards aggregating agents.     

A new cGMP phosphodiesterase isolated from bovine platelets is substrate for cAMP- and cGMP-dependent protein kinases: evidence for a key role in the process of platelet activation.

The biochemical differences among cGMP phosphodiesterases in platelets have not been thoroughly examined, primarily due to the lack of sufficient purified material. This report describes a simple method developed to isolate a specific bovine platelet cGMP phosphodiesterase. This enzyme is cytosolic in its native form and was purified to an apparent homogeneity by ion-exchange chromatography, affinity chromatography, and density gradient centrifugation. Cyclic GMP binds to a "pseudo-site" when the catalytic site is deprived of Mg++. The affinity for cGMP at alkaline pH in presence of EDTA and IBMX (Kd = 60 nM) suggests that the removal of Mg++ by EDTA converts the catalytic site to a binding site. A ligand affinity chromatography was designed to take advantage of these features. The core enzyme has a molecular weight 190,000 composed of 2 subunits (MW 95,000) and has a specific activity of 2.5 mumol/min/mg. Moreover, this enzyme was phosphorylated by cAMP- and cGMP-dependent protein kinases, suggesting that its activity could be indirectly regulated by cyclic nucleotides. Agents elevating cGMP and cAMP inhibit platelet activation by inhibiting protein kinase C and thrombin induced hydrolysis of phosphatidylinositol 4,5 diphosphate. The antiaggregating properties of some of these agents might therefore be attributed to the fact that they are inhibitors of phosphodiesterases.     

Conditions of activation of guanylate cyclase from human platelets

Preincubation (50 min, 0 degree C) with nitroprusside increases 12-fold the activity of human platelet guanylate cyclase. The stimulating effect of nitroprusside is enhanced two-fold by dithiothreitol (2 mM) and by 60% by hemoglobin (20 micrograms/ml). Storage of guanylate cyclase preparations (105000 g supernatant) for 2-3 days at 4 degrees C causes a progressive increase of the enzyme activity and diminishes the stimulating effect of nitroprusside. After storage of guanylate cyclase preparations for 3 days, hemoglobin (20 micrograms/ml) augments the stimulating effect of nitroprusside by 130%. It is concluded that the degree of activation of guanylate cyclase by nitroprusside reflects the functional state of the enzyme.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d = 6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 °C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 °C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Binding of adenosine diphosphate to human blood platelets and to isolated blood platelet membranes

The equilibrium binding of 14C-labeled ADP to intact washed human blood platelets and to platelet membranes was investigated. With both intact platelets and platelet membranes a similar concentration dependence curve was found. It consisted of a curvilinear part below 20 microM and a rectilinear part above this concentration. At high ADP concentrations, the rectilinear part appeared to be saturable. Because of this, two classes of saturable ADP binding sites were proposed. ADP was partly converted to ATP and AMP with intact platelets while this conversion was virtually absent in isolated platelet membranes. ADP was bound to platelet membranes with the same type of curves found for intact platelets. The ADP binding to the high affinity system, which was stimulated by calcium ions, was nearly independent of temperature and had a pH optimum at 7.8. A number of agents were investigated for inhibiting properties. Of the sulfhydryl reagents only p-chloromercuribenzene sulfonate inhibited both high and low affinity binding systems while iodoacetamide and N-ethylmaleimide were without effect. Compounds acting via cyclic AMP on platelet aggregation, such as adenosine and cyclic AMP itself, had no influence on binding. Some nucleosidediphosphates and nucleotide analogs at a concentration of 100 microM had no, or only a slight, effect on high affinity ADP binding. For some other nucleotides inhibitor constants were determined for both platelet ADP aggregation and ADP binding. The inhibitor constants of ATP, adenyl-5'-yl-(beta,gamma-methylene)diphosphate, IDP, adenosine-5'(2-O-thio)diphosphate, for aggregation and high affinity binding were in good correlation with each other. Exceptions formed fluorosulfonylbenzoyl adenosine and AMP. The ATP formation found with intact platelets could be attributed to a nucleosidediphosphate kinase. It was investigated in some detail. The enzyme was magnesium dependent, had a Q10 value of 1.41, a pH optimum at 8.0, was competitively inhibited by AMP and reacted via a ping pong mechanism. All findings described in this paper indicate that platelets as well as platelet membranes bind ADP with the same characteristics and they suggest that the high affinity binding of ADP is involved in platelet aggregation induced by ADP. The results on nucleosidediphosphate kinase did not permit a firm conclusion about the role of the enzyme in induction of platelet aggregation by ADP.     

Comparative lipid analysis and structure of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

The lipid composition and structure of detergent-resistant membrane rafts from human, goat, and sheep erythrocytes is investigated. While the sphingomyelin:cholesterol ratio varied from about 1:5 in human to 1:1 in sheep erythrocytes a ratio of 1:1 was found in all raft preparations insoluble in Triton X-100 at 4 degrees C. Excess cholesterol is excluded from rafts and saturated molecular species of sphingomyelin assayed by gas chromatography-mass spectrometry determines the solubility of cholesterol in the detergent. Freeze-fracture electron microscopy shows that vesicles and multilamellar structures formed by membrane rafts have undergone considerable rearrangement from the original membrane. No membrane-associated particles are observed. Synchrotron X-ray diffraction studies showed that d spacings of vesicle preparations of rafts cannot be distinguished from ghost membranes from which they are derived. Dispersions of total polar lipid extracts of sheep rafts show phase separation of inverted hexagonal structure upon heating and this phase coexists with multilamellar structures at 37 degrees C.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d=6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 degrees C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 degrees C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Lipoxygenase activities of human platelets and their subcellular fractions: Comparison between lipoxygenase-deficient platelets and normal platelets

Lipoxygenase activities were estimated in washed platelets (intact platelets) and their subcellular fractions obtained from 7 patients with deficient platelet lipoxygenase activities and 9 normal subjects. From sonicated platelet preparations, 12,000 g supernatant (F-I), cytosol (F-II) and microsomal fractions (F-III) were prepared by differential centrifugation. When 12-hydroxyeicosatetraenoic acid (12-HETE) produced by the incubation of arachidonic acid with intact platelets or each of their subcellular fractions from normal subjects was measured by reversed-phase high-performance liquid chromatography analysis, the lipoxygenase activities of F-I, F-II and F-III were 87%, 31% and 17%, respectively, of the enzyme activity of intact platelets. One of the patients showed no detectable lipoxygenase activity in any preparation, while the other patients showed reduced enzyme activities in all preparations. The addition of CaCl2 significantly increased 12-HETE synthesis solely by F-I from these patients. In most of these patients, contrary to normal subjects, it appeared that the lipoxygenase activity was not fully expressed in intact platelets, since the F-I produced more 12-HETE than the intact platelets.     

Met identification on human platelets: role of hepatocyte growth factor in the modulation of platelet activation

Circulating HGF is significantly increased in a number of thrombus-associated disorders. Since platelets play a pivotal role in thrombogenesis, the ability of HGF to interact with human platelets was investigated. This paper shows for the first time that human platelets express HGF receptor, the tyrosine kinase encoded by c-MET gene. At physiological concentrations HGF was found to inhibit both glycoprotein (alpha)IIb(beta)3 activation and thrombin-dependent platelet aggregation in a dose- and time-dependent manner. These results suggest that circulating HGF may counteract thrombogenesis by negatively modulating platelet functions.     

Isolation and properties of mesosomal membrane fractions from Micrococcus lysodeikticus

1. A method is described for the isolation of pure mesosomal membrane fractions from Micrococcus lysodeikticus. 2. Plasmolysis of cells, before wall digestion, was necessary for effective mesosome release. 3. The effect of mild shearing forces, temperature and time upon the release of mesosomal membrane from protoplasts was investigated. 4. The optimum yield of mesosomal membranes from stable protoplasts was achieved at 10mm-Mg(2+). 5. Mesosomal membrane vesicle fractions prepared at differing Mg(2+) concentrations above 10mm were similar in chemical composition. 6. Comparison of the properties of peripheral and mesosomal membrane fractions revealed major differences in the distribution of protein components, membrane phosphorus, mannose and dehydrogenase activities between the two fractions. 7. Only cytochrome b(556) was detected in mesosomal membranes, whereas peripheral membranes contained a full complement of cytochromes. 8. Preliminary investigations suggested the localization of an autolytic enzyme(s) in the mesosomal vesicles. 9. The anatomy of mesosomal and peripheral membrane have been compared by the negative-staining and freeze-fracture technique. 10. The results are discussed in relation to a plausible role for the mesosome.