Highly sequential binding of protein kinase C and related proteins to membranes

Protein kinase C belongs to a class of proteins that displays simultaneous interaction with calcium and phospholipids. Other members of this class include two proteins (Mr 64K and 32K) isolated from bovine brain. The association of these proteins with membranes exhibited highly unusual properties that were not consistent with a simple equilibrium. Titration of protein-phospholipid binding as a function of calcium showed an apparently normal curve with a low degree of cooperativity. The binding was rapid and quickly adjusted to changes in the calcium concentration. Calcium was readily exchanged from the protein-phospholipid complex. However, at each calcium concentration, membrane-bound protein was not in rapid equilibrium with free protein in solution; the half-time for dissociation exceeded 24 h. Titration of phospholipid vesicles with proteins showed different saturation levels of bound protein at different calcium concentrations. The amount of protein bound was almost entirely determined by the concentration of calcium and was virtually unaffected by the free protein concentration. These properties suggested that protein-phospholipid binding involved a sequence of steps that were each irreversible upon completion. These binding properties were consistent with high-affinity interaction between protein and phospholipid, high cooperativity with respect to calcium (N greater than or equal to 10), clustering of acidic phospholipids, and negative cooperativity with respect to protein density on the membrane. A major apparent problem with the complete titration of PKC-membrane interaction was a requirement for calcium in excess of intracellular levels. However, a highly sequential binding process showed that a number of protein-binding sites on the membrane would be saturated with calcium at physiological levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteins that bind calcium in a phospholipid-dependent manner

Three proteins (Mr = 64K, 32K, and 22K) that bind to phospholipids in a calcium-dependent manner were purified from bovine brain. The calcium-binding properties of these proteins were investigated by equilibrium dialysis and by gel filtration chromatography. The 64- and 32-kDa proteins were found to have calcium- and phospholipid-binding properties strikingly similar to those of protein kinase C [Bazzi, M.D., & Nelsestuen, G.L. (1990) Biochemistry 29, 7624]. The free proteins bound limited divalent metal ion even at 200 microM calcium. However, they bound eight to nine calcium ions per protein in the presence of membranes containing acidic phospholipids. The calcium concentrations needed for protein-phospholipid binding were different for these two proteins and were strongly influenced by the phospholipid composition of the vesicles; vesicles of higher phosphatidylserine content required lower concentrations of calcium for protein-membrane association. These properties described a general type of calcium-interacting system where simultaneous interaction of all three components (protein, phospholipids, and calcium) is required. The free proteins may provide only partial coordinate bonds to each calcium ion, but complete calcium-binding sites could be generated at the protein-phospholipid interface. In contrast to the 64- and 32-kDa proteins, the 22-kDa protein bound similar amounts of calcium (two to three ions/protein) in the presence or the absence of phospholipids. The 22-kDa protein had the lowest affinity for phospholipid and the highest affinity for calcium of the three proteins tested. Thus, calcium-dependent phospholipid-binding proteins consist of several types. For example, the 64- and 32-kDa proteins appear to be quite abundant and may even function as a calcium buffer to modulate signaling events.     

Extensive segregation of acidic phospholipids in membranes induced by protein kinase C and related proteins

Protein kinase C and two other proteins with molecular masses of 64 and 32 kDa, purified from bovine brain, constitute a type of protein that binds a large number of calcium ions in a phospholipid-dependent manner. This study suggested that these proteins also induced extensive clustering of acidic phospholipids in the membranes. Clustering of acidic phospholipids was detected by the self-quenching of a fluorescence probe that was attached to acidic phospholipids (phosphatidic acid or phosphatidylglycerol). Addition of these proteins to phospholipid vesicles containing 15% fluorescently labeled phosphatidic acid dispersed in neutral phosphatidylcholine resulted in extensive, rapid, and calcium-dependent quenching of the fluorescence signal. Fluorescence-quenching requirements coincided with protein-membrane binding characteristics. As expected, the addition of these proteins to phospholipid vesicles containing fluorescent phospholipids dispersed with large excess of acidic phospholipids produced only small fluorescence changes. In addition, association of these proteins with vesicles composed of 100% fluorescent phospholipids resulted in no fluorescence quenching. Protein binding to vesicles containing 5-50% fluorescent phospholipid showed different levels of fluorescence quenching that closely resemble the behavior expected for extensive segregation of the acidic phospholipids in the outer layer of the vesicles. Thus, the fluorescence quenching appeared to result from self-quenching of the fluorophores that become clustered upon protein-membrane binding. These results were consistent with protein-membrane binding that was maintained by calcium bridges between the proteins and acidic phospholipids in the membrane. Since each protein bound eight or more calcium ions in the presence of phospholipid, they may each induce clustering of a related number of acidic phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of the protein kinase C-phorbol ester interaction

The properties of the protein kinase C (PKC)-phorbol ester interaction were highly dependent on assay methods and conditions. Binding to cation-exchange materials or adsorption to gel matrices resulted in PKC that was capable of binding phorbol 12,13-dibutyrate (PDBu). The extraneous interactions were eliminated by measuring phorbol ester binding with a gel filtration chromatography assay in the presence of bovine serum albumin (BSA). In the absence of calcium, free PKC did not bind PDBu or phospholipids. Calcium caused structural changes in PKC which enhanced its interaction with surfaces such as the gel chromatography matrix. While BSA prevented this interaction, it did not interfere with PKC association with acidic phospholipids. Interaction of PKC with phospholipid resulted in two forms of membrane-associated PKC. The initial calcium-dependent and reversible form of membrane-associated PKC was capable of binding PDBu. Both PKC and PDBu were released from this complex by calcium chelation. Sustained interaction with phospholipid vesicles resulted in a PKC-membrane complex that could not be dissociated by calcium chelation and appeared to result from insertion of PKC into the hydrocarbon portion of the phospholipid bilayer. Membrane insertion was observed at calcium concentrations of 2-500 microM and with membrane compositions of 10-50% acidic phospholipid. However, the extent of insertion was dependent on the binding conditions and was promoted by high phospholipid to PKC ratios, high calcium, the presence of phorbol esters, high membrane charge, and long incubations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

Studies of protein-phospholipid interaction in isolated mitochondrial ubiquinone-cytochrome c reductase

The interaction between phospholipids, ubiquinone and highly purified ubiquinol-cytochrome c reductase was studied using differential scanning calorimetry. The enzyme complex and its delipidated forms undergo thermodenaturation at 337.3 and 322.7 K, respectively. The reduced reductase is more stable toward thermodenaturation than is the oxidized enzyme. While phospholipids restored enzymatic activity to the delipidated enzyme complex and stabilized the enzyme toward thermodenaturation, ubiquinone showed little effect on the thermostability of ubiquinol-cytochrome c reductase. The effect of phospholipids on the thermotropic properties of ubiquinol-cytochrome c reductase is dependent upon the molecular properties of the phospholipid. When ubiquinol-cytochrome c reductase was embedded in closed asolectin vesicles, an exothermic transition peak was observed upon thermodenaturation. When the asolectin concentration in the reconstituted preparation was less than 0.3 mg/mg protein, an amorphous structure was observed in the electron micrograph and the preparation showed an endothermic transition upon thermodenaturation. The thermotropic properties of the enzyme-phospholipid vesicles were affected by the phospholipid head groups as well as the fatty-acyl chains, with those phospholipids having the most highly unsaturated fatty-acyl chains having the greatest effect. The energy for the exothermic transition may be derived from the collapse, upon thermodenaturation, of a strained interaction between the unsaturated fatty-acyl groups of phospholipids and protein molecules resulting from vesicle formation. The exothermic transition of the enzyme-phospholipid vesicle was abolished when cholesterol was included in the vesicles and when reductase was treated with a proteolytic enzyme prior to incorporation into the phospholipid vesicles.     

Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles

Thrombin-activated factor Va and factor Va subunit binding to large-volume vesicles was investigated by a technique based on the separation by centrifugation of phospholipid-bound protein from the bulk solution. This technique allows the direct measurement of free-protein concentration. It is concluded that the phospholipid binding site on factor Va is located on a basic factor Va subunit with Mr 80 000 (factor Va-LC). The effects of phospholipid vesicle composition, calcium concentration, pH, and ionic strength on the equilibrium constants of factor Va- and factor Va-LC-phospholipid interaction were studied. Factor Va and factor Va-LC binding to phospholipid requires the presence of negatively charged phospholipids. It is further demonstrated that the following occur: (a) Calcium ions compete with factor Va and factor Va-LC for phospholipid-binding sites. (b) The dissociation constant of protein-phospholipid interaction increases with the ionic strength, whereas the maximum protein-binding capacity of the phospholipid vesicle was not affected by ionic strength. (c) The dissociation constant for factor Va-phospholipid interaction depends on pH when the vesicle consists of phosphatidic acid. It is concluded that factor Va-phospholipid interaction is primarily electrostatic in nature, where positively charged groups on the protein directly interact with the phosphate group of net negatively charged phospholipids. The results suggest that factor Va, like factor Xa and prothrombin, has the characteristics of an extrinsic membrane protein.     

Restoration by phospholipids of dolichol pyrophosphate N-acetylglucosamine synthesis in delipidated rat lung microsomes

The effects of phospholipids on the reaction catalyzed by UDP-GlcNAc:dolichol phosphate GlcNAc-1-phosphate transferase have been studied with delipidated rat lung microsomes. Deoxycholate-solubilized enzyme was depleted of measurable phospholipid by either gel filtration on Sephadex G-100 or affinity chromatography on pentyl-agarose. The latter procedure also removed nucleotide and sugar nucleotide hydrolases. Delipidated protein fractions were devoid of GlcNAc-1-phosphate transferase activity unless supplemented with phospholipids. Maximal recovery of enzyme activity was obtained with an approximate 1:1 weight ratio of phosphatidylglycerol:phosphatidylcholine, with the observed rate being synergistic as compared to rates observed for each individual phospholipid. Variable recoveries of enzyme activity were obtained with mixtures containing other acidic phospholipids and phosphatidylcholine. Enzyme activity in the fraction eluted from pentyl-agarose could be recovered, after removal of Triton X-100, with sedimented phospholipid vesicles. Significant stabilization of enzyme activity associated with the phospholipid vesicles was obtained by the inclusion of dolichol phosphate.     

Influence of proteins on the reorganization of phospholipid bilayers into large domains.

Using large (5-10 microns) vesicles formed in the presence of phospholipids fluorescently labeled on the acyl chain and visualized using a fluorescence microscope, charge-coupled-device camera, and digital image processor, we examined the effects of membrane proteins on phospholipid domain formation. In vesicles composed of phosphatidic acid and phosphatidylcholine, incubation with cytochrome c induced the reorganization of phospholipids into large phosphatidic acid-enriched domains with the exclusion of phosphatidylcholine. Cytochrome c binding was demonstrated to be highest in the phosphatidic acid-enriched domain of the vesicle using the absorbance of the heme moiety for visualization. Both binding of cytochrome c and phospholipid reorganization were blocked by pretreatment of the vesicles with 0.1 M NaCl. The pore forming peptide gramicidin was examined for the effects of an integral protein on domain formation. Initially, gramicidin distributed randomly within the vesicle and showed no phospholipid specificity. Phosphatidic acid domain formation in the presence of 2.0 mM CaCl2 or 100 microM cytochrome c was not affected by the presence of 5 mol % gramicidin within the vesicles. In both cases, gramicidin was preferentially excluded from the phosphatidic acid-enriched domain and became associated with phosphatidylcholine-enriched areas of the vesicle. Thus, cytochrome c caused a major reorganization of both the phospholipids and the proteins in the bilayer.     

Association of Pseudomonas cytochrome oxidase with liposomes.

Purified Pseudomonas cytochrome oxidase has been associated with asolectin liposomes by two different methods. Firstly, the enzyme was attached to liposomic membranes by adding it to a cholate-phospholipid dispersion and subsequently dialyzing the detergent out of suspension. In the second case the enzyme was adsorbed on the preformed liposomes when added to them after the dialysis. A stimulation of the cytochrome oxidase activity approximately twenty-fold was observed by the first method. In contrast, the activation was absent in the second type of preparation, indicating that interaction between the enzyme and phospholipids is very different in the two types of vesicles. The cholate-dialysis method for reconstitution of protein-phospholipid vesicles seems to lead to rather heterogenous preparations. These can be further fractionated, not only according to their size but also to the protein/phospholipid ratio, by gel chromatography.     

Interaction of d-β-hydroxybutyrate dehydrogenase with lecithin vesicles

Rat liver mitochondrial d-β-hydroxybutyrate dehydrogenase has an absolute requirement for lecithin. The nature of the interaction between the enzyme and phospholipid has been investigated. Single bilayer lecithin liposomes of shell-like structure bring about maximal enzyme activation, whereas the interaction with larger vesicles leads to enzyme inactivation. The strong binding of the enzyme to lecithin confers great stability to the enzyme activity as compared with the nonlipid-activated enzyme, and permits the isolation of a lipoprotein complex by chromatography on Sephadex G-200. Only 20% of the proteins solubilized with d-β-hydroxybutyrate dehydrogenase from mitochondrial membranes bind to lecithin liposomes, thus a 5-fold purification of the enzyme is achieved. The liposome-bound proteins had a significantly lower polarity than the remaining 80% of solubilized mitochondrial membrane proteins.     

Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA.

Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2-dioleoyl-sn-glycerol is observed in the absence of added Ca2+. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2-Dioleoyl-sn-glycerol eliminates the Ca(2+)-dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2-dioleoyl-sn-glycerol in this respect. This suggests that the 1,2-dioleoyl-sn-glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2-dioleoyl-sn-glycerol on the phosphatidic-acid-stimulated protein kinase C activity is dependent on the molar fraction of 1,2-dioleoyl-sn-glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2-dioleoyl-sn-glycerol concentrations to calcium inhibition at higher concentrations of 1,2-dioleoyl-sn-glycerol. Phosphatidylserine-stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2-dioleoyl-sn-glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2+ to high affinity sites on the enzyme. Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2-dioleoyl-sn-glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2+. There is no isozyme specificity in this binding. These results suggest that a less-tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.     

Transbilayer distributions of red cell membrane phospholipids in unilamellar vesicles

The phospholipid organization in unilamellar vesicles comprised of various purified phospholipid components of monkey erythrocyte membrane was ascertained using phospholipase A2 and trinitrobenzenesulfonic acid as external membrane probes. The vesicles were formed by sonication or detergent dialysis and fractionated by centrifugation or gel permeation chromatography. Experiments were done to confirm that the phospholipase A2 treatments did not cause lysis or induce fusion of the vesicles. This enzyme hydrolysed only the glycerophospholipids in the outer surface of the vesicles. The amounts of the external phospholipids determined by this enzymatic method were verified using the chemical probe, trinitrobenzenesulfonic acid. The choline-containing phospholipids and phosphatidylethanolamine localized randomly in the two surfaces of sonicated vesicles (outer diameter, about 30 nm), whereas phosphatidylserine preferentially distributed in the inner monolayer. This phosphatidylserine asymmetry virtually disappeared in detergent dialysed vesicles (outer diameter, about 45 nm). Furthermore, inclusion of cholesterol in both the types of vesicles resulted in more random glycerophospholipid distributions across the plane of vesicles bilayer, presumably due to the cholesterol-induced increases in the size of vesicles. These results demonstrate that the transbilayer distribution of erythrocyte membrane phospholipids in unilamellar vesicles are controlled mainly by the surface curvature rather than by interlipid interactions, and therefore suggest that phospholipid-phospholipid and phospholipid-cholesterol interactions should not play any significant role in determining the membrane phospholipid asymmetry in red cells. It is proposed that this asymmetry primarily originates from differential bindings of phospholipids with membrane proteins in the two leaflets of the membrane bilayer.     

Molecular interactions of the intrinsic activation complex of coagulation: binding of native and activated human factors IX and X to defined phospholipid vesicles

The assembly of proteins of the intrinsic activation complex has been partially elucidated. In the present study we examine the association of gamma-carboxylated serine proteinase zymogens factors IX and X, and their proteolytically activated counterparts factors IXa and Xa to unilamellar lipid vesicles of defined composition using three types of physical measurement. Utilizing relative light scatter to estimate the dissociation constants for binding in the presence of calcium ions, it appears that factor IXa (0.93 +/- 0.37 microM) may preferentially associate with phospholipids relative to factor IX (0.35 +/- 0.08 microM). In contrast, factor X (0.34 +/- 0.14 microM), the substrate for factor IXa, appears to bind to phospholipid with a higher affinity than factor Xa (0.58 +/- 0.13 microM). These observations are compatible with the hypothesized dynamics where the forward 'traffic' is facilitated by favoring the association of factor IXa with factor X. The dissociation constants were estimated by molecular exclusion chromatography (1.1 - 2.5 microM) and do not reflect these relative and ordered differences in association with lipid vesicles. Quasi-elastic light scatter analyses indicate that each protein appears to saturate the same vesicle surface, consistent with competition for similar surface lipids, although the molecular shell formed by factor Xa (36 A) is smaller, suggesting that it has a different packing on the phospholipid surface than the other proteins (64-79 A). The pattern of preferential affinities for phospholipid is consistent with a kinetically functional forward traffic through the reaction precursors to products, and suggests that these preferential affinities may assist in the ordering of the four proteins in the intrinsic activation complex.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

The interaction of protein kinase C and other specific cytoplasmic proteins with phospholipid bilayers

The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.     

Double C2 protein. A review

Concerted effort has led to the identification of dozens of synaptic proteins and has thereby opened the door for the characterisation of the molecular mechanisms underlying regulated exocytosis. Calcium is known to play a number of roles in regulated exocytosis, acting as the trigger for fast synaptic transmission and also acting at some of the steps preceding vesicle fusion. Investigators have therefore focussed considerable attention on possible calcium sensors. What many of the candidate proteins have in common is a C2 domain, one of the four conserved domains originally described in protein kinase C. Such domains have been shown to bind calcium and phospholipid in a large number of intracellular proteins. Synaptotagmin, a C2-domain protein, is a very strong candidate for the protein involved in triggering fast calcium-dependent vesicle fusion. Recent attention has also concerned the other calcium sensors, which may play roles in the 'priming' or transport of vesicles. This review concerns one of these tentative calcium-binding proteins, double C2 or DOC2. DOC2 was originally isolated from nervous tissue but subsequently has been found to be more widely expressed. DOC2 is a vesicular protein that may be involved in the early stages of preparing vesicles for exocytosis.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values <6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

Nonspecific lipid transfer protein (sterol carrier protein 2) is bound to rat liver mitochondria: its role in spontaneous intermembrane phospholipid transfer

In the present study we have investigated the transfer of phospholipids between vesicles and rat liver mitochondria. Transfer was measured by electron paramagnetic resonance spectroscopy using vesicles that contained spin-labeled phospholipids. A spontaneous transfer was observed which could be strongly inhibited by treating the mitochondria with the thiol reagent mersalyl. Transfer was also greatly reduced after a saline wash of the mitochondria; the transfer activity was then recovered in the wash. This activity was inhibited by tryptic digestion and mersalyl. By gel chromatography, enzyme immunoassay and immunoblotting it was demonstrated that the activity in the wash was due to the nonspecific lipid transfer protein (sterol carrier protein 2). We could estimate that up to 85% of the spontaneous phospholipid transfer between vesicles and rat liver mitochondria was mediated by this transfer protein.     

Association of Pseudomonas cytochrome oxidase with liposomes

Purified Pseudomonas cytochrome oxidase has been associated with asolectin liposomes by two different methods. Firstly, the enzyme was attached to liposomic membranes by adding it to a cholate-phospholipid dispersion and subsequently dialyzing the detergent out of suspension. In the second case the enzyme was adsorbed on the preformed liposomes when added to them after the dialysis.A stimulation of the cytochrome oxidase activity approximately twenty-fold was observed by the first method. In contrast, the activation was absent in the second type of preparation, indicating that interaction between the enzyme and phospholipids is very different in the two types of vesicles.The cholate-dialysis method for reconstitution of protein-phospholipid vesicles seems to lead to rather heterogeneous preparations. These can be further fractionated, not only according to their size but also to the protein/phospholipid ratio, by gel chromatography.