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PubMed1989

Characteristics of an adenosine A1 binding site in human placental membranes.

Binding sites were solubilized from human placental membrane using 1.5% sodium cholate and were assayed using polyethylene glycol precipitation. These soluble binding sites had properties of an adenosine A1 binding site. 2-[3H]Chloroadenosine and N-[3H]-ethylcarboxamidoadenosine (NECA) binding were time dependent and reversible. Scatchard plots indicate two classes of binding sites with Kd values of 6 and 357 nM for 2-chloro[8-3H]adenosine and 0.1 and 26 nM with [3H]NECA. The specificity of [3H]NECA binding was assessed by the ability of adenosine analogs to complete for binding sites. Using this approach the estimated IC50 values were 60 nM for (R-PIA), 160 nM for S-PIA, 80 nM for NECA, and 20 nM for 2-chloroadenosine. Binding of [3H]NECA to the soluble sites is inhibited to 48% of the control value by 100 microM guanylyl-5'-imidodiphosphate (Gpp(NH)p). The IC50 value for NECA binding to the soluble binding site was increased from 80 nM to 1500 by Gpp(NH)p. There was a shift of binding affinity from a mixture of high and low affinity to only low affinity with 100 microM Gpp(NH)p. Despite these alterations a NECA prelabeled molecular species of 150 kDa did not decrease in molecular weight upon the addition of 100 microM Gpp(NH)p during high-performance liquid chromatography on a Superose 12 column. Other evidence to support the concept of preferential solubilization and assay of a small population of A1 binding sites was obtained. Following solubilization adenosine A2-like binding sites could be detected only in reconstituted vesicles. The existence of small amounts of A1 binding sites in intact human placental membranes was directly demonstrated using the A1 agonist ligand N6-[3H]cyclohexyladenosine and the A1 antagonist ligand 8-[3H]cyclopentyl-1,3-dipropylxanthine. JAR choriocarcinoma cells have "A2-like" membrane binding sites. In contrast to placental membranes, only A2-like binding sites could be solubilized from JAR choriocarcinoma cells. These observations indicate that human placental membranes contain adenosine A1 binding sites in addition to A2-like binding sites. These sites are guanine nucleotide sensitive, but do not shift to a lower molecular weight form upon assumption of a low affinity state.

2-Chloroadenosine↗

PubMed1978

Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor o.

The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission. Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights 0f 32 000, 23 000 and 15 000. The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF1-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark difference in labeling are observed in CF1-depleted chloroplasts which have been treated with N,N'-dicyclohexylcarbodiimide. The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CFo) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CFo, and the light/dark labeling differences may indicate conformational changes within CFo. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.

Cell Membrane↗

PubMed1978

Resonance Raman spectra of cytochrome oxidase. Evidence for photoreduction by laser photons in resonance with the Soret band.

Resonance Raman spectra of cytochrome oxidase solubilized in Tween 20 and sodium cholate, and excited at 413.1 nm have been recorded. Differences in the resonance Raman spectra of the two preparations are minimal indicating that the local environment of the hemes is similar in the two preparations. As in the work of Salmeen, et al. (1973) (Biochem. Biophys. Res. Commun. 52, 1100) the strongest band appears at 1358 cm-1. Some of the other bands differ slightly in their band shapes and frequencies when compared to their spectra; these differences can be accounted for by differences in resonance enhancement of the various bands wnen exciting at 441.6 and 413.1 nm. A study of the region from 1350 to 1380 cm-1 as a function of laser intensity (10--130 mW on sample) indicate that the doublet reported by Salmeen, et al. at 1358 and 1372 cm-1 is a result of photoreduction of the preparations. In samples to which potassium ferricyanide had been added, broad luminescence bands appear at 476 and 641 nm from which it is inferred that catalytic amounts of flavin in the preparations are photoreduced providing reducing equivalents to cytochrome oxidase.

Electron Transport Complex IV↗

PubMed1979

The reconstitution of energy transfer in membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas sphaeroides by addition of light-harvesting and reaction centre pigment-protein complexes.

Antenna and reaction centre complexes purified from photosynthetically-grown cells of Rhodopseudomonas sphaeroides have been mixed with cytoplasmic membranes prepared from an aerobically-grown bacteriochlorophyll-less mutant of Rp. sphaeroides (designated 01) in the presence of 1% sodium cholate. After removal of the cholate by dislysis, the dislysate was subjected to isopycnic centrifugation. Reconstituted cytochrome c2 photooxidation and cytochrome b photoreduction was demonstrated in a pigmented fraction recovered from the sucrose gradient, suggesting that the pigment-proteins were incorporated into the 01 membrane. The fluorescence properties of the system were examined. The appearance of a variable component after the initial fast fluorescence rise indicated that energy transfer occurred between the antenna and reaction centre proteins in the presence of 01 membrane. The order in which the system was assembled was important. Reconstituted energy transfer with a pre-dialysed reaction centre-antenna complex was more effective than when all the components were mixed at once. Energy transfer was also reconstituted between added reaction centre protein and the endogenous antenna present in membranes from the pigmented, but aerobically-grown reaction centre-less mutant PM8dp of Rp. sphaeroides. Preparations of 01 membranes reconstituted with reaction centre exhibited a light intensity dependent cytochrome c2 photooxidation. At low exciting light intensities, preparations containing reconstituted antenna protein in addition to reaction centres showed greated membrane cytochrome c2 photooxidation than preparations with the antenna omitted; this improvement was maximal when a pre-dialysed antenna-reaction centre complex was used.

Aerobiosis↗

PubMed1979

The incorporation of reaction centres into membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas sphaeroides.

Reaction centres purified from a blue-green mutant R-26 of Rhodopseudomonas sphaeroides can be incorporated into bacteriochlorophyll-less membranes purified from an aerobically-grown bacteriochlorophyll-less mutant 01 of R. sphaeroides. This can be accomplished by raising the temperature of the mixture or by addition of the detergent sodium cholate and its subsequent removal by dilution or dialysis. Optimum conditions for the reconstitution are at 4 degrees C in the presence of 1% cholate and soybean phospholipid (2 : 1, w/w, with membrane protein). Isopycnic sucrose density gradient centrifugation of such preparations shows that reaction centres and light-harvesting pigment-protein complex bind to the membranes. Reconstituted membranes exhibit light-induced steady-state cytochrome absorbance changes resembling those observed in chromatophores prepared from the photosynthetically-grown mutant R-26. The effect on these absorbance changes of varying reaction centre content in the membrane has been studied, and the time course of the interaction between 01 membrane cytochrome c2 and added reaction centre examined. Cytochrome b photoreduction and cytochrome c2 photo-oxidation were observed in the reconstituted preparation; each increased following the addition of antimycin A, suggesting that a cyclic light-driven system had been reconstituted.

Bacterial Chromatophores↗

PubMed1982

ATP-dependent phosphate transport in sarcoplasmic reticulum and reconstituted proteoliposomes.

During uptake of Ca2+ by rabbit sarcoplasmic reticulum, about 1 mumol of 32Pi was taken up per mumol 45Ca2+ transported. The uptake of Pi was dependent on external Ca2+, Mg2+ and ATP. Intravesicular Ca2+ did not substitute for external Ca2+. In contrast to the accumulation of Ca2+ which was abolished by the ionophore A23187, the uptake of Pi continued to take place provided sufficient Ca2+ was present in the medium. Thus, a Ca2+ gradient did not seem to be required. Similar observations were made with proteoliposomes reconstituted with membrane preparations of sarcoplasmic reticulum and soybean phospholipids. However, when purified Ca2+ -ATPase was used for reconstitution, there was ATP-dependent Ca2+ uptake but no ATP-dependent Pi transport was observed. These data show that the mechanism of Pi transport cannot be a passive movement in response to a Ca2+ gradient but appears to be catalyzed by a specific protein, which is inactivated during purification of the Ca2+ -ATPase. A protein that catalyzes Pi transport in reconstituted vesicles has been solubilized by extraction of sarcoplasmic reticulum with sodium cholate.

Adenosine Triphosphate↗

PubMed1986

Identification of ubiquinone-binding proteins in yeast mitochondrial ubiquinol-cytochrome c reductase using an azido-ubiquinone derivative.

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone, was used to study the ubiquinone-protein interaction and to identify the ubiquinone-binding proteins in yeast mitochondrial ubiquinone-cytochrome c reductase. The phospholipids and Q6 in purified reductase were removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The resulting phospholipid- and ubiquinone-depleted reductase shows no enzymatic activity; activity can be completely restored by the addition of phospholipids and Q6 or Q2. The ubiquinone- and phospholipid-replenished ubiquinonol-cytochrome c reductase is also fully active upon reconstituting with bovine succinate-ubiquinone reductase to form succinate-cytochrome c reductase. When an azido-ubiquinone derivative was added to the ubiquinone and phospholipid-depleted reductase in the dark, followed by the addition of phospholipids, partial reconstitutive activity was restored, while full ubiquinol-cytochrome c reductase activity was observed when Q2H2 was used as substrate in the assay mixture. Apparently, the large amount of Q2H2 present in the assay mixture displaces the azido-ubiquinone in the system. Photolysis of the azido-Q-treated reductase with long-wavelength ultraviolet light abolishes about 70% of both the restored reconstitutive activity and Q2H2-cytochrome c reductase activity. The activity loss is directly proportional to the covalent binding of [3H]azido-ubiquinone to the reductase protein. When the photolyzed, [3H]azido-ubiquinone-treated sample was subjected to SDS-polyacrylamide gel electrophoresis followed by analysis of the distribution of radioactivity among the subunits, the cytochrome b protein and a protein with an apparent molecular weight of 14 000 were heavily labeled. The amount of radioactive labeling in both these proteins was affected by the presence of phospholipids.

Affinity Labels↗

PubMed1976

Single bilayer vesicles prepared without sonication. Physico-chemical properties.

Single shelled lecithin vesicles of uniform size (diameter = 300 A) are prepared without sonication by solubilizing unsonicated lecithin dispersions with sodium cholate and removing the detergent from the mixed lecithin - cholate micelles by gel filtration on Sephadex G-50. A homogeneous population of pure lecithin single-bilayer vesicles free of multilamellar structures is obtained. The vesicle diameter is somewhat larger than the average diameter of sonicated vesicles. The curvature of the bilayer seems to be sufficiently large to allow for similar packing densities (areas/molecule) on the outer and inner layer of the bilayer. The morphology and some physico-chemical properties of these vesicles are described and compared with those of sonicated vesicles.

Cholic Acids↗

PubMed1978

The preparation of large single bilayer liposomes by a fast and controlled dialysis.

A new method is described for the preparation of large, homogeneously sized, single bilayer phospholipid vesicles. This method, based on a fast and controlled dialysis of sodium cholate from phosphatidylcholine/cholate mixed micelles, has the advantage of high yield of homogeneous vesicles avoiding any dilution and mechanical stress during preparation. Physicochemical properties of these vesicles are examined by several techniques and compared with those prepared with other methods.

Cholic Acids↗

PubMed1979

Effect of different physical states of phospholipid substrates on partially purified platelet phospholipase A2 activity.

Partial purification of alkaline phospholipase A2 (EC 3.1.1.4) from rabbit platelets was carried out and the effect of different physical states of the substrate phosphatidylcholine on the activity was investigated. (1) The enzyme was purified about 1020-fold by means of Sephadex gel chromatography after extraction from a particulate fraction of rabbit platelets, followed by CM-cellulose chromatography, and had a molecular weight of approx. 12 000 as determined by gel chromatography. (2) The activity of the purified enzyme was enhanced by the addition of detergents. Sodium deoxycholate and sodium cholate markedly stimulated the activity, and the effect of these substances was observed well below the critical micelle concentrations. Triton X-100 stimulated the activity moderately, and the activation was observed only above the critical micelle concentration. (3) The addition of negatively charged phospholipids to the substrate egg phosphatidylcholine induced a moderate activation of hydrolysis. (4) The addition of long-chain cation to the substrate induced an inhibition of the activity, whereas the addition of long-chain anion activated the hydrolysis of egg phosphatidylcholine, but did not activate the hydrolysis of phosphatidylcholine in the total lipid extract of rabbit platelets. (5) Hydrolysis of dimyristoyl phosphatidylcholine increased in the temperature region of the phase transition of the substrate. Addition of cholesterol at the concentration of 20 mol% diminished the effect of phase transition. (6) Release of [1-14C]arachidonic acid from an equimolar mixture of egg phosphatidylcholine with dipalmitoyl or distearoyl phosphatidylcholine was activated at the temperature of 0 degrees C or 20 degrees C, respectively. From these results, we suggest that platelet phospholipase A2 can be activated to release fatty acids from the platelet phospholipids at the domains within membranes, where exist the structural irregularities and/or accumulation of negative charge within the bilayers.

Animals↗

PubMed1981

The role of phospholipids in the modulation of enzyme activities in the chromaffin granule membrane.

(1) 93% of protein of chromaffin granule membranes can be solubilized by 1.3% (w/v) sodium cholate. The solubilized material can be substantially delipidated by ammonium sulphate precipitation. After three such cycles less than 2% of the endogenous phospholipids remain. (2) The chromaffin granule membrane Mg2+-ATPase depends on the presence of phospholipids for retention of its full activity. Soybean and extracted chromaffin granule phospholipids fully reactivate the delipidated enzyme provided only one delipidation step is used. (3) Successive ammonium sulphate precipitation steps result in a delipidated, and deactivated ATPase preparation which can be only partially reactivated on re-addition of phospholipids. (4) The phospholipid specificity for reactivation of the Mg2+-ATPase is broad. Although acidic phospholipids allow higher activities than neutral phospholipids, the main requirement appears to be the hydrophobic environment provided by the phospholipid hydrocarbon chains. (5) Correlations between changes in slope in the Arrhenius plot of the Mg2+-ATPase, and phase transitions in the phospholipid used for reactivation suggest that the 'fluidity' of the hydrocarbon chains modulates the activity of the enzyme.

Adenosine Triphosphatases↗

PubMed1981

Rapid transmembrane movement of phosphatidylcholine in small unilamellar lipid vesicles formed by detergent removal.

Small unilamellar phosphatidylcholine vesicles, formed by solubilizing phosphatidylcholine with sodium cholate and removing the detergent by gel filtration, have been studied in their interaction with phospholipid exchange protein. The exchange of phosphatidylcholine between the vesicles and erythrocyte ghosts was greatly stimulated by the phosphatidylcholine-specific exchange protein from bovine liver. It was found that 95% of the phosphatidylcholine was readily available for exchange within 3 h at 37 degrees C. In similar vesicles prepared by sonication only 70% of the phosphatidylcholine was rapidly exchangeable. Our results indicate that the transmembrane movement of phosphatidylcholine across the bilayer of vesicles prepared by the cholate technique is a relatively fast process. The results are discussed with respect to the presence of trace amounts of lipid-associated cholate which may facilitate the transbilayer exchange of phosphatidylcholine.

Animals↗

PubMed1985

Preparation of a highly concentrated, completely monomeric, active sarcoplasmic reticulum Ca2+-ATPase.

Sarcoplasmic reticulum vesicles from fast skeletal muscle were partially delipidated with sodium cholate at high ionic strength and sedimented in a discontinuous sucrose gradient. Phospholipid content was reduced from 0.777 mumol/mg protein to 0.242 mumol/mg protein. As judged from gel electrophoresis and high pressure liquid gel chromatography, accessory proteins were removed during centrifugation and the Ca2+-ATPase was obtained in an almost pure form. Addition of myristoylglycerophosphocholine (1 mg/mg protein) reactivates ATPase and dinitrophenylphosphatase activity to the same degree obtained with native vesicles. Using the analytical ultracentrifuge it could be demonstrated that the reactivated Ca2+-ATPase was present exclusively in a monomeric state. These results were obtained at high and low ionic strength and up to a protein concentration of 10 mg/ml. Therefore this preparation should be very useful to investigate differences between oligomeric and monomeric Ca2+-ATPase.

Animals↗

PubMed1987

Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles.

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.

Animals↗

PubMed1989

Study of the transcortin binding to human endometrium plasma membrane.

Transcortin complexed with progesterone was shown to bind specifically to the plasma membrane of human decidual endometrium. The binding reaction was characterized by a high affinity (an apparent Kd value was (1.0 +/- 0.2).10(-10) mol/l) and high selectivity: such human serum proteins as albumin, orosomucoid, transferrin, thyroxine-binding globulin and sex hormone-binding globulin did not compete with transcortin for the membrane binding sites. Transcortin binding to the membrane was steroid-dependent: transcortin-cortisol complex bound to the membranes substantially more weakly than transcortin-progesterone, and specific binding of transcortin devoid of steroid was not detected. Using a radioimmunoassay, we have measured the concentration of endogenous transcortin in highly purified membrane preparations solubilized with sodium cholate. It was found that an extensive washing of decidual strips with a physiological buffer prior to the membrane isolation resulted in a decrease of the endogenous transcortin level along with an increase of the specific membrane binding of exogenous 125I-labeled transcortin. Affinity chromatography on immobilized transcortin was used to isolate transcortin-binding components from 125I-labeled, cholate-solubilized plasma membrane of decidual endometrium. Along with lipid components, the structure of which was not investigated, a 125I-labeled transcortin-binding sialoglycoprotein with a minimal Mr of 20.0 +/- 1.5 kDa and a pI of approx. 3.3 was detected. In the presence of transcortin, this sialoglycoprotein could be precipitated with a monospecific antitranscortin antiserum. Using hydroxylapatite as a separating agent, the interaction of transcortin and the membrane sialoglycoprotein in model systems containing the two proteins and various steroid hormones was studied. It was found that the membrane sialoglycoprotein displayed a higher affinity for transcortin-progesterone than for transcortin-cortisol (the Kd values were, respectively, 2.10(-11) and 7.10(-11) mol/l) and it did not bind transcortin complexed with testosterone.

Blood Proteins↗

PubMed1992

Reconstitution of the sarcoplasmic reticulum Ca(2+)-ATPase: mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents.

The Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum was reconstituted into sealed phospholipid vesicles using the method recently developed for bacteriorhodopsin (Rigaud, J.L., Paternostre, M.T. and Bluzat, A. (1988) Biochemistry 27, 2677-2688). Liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, sodium cholate or dodecyl octa(oxyethylene) glycol ether (C12E8) and protein incorporation was studied at each step of the liposome solubilization process by each of these detergents. After detergent removal by SM-2 Bio-Beads the resulting vesicles were analyzed with respect to protein incorporation by freeze-fracture electron microscopy, sucrose density gradients and Ca2+ pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With octyl glucoside, direct incorporation of Ca(2+)-ATPase into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes homogeneous in regard to protein distribution. With the other detergents, optimal Ca(2+)-ATPase pumping activities were obtained when starting from Ca(2+)-ATPase/detergent/phospholipid micellar solutions. However, the homogeneity of the resulting recombinants was shown to be dependent upon the detergent used and in the presence of Triton X-100 or C12E8 different populations were clearly evidenced. It was further demonstrated that the rate of detergent removal drastically influenced the composition of resulting proteoliposomes: upon slow detergent removal from samples solubilized with Triton X-100 or C12E8, Ca(2+)-ATPase was found seggregated and/or aggregated in very few liposomes while upon rapid detergent removal compositionally homogeneous proteoliposomes were obtained with high Ca2+ pumping activities. The reconstitution process was further analyzed by centrifugation experiments and the results demonstrated that the different mechanisms of reconstitution were driven predominantly by the tendency for self-aggregation of the Ca(2+)-ATPase. A model for Ca(2+)-ATPase reconstitution was proposed which accounted for all our results. In summary, the advantage of the systematic studies reported in this paper was to allow a rapid and easy determination of the experimental conditions for optimal detergent-mediated reconstitution of Ca(2+)-ATPase. Proteoliposomes prepared by the present simple method exhibited the highest Ca2+ pumping activities reported to date in Ca(2+)-ATPase reconstitution experiments performed in the absence of Ca2+ precipitating agents.

Calcium↗

PubMed1993

Conjugation of apolipoprotein B with liposomes and targeting to cells in culture.

Mixed phospholipid/cholesterol (2:1 molar ratio) liposomes were conjugated with native and acetylated apolipoprotein B (apoB), the protein part of low density lipoprotein (LDL). The objective was to increase the specificity of the cellular uptake of liposomes by utilization of the LDL and scavenger receptor pathways. The method of choice for the conjugation of liposomes with apoB proved to be the detergent solubilization and removal procedure. Two detergents were tested;sodium cholate (NaC) and octyl glucoside (OG). The integrity of the resulting complexes was demonstrated by Sepharose CL-4B gel chromatography and Metrizamide gradient centrifugation. The conjugates showed a good physical stability and the leakiness was only marginally larger than for unconjugated liposomes. The interaction of apoB- and acetyl apoB-liposome conjugates with CV-1 and J774 cells, respectively, was monitored by an encapsulated pH-sensitive fluorophore, pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS)). This dye provides means of detecting binding and endocytosis of conjugates in living cells. The internalization was a fast process and about 10-times faster for the OG-conjugates than for the corresponding unconjugated liposomes. The conjugates showed a clear concentration-dependent association of dye with cells, while this was less prominent with liposomes. The uptake was nearly an order of magnitude faster with CV-1 cells than with J774 cells. Acidification of intracellular conjugates proceeded fast during the first 30 min of incubation and reached a minimum value of approx. pH 6 after 3 h. The specificity of binding of apoB-liposome conjugates to CV-1 cells was demonstrated by displacement experiments with native LDL. The results indicate that apoB-liposome conjugates may be used as a delivery vehicle for bioactive subtsances to cells.

Animals↗

PubMed1976

A phospholipase A2 with anticoagulant activity. I. Isolation from Vipera berus venom and properties.

An anticoagulant protein has been isolated by DEAE cellulose chromatography and gel filtration from the venom of the Vipera berus orientale (Eastern Europe). Purification has been completed by elution on carboxymethyl cellulose with continuous gradient at constant pH. The inhibitor of coagulation was separated from the other venom enzymes, e.g. procoagulant, fibrinogenolytic, aminoesterase and amino acid oxidase activities. It was also separated from other phospholipase components which were not related to the anticoagulant property. The inhibitor appeared as a simgle polypeptidic chain protein, formed by 119 amino acid residues, with a molecular weight of 13400 and an isoelectric point of 9.2. At low saline molarity, a monomer-trimer transition of this protein was observed. Both forms had the same amino acid composition. There were six disulfide bridges without free SH groups per phospholipase molecule. Deprived of any proteolytic activity, the clotting inhibitor displayed a high phospholipase activity in the presence of calcium. Activity did no appear with EDTA buffer deprived of cation. Finely dispersed micellar suspensions were found suitable for obtaining the highest phospholipase activity. High sodium cholate concentration or methanol/chloroform/ether solvent were effective without loss of enzymatic activity. As characteristis of phospholipase A2 (EC 3.1.1.4), the degradation products identified on thin-layer chromatography induced hemolysis of human erythrocytes. The apparent Km value 1.25 - 10(-3) M was determined on phosphatidylcholine isolated from ovolecithin. This purified berus inhibitor would be of value for investigating the involvement of phospholipids in the clotting mechanism.

Amino Acids↗