Peter G. Barton

Osmometric and microscopic studies on bilayers of polar lipids from the extreme halophile, halobacterium cutirubrum

Abstract The major membrane polar lipid components in Halobacterium cutirubrum are the diphytanyl ether analogues of phosphatidylglycerol phosphate, glycolipid sulfate, phosphatidylglycerol and phosphatidylglycerol sulfate. Dispersions of total polar lipids in water formed large birefringent liposomes showing concentric lipid bilayers in the elctron microscope; they behaved as ideal osmometers in KCl or NaCl solutions in the concentration range 0.005–0.2 M. At concentrations above 0.2 M KCl the liposomes shrank to spherical particles which were much less birefringent, showed no distinct bilayer structures by electron microscopy, and no longer behaved as ideal osmometers. Dispersions of phosphatidylglycerol phosphate, phosphatidylglycerol or phosphatidylglycerol sulfate alone did not behave as osmometers at any concentration of KCl or NaCl, but glycolipid sulfate alone or mixed with phosphatidylglycerol phosphate or phosphatidylglycerol phosphate + phosphatidylglycerol sulfate showed ideal osmometer behavior in 0.005–0.2 M KCl or NaCl. The highly negatively charged total polar lipids of H. cutirubrum thus can form stable lipid bilayers only at low ionic concentrations (0.005–0.2 M), much lower than the salt concentration (4 M) of the growth medium, and the presence of glycolipid sulfate is essential. Stability of the membrane in 4 M salt appears to require direct participation of the protein components.

Mesomorphic behaviour of some phospholip1ds with aliphatic alcohols and other non-ionic substances

The mesomorphic behaviour of some ternary systems, each containing a diether or diester phospholipid, water and a non-ionic substance, has been investigated principally by differential thermal analysis and polarization microscopy. Trace amounts of n-butyl acetate and diethyl ketone lower the transition temperature of partially hydrated phospholipid to a value close to the limiting value for a gel to liquid-crystalline phase change. The influence of n-alkanols is dependent on their chain length and can be interpreted, in part, on the basis of their distribution coefficients between water and n-alkane. Dodecanol and tetradecanol form “association complexes” of definite stoichiometry with 1,2-ditetradecanoyl-sn-glycero-3-phosphorylcholine

Studies on the stability of Bovine plasma factor V

Abstract (NH4)2SO4 precipitation and cellulose phosphate chromatography, commonly used to prepare plasma Factor V, have been found to produce degradation products of lower molecular weight and higher specific activity than the native protein. The so-called forms L and C ( G. Philip, J. Moran and R. W. Colman , Biochemistry, 9 (1970) 2212) were shown not to be constituents of native Factor V but instead artifacts of preparative procedures. The inclusion of Ca2+ in the eluting buffer during gel filtration of bovine plasma Factor V allowed retention of most of the biological activity generally lost by this procedure. Concentration of Ca2+ higher than 0.001 M depressed the activity of factor V as measured in the one-stage assay.

Effects of α,β-methylene-adenosine-5′-diphosphate on blood platelet aggregation

The effects on platelet aggregation of α,β-methylene-adenosine-5′-diphosphate (Ado-PCP) have been investigated. Using human citrated platelet-rich plasma it has been shown that: (i) at concentrations of 10−3 M or higher Ado-PCP is able to induce platelet aggregation; (ii) the rate of Ado-PCP-induced aggregation increases on raising the pH of platelet-rich plasma above the pKa for the secondary phosphonyl dissociation of Ado-PCP; (iii) at concentrations from 1 · 10−4 to 5 · 10−4 M Ado-PCP does not cause platelet aggregation itself, but it inhibits ADP-induced aggregation. This inhibition is also observed in washed platelet suspensions. The data suggest that Ado-PCP acts at the same site on the platelet membrane as does ADP and that ADP to AMP transformation is not a prerequisite for the process of aggregation. The observed effect of pH on the rate of Ado-PCP induced aggregation suggests that the ionization state of a nucleotide terminal acid group is important in the process of aggregation.

The influence of cholesterol on the activity of phospholipids in blood coagulation: Requirement for a liquid-crystalline lipid phase

Abstract A series of blood clotting tests including a specific activated factor X assay ∗∗ , a thromboplastin generation test (TGT) † and a partial thromboplastin time (PTT) have been used to examine the activity of phosphatidyl serine (PS) in the presence and absence of cholesterol. In all the tests, PS from bovine brains showed a high level of activity which was potentiated by cholesterol to varying degrees. Cholesterol alone had no effect. Hydrogenated PS alone showed no activity but in all cases activity was largely restored by addition of cholesterol. The restoration of activity was shown to be related to the transition temperatures of the lipid mixtures in water as measured by differential thermal analysis. It was concluded that a liquid-crystalline phase is essential for the activities observed. Cholesterol may also act as an inert spacer for the acidic phospholipid molecules.

Interactions of phosphatides with some paraffin-chain salts☆

Abstract The interactions of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidic acid and hexadecylamine hydrochloride with cetyltrimethylammonium bromide and sodium dodecylsulphate have been examined by means of acid-base titration and microelectrophoresis of the dispersed particles. The data obtained can be interpreted satisfactorily by a combination of four effects: 1. a.|a Boltzmann distribution of hydrogen counterions from a charged surface into the bulk aqueous phase. 2. b.|small shifts in pK related to changes in the continuous and molecular environment in the interface. 3. c.|restricted access of titrant to functional groups in coarse dispersions 4. d.|intermolecular salt formation between adjacent ions in the interface The significance of these findings in connection with the role of phospholipids in blood coagulation is discussed.

13C Nuclear magnetic resonance spectra of 1,2-dioctadec-cis-enoyl-sn-glycero-3-phosphoryl-cholines

Abstract The 13 C NMR spectra of all sixteen 1,2-dioctadec- cis -enoyl- sn -glycero-3-phosphorylcholines have been obtained. Resonance lines of the olefinic, methylene, methyl and carboxyl carbon nuclei are sufficiently characteristic to permit unequivocal designation of double bond position for each isomer. Two resonances of the sn -glycero-3-phosphorylcholine structure have been reassigned.

Effects of polyoxyethylene detergent (Brij 58) on platelet aggregation, release and clotting activity

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet-rich plasma but had no effect on primary aggregation. Thrombin-induced aggregation of washed human platelets suspended in Tyrodes buffer was inhibited after incubation of cells with 4.10(-6) M detergent. Efflux of [14C]serotonin, 45Ca2+ and labile aorta contracting substance (thromboxane A2) and development of prothrombin-converting activity (platelet factor 3) were abolished concomitantly. Aggregation of washed platelets either by sodium arachidonate or by collagen was also inhibited by the same concentration of Brij 58 which inhibited thrombin aggregation. This concentration did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 4.10(-5) M, lysed the cells liberating lactate dehydrogenase, serotonin and Ca2+. When albumin was included as a platelet stabilizer in the suspending medium the concentration of detergent required for the inhibitory effects was increased ten-fold. This could be attributed to competitive binding of the detergent to albumin, demonstrated with [14C]acetylated Brij 58. A variety of other polyoxyethylene detergents, at concentrations from 8.10(-4) to 5.10(-3) M, also inhibited platelet aggregation induced by thrombin. It is concluded that low concentrations of Brij 58 stabilize the platelets against the action of aggregating agents, while higher concentrations produce membrane destabilization and cell lysis.

Phase transitions of sterols and sterol—lecithin sterol—lecithin mixtures

Abstract Sterols exhibit reversible htermal transitions below their melting points which are dependent on the state of hydration and on the structure of the aliphatic substituent at C 17 . The endotherm exhibited by cholesterol can be abolished by mixing with hydrated phospholipids at molar ratios below 1 : 1 but reappears in a metastable form at molar ratios between 1 : 1 and 2 : 1.

Molecular characteristics of bovine factor X activated in concentrated sodium citrate solution.

Abstract Preparations of the zymogen form of bovine factor X were incubated in 25% w v sodium citrate at room temperature. The rate of activation of factor X was dependent on the extent of contamination with factor VII, prothrombin, and thrombin. The activated factor X was isolated by DEAE-cellulose chromatography. Analysis of the final product by sedimentation velocity centrifugation coupled with measurements of the rate of boundary spreading, high-speed sedimentation equilibrium, and gel filtration chromatography provided evidence for a single molecular species undergoing reversible association-dissociation with a monomeric molecular weight of 48,000. In the absence of mercaptoethanol a single band was seen by disc electrophoresis and by SDS-acrylamide electrophoresis but after disulfide reduction two components of molecular weights 30,000 and 17,000 were visible. The protein contained large amounts of acidic amino acids but no carbohydrate. The N -terminal amino acids were alanine and isoleucine and 1 mole C -terminal arginine per mole protein was found. These characteristics are very similar to those of factor X activated with Russells viper venom. When a BaSO 4 eluate of bovine plasma rich in prothrombin was allowed to stand in 25% sodium citrate both thrombin and activated factor X were generated. Chromatography of the isolated activated factor X on Sephadex G-200 as well as disc electrophoresis showed that it behaved identically with the enzyme obtained from purified zymogen and was clearly distinguishable from autoprothrombin c, a glycoprotein possessing qualitatively similar biological activity (Seegers, W. H., Cole, E. R., Harmison, C. R., and Marciniak, E. (1963) Can. J. Biochem. Physiol. 41 , 1047).