Application of high-performance liquid chromatography for the study of the microheterogeneity changes of mouse alpha-fetoprotein in fetal development.

Changes in the microheterogeneity of mouse alpha-fetoprotein (MAFP) during fetal development were investigated by high-performance liquid chromatography (HPLC). A total of six distinct isoforms (Iso-1, Iso-2, Iso-3, Iso-4, Iso-5, Iso-6) of the heterogeneous MAFP were resolved from mouse amniotic fluid (MAF). Analysis of MAF collected at various times revealed that these isoforms were accumulated at different stages of the fetal development.

Purification and properties of two phospholipid transfer proteins from yeast.

Several intracellular proteins of low and intermediate molecular weights have been isolated from a variety of mammalian and plant tissues that possess an ability to catalyze the transfer or exchange of intact phospholipid molecules between different membrane systems. The soluble cytosolic fraction of the yeast Saccharomyces cerevisiae also contains phospholipid transfer activity that varies with both the state of cellular growth and the type of metabolic carbon source. This activity is protein in nature and very unstable, and requires powerful separation techniques for its purification. Here we report the isolation and characterization of two phospholipid transfer proteins from yeast, one of which we believe represents a partial proteolytic product of the other. The two proteins were purified to near homogeneity through a combination of dye-ligand and high performance ion-exchange chromatographic techniques. Transfer protein I (TP-I) is eluted at a lower ionic strength from an anion-exchange column than transfer protein II (TP-II), which reflects the difference in their isoelectric points; TP-I has a pI of 6.3, while that for TP-II is 6.1. Both species have the same apparent molecular weight of 33,400 and virtually identical substrate specificities. The order of the relative rates of phospholipid transfer are phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol greater than phosphatidylserine.

Catalytic properties of the yeast phospholipid transfer protein.

The properties of the phosphatidylcholine (PC) transfer reaction catalyzed by the yeast phospholipid transfer protein (TP-I) were examined in vitro. Donor and acceptor membranes consisted of unilamellar (ULV) and multilamellar (MLV) vesicles, respectively. The phospholipid composition of the membranes participating in the transfer reaction, and in particular that of the MLV acceptors, have a tremendous effect upon the rate of PC-catalyzed transfer. Phosphatidylethanolamine (PE) is an essential component of the acceptor membrane, but it alone is not sufficient to sustain appreciable transfer rates. If combined in an equimolar ratio with PC, there is only a modest increase in transfer rates. On the other hand, when combined with alternate substrates such as phosphatidylinositol (PI) or phosphatidylserine (PS), very high rates of PC transfer occur. The measurement of transfer rates is not affected by the molecular species of PC used as the radioactive tracer. Evidence is also presented to indicate that the two forms of the transfer protein (TP-I and TP-II) are not identical in terms of their interactions with a membrane surface: differences occur in the levels of transfer of PC, PE, PI, and PS at equilibrium. Finally, by kinetic analysis, the mechanism of the protein-catalyzed transfer of PC is shown to conform to a ping-pong bibi model with excess substrate inhibition, analogous to ordinary two-substrate enzyme-catalyzed reactions. Both the rates of desorption and adsorption of the protein from the surface of the ULV are much greater than those describing the similar interactions of the protein with MLV.(ABSTRACT TRUNCATED AT 250 WORDS)

Unsaturated fatty acids inhibit ADP- arachidonate-induced platelet aggregation without affecting thromboxane synthesis.

The inhibitory effects of three cis-unsaturated C18 fatty acids (oleic, linoleic, and linolenic acids, sodium salts) on ADP- and sodium-arachidonate-induced aggregation of washed rabbit platelets were investigated. When the platelets were suspended in protein-free medium containing dextran, it was found that these fatty acids at very low concentrations (2-45 microM) were potent inhibitors of platelet responsiveness and the inhibitory effect occurred within seconds. The inhibition of ADP-induced aggregation was not affected by abolishing the activity of platelet cyclooxygenase using aspirin. Human serum albumin relieved the inhibition caused by fatty acids for both ADP- and arachidonate-induced aggregation. The inhibitory effect of fatty acids does not seem to be due to decreased thromboxane formation (except possibly in the case of linolenate), and the relief of fatty acid inhibition by albumin does not potentiate thromboxane B2 formation from exogenous arachidonate. It is suggested that the inhibitory effect of polyunsaturated fatty acids on platelet aggregation is specific and not related to a general surfactant effect, since inhibition occurs far below the critical micelle concentration of fatty acid soaps.

The modulation by serum albumin of rabbit platelet aggregation in response to exogenous sodium arachidonate.

This work presents a quantitative study of the modulation of platelet responsiveness to sodium arachidonate by serum albumin. Rabbit platelets suspended in protein-free buffer containing dextran aggregate reversibly in response to micromolar amounts of ADP and sodium arachidonate. The optimal concentration of arachidonate for aggregation response is 5 microM. Inhibition occurs at higher concentrations and is not related to thromboxane A2 formation since arachidonate inhibits ADP-induced aggregation of aspirin-treated platelets. Thin-layer chromatographic studies show that, at the high arachidonate levels sufficient to almost completely abolish platelet aggregation, the synthesis of thromboxane A2 persists. Albumin relieves the inhibition caused by excess arachidonate, whether the stimulus is arachidonate itself or ADP. This effect is due to arachidonate binding and is optimal at fatty acid/protein ratios near 4; no stimulation of platelets was observed at ratios less than 2. The optimal concentration of arachidonate for stimulation of platelet aggregation occurs in the range where albumin buffers the free arachidonate concentration most effectively, hence the extremely narrow range of total arachidonate concentrations effective for platelet response seen in the absence of albumin is enormously broadened in the presence of albumin. Albumin inhibits conversion of arachidonate to thromboxane A2 and hydroxy acids, especially at ratios of arachidonate/albumin below 10. Bilirubin (an albumin ligand) has no effect on albumin modulation of platelet response until the bilirubin/protein ratio exceeds 2. Palmitate progressively displaces arachidonate from albumin and affects the range of effective arachidonate concentrations but not the maximum response.

Bovine serum albumin: characterization of a fatty acid binding site on the N-terminal peptic fragment using a new spin-label.

A new spin-label, 4-(L-glutamo)-4'-[(1-oxy-2,2,5,5-tetramethyl-3L-pyrrolidinyl )amino]-3, 3'-dinitrodiphenyl sulfone, is shown to bind to one high-affinity binding site on bovine serum albumin (K = 5 X 10(4) M-1, n = 1). Analysis of the binding of the spin-label to the amino-terminal half (peptic fragment PB) and the carboxy-terminal half (peptic fragment PA) of BSA, and their complex (PA-PB), indicates that the spin-label binds to a long-chain fatty acid binding site located on PB. The usefulness of the novel specificity of the spin-label in characterizing this binding site is discussed.

Retinoic acid stimulates 1,25-dihydroxyvitamin D3 binding in rat osteosarcoma cells.

Since several aspects of the effects of vitamin A and 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on bone metabolism are quite similar, we examined the possibility that vitamin A effects on bone were mediated through the regulation of cytosolic 1,25-(OH)2D3 receptors. A clonal osteoblast-like cell line derived from rat osteosarcoma (ROS 17/2) was used as a model system. Vitamin A acid (retinoic acid) in concentrations ranging from 10(-8) to 10(-5) M was found to elicit a dose-dependent increase in 1,25-(OH)2D3 binding in these cells. This effect was maximal after 24 h, was independent of cell density, and was inhibited by actinomycin D (0.05-0.5 microgram/ml). The 1,25-(OH)2D3 binding macromolecule in cytosol preparations from both vehicle- and retinoic acid-treated cells had a sedimentation coefficient of 3.2 S and binding specificities for vitamin D3 metabolites in the order: 1,25-(OH)2D3 greater than 25-(OH)-D3 greater than 24,25-(OH)2D3. Sucrose density gradient analysis, vitamin D3 metabolite displacement studies, and saturation and Scatchard analyses all indicated that the specific increase in 1,25-(OH)2[3H]D3 binding in these cells was the result of a selective increase in the number of specific 1,25-(OH)2D3 receptors.

Thermodynamics of mixing of dipalmitoyl phosphatidylcholine and egg phosphatidylcholine in hydrated bilayers.

Dipalmitoyl phosphatidylcholine (DPPC) and egg phosphatidylcholine (egg PC) are not completely miscible at all temperatures. Their phase diagram was determined by differential scanning calorimetry (DSC) of aqueous mixtures of the two. From the integrated DSX curves we obtained the enthalpy of solution of DPPC in egg PC delta hs, as a function of the mole fraction of DPPC, X, and using the empirical relationship between delta hs and X, the solubility Xsat as a function of temperature, T. The latter could be described by the semiempirical relationship: R1nXsat = a + blnT - c/T, where a = 6.57 X 10(-2) kcal.mol-1. degree -1 and c = 20.5 kcal. mol-1 (1 cal = 4.1868 J); the coefficient b was very small and could be ignored. The quantity delta hs can be given as XdeltahDPPC + deltah mix, where deltah DPPC is the gel - liquid crystalline transition enthalpy of DPPC (8.74 kcal.mol-1) and deltah mix is the enthalpy of mixing the two liquid crystalline lipids. Deltahmix depends on X in approximately a parabolic fashion, having a maximal value of 4.8 kcal.mol-1 at X = 0.6. It was shown that both the solubility and mixing enthalpy data can be described by the theory of regular solutions (RST). In RST, the activity coefficient of the solute (component 2) of a binary solution is given by RTIngamma 2 = (1 - Theta2)2deltaU, while the mixing enthalpy is given by delta hmix = Theta1 Theta2 delta U/v2, where Theta1 and Theta2 are the volume fractions of solvent and solute (egg PC and DPPC, respectively), v2 is the partial molar volume of DPPC, and deltaU is the energy change per mole on interchanging a DPPC and an egg PC molecule between their respective liquid crystalline phases. The thermodynamic data are accurately described by RST, the molar volume of DPPC being found to be about half that of egg PC solution and the interchange energy deltaU having a value of 10-11 kcal.mol-1. There was some evidence that deltaU may be an increasing function of temperature. The large value of the deltaU accounts for the pronounced temperature dependence of the solubility Xsat, which decreases from 0.35 at 35 degrees C to 0.02 at 10 degrees C. The presence of cholesterol in the mixtures decreases both the transition enthalpy of DPPC and the mixing enthalpy in a linear fashion, so that deltahs is zero at Xcholesterol greater than or equal to 0.2. The results are consistent with recent data including the formation of a PC-cholesterol complex oc stoichiometry approximately 4:1.

Heterogeneous catalysis by phospholipase A2: mechanism of hydrolysis of gel phase phosphatidylcholine.

Hydrolysis of gel phase dipalmitoylphosphatidylcholine (DPPC) at 37 degrees C catalysed by Crotalus atrox phospholipase A2 (PLA) is described extremely well by the "path 1" kinetic mechanism of Tinker and Wei (1979) (Can. J. Biochem. 57, 97-106), if reversible adsorption is allowed as a side reaction. Progress curves show an initial rapid phase, the initial velocity being a Michaelis-Menten function dependent on the catalytic properties of the enzyme (kcat approximately equal to 9200 min-1, Km approximately equal to 0.12 mM), then level off to a slower rate determined by the desorption equilibrium constant (KD approximately equal to 0.01 mM) and desorption rate constant (kD approximately equal to 0.15 min-1). The relaxation time, tau, for the transition to the desorption-limited reaction is approximately 0.5 min; this large value of tau probably arises from a slow conversion of active, dimeric enzyme to an inactive protein species adsorbed to the lipid surface. At later times in the reaction there is an increase in the rate of hydrolysis, attributed to a stimulation of desorption by the products. The desorption equilibrium constant KD is a quadratic function of the surface concentration of products and increases 20- to 30-fold when all accessible substrate is hydrolysed. Both lysophosphatidylcholine (lyso-PC) and fatty acid were found to stimulate the desorption, but lyso-PC was also found to be a competitive inhibitor of the catalysis. Adsorption of PLA to DPPC and egg PC vesicles was directly measured using a gel partition technique. Strong binding to egg PC was observed, which was not dependent on the presence of calcium ion (essential for catalysis); PLA inhibited by acylation of up to four lysine residues per mole of monomeric enzyme with ethoxyformic anhydride was equally strongly adsorbed, indicating that lipid binding is not dependent on catalytic activity. Reaction products greatly weakened the binding of PLA to the lipid surface as expected. Cholesterol had two effects on the hydrolytic reaction: there was a striking decrease in the rate of the slower, desorption-limited phase, the rate of which decrease to almost zero at 15 mol% cholesterol, but there was also evidence for the formation of a complex with stoichiometry 1 cholesterol: 2 DPPC in which DPPC is no longer a substrate for the enzyme. Implications of the proposed mechanism for specificity and control of surface catalysis by PLA are discussed.

Heterogeneous catalysis by phospholipase A2: formulation of a kinetic description of surface effects.

The kinetics of hydrolysis of aqueous dispersion of long-chain, saturated phosphatidylcholines (PC) catalysed by Crotalus atrox phospholipase A2 (PLA) have been analyzed, and a reaction mechanism proposed which takes surface effects into account. PLA is proposed to form an enzyme-substrate complex with surface substrate molecules, thereby undergoing a conformational change which exposes sites that interact with the lipid surface. After a hydrolytic event, the enzyme can either desorb from the surface (path 1), or diffuse along the surface to an adjacent substrate molecule (path 2). The path 1 dominated mechanism leads to Michaelis-Menton steady-state kinetics, and characterizes hydrolysis of gel phase PC. Evidence for saturation of the surface with PLA was obtained at high enzyme concentrations. The path 2 mechanism dominates when the desorption rate is very small; this mechanism describes hydrolysis of liquid crystalline phase PC and is characterized by an initial burst of hydrolysis followed by a very slow reaction. The velocities in these two phases of the reaction are independent of bulk PC concentration. When gel and liquid crystalline PC phases coexist, as in mixtures of dimyristoyl- and distearoyl-PC, the liquid crystalline phase is preferentially hydrolysed. Products of the reaction (lyso-PC and fatty acid) stimulated hydrolysis, apparently by stimulating desorption of PLA. The desorption rate constant appears to be a linear function of the surface concentrations of lyso-PC and fatty acid. The proposed model describes hydrolysis progress curves extremely well and is consistent with current ideas on the mechanism of catalysis by this enzyme.

Kinetics of hydrolysis of dispersions of saturated phosphatidylcholines by Crotalus atrox phospholipase A2.

Dispersions of lamellar phase dipalmitoyl phosphatidylcholine (DDPC) and dimyristoyl phosphatidylcholine (DMPC) in 0.01 M CaCl2 were subjected to hydrolysis by phospholipase A2 (EC 3.1.1.4) from Crotalus atrox venom. The reaction was followed continuously by titrating the released fatty acids. For hydrolysis of gel phase phosphatides, the steady-state initial velocities were hyperbolic functions of bulk lipid concentrations. At the 'pre-transition' temperature (34 degrees C for DPPC, 15 degrees C for DMPC), there was a large increase in the Michaelis parameter Vmax but no change in the parameter Km. A model was devised to account for these observations, in which the enzyme desorbs from the lipid surface after hydrolysis. The desorption rate constant is postulated to increase above the pretransition temperature. For hydrolysis of liquid crystalline phosphatides, the reaction consisted of a short initial burst of hydrolysis, a long 'lag' period of very slow reaction, followed by a dramatic increase in the reaction rate. Addition of 10 mol% lysolecithin or fatty acid abolished the 'lag' period. It was postulated that the enzyme adsorbs irreversibly to the surface of the liquid crystalline phase. Reaction products are postulated to stimulate desorption of enzyme from the surface. Thus, temperature-dependent changes in the rate of hydrolysis of dispersed phosphatidylcholines are attributed to changes in the rate of desorption of the enzyme from the lipid surface.

Thermodynamic and conformational studies on an immunoglobulin light chain which reversibly precipitates at low temperatures.

A lambda light chain, isolated from an immunoglobulin G molecule, was found to reversibly precipitate at low temperatures. This cryoprecipitation was a function of pH, ionic strength, protein concentration, and time as well as temperature. The lambda chain underwent a cooperative conformational change as the temperature was lowered from 26 to 0 degrees C as judged by ultraviolet difference spectroscopy and circular dichroism. Normal lambda chains showed no conformational change. By difference spectroscopy it was possible to calculate the equilibrium constant governing the conformational change. The change was strongly exothermic (delta H approximately -80 kcal mol-1) and accompanied by a large decrease in entropy (delta S approximately -280 eu). The midpoint of the transition was dependent on the initial protein concentration, suggesting that only the noncovalent dimer of the lambda chain exhibited the conformational change. The existence of a monomer-dimer eqiulibrium (KA approximately 4 X 10(5) M-1) was confirmed by sedimentation velocity. No conformational change was observed by circular dichroism at concentrations where greater than 95% of lambda chain was in the form of a monomer. Although high ionic strength inhibited cryoprecipitation, it had no effect on the conformational change. Stabilization of the dimer by forming an interchain disulfide bond between two monomers abolished both the conformational change and cryoprecipitation. A fragment corresponding to the constant region was isolated from both peptic and tryptic digests of the lambda chain. This fragment neither cryoprecipitated nor showed temperature dependence conformational changes. It proved impossible to isolate a fragment corresponding to the variable region. Both qualitative and quantitative models are presented to account for the behavior of the lambda chain at low temperatures.

Detection of lipid phase transitions by surface tensiometry.

A technique for the detection of lipid phase transitions is described, which involves measurement of the surface tension as a function of temperature. In the case of insoluble lipids, such as dipalmitoylphosphatidylcholine (DPPC) the lipid is spread as a multibilayer film on an aqueous substrate, while in the case of water-soluble lipids such as lysophosphatidylcholine (LPC) the surface tension of aqueous sols is measured. Surface tension at the interface, is monitored using a Wilhelmy plate while the temperature is continuously varied. Discontinuities or changes in slope in the surface tension-temperature (gamma-T) curve reflect phase transitions in the lipid. In the case of DPPC, the technique has been used to demonstrate the well-known gel-liquid crystalline thermal transition. This occurs at 36-38 degrees C in the multibilayer films; in bulk DPPC-water dispersions the transition is at 41 degrees. Cholesterol has the effect of lowering the thermal transition and broadening the temperature range. In films containing DPPC-cholesterol at a molar ratio of 2:1 or less, the transition is not present. These results are in agreement with a large number of previous studies of this system. In the case of LPC sols, a phase transition at about 70 degrees was detected when the concentration of SPC was close to the critical micelle concentration (CMC) at 70 degrees. This transition appears to reflect an increase in the equilibrium constant for micelle formation at this temperature. At higher concentrations of LPC a transition at 30 degrees, corresponding to a gel-liquid crystalline transition, was also detected. A complete description of gamma as a function of concentration and temperature in the range 10(-7) to 10(-3) g cm-3 and 20 degrees to 80 degrees has been obtained for LPC sols. The CMC varies from 6 X 10(-6) g cm-3 at 20 degrees to 10(-5) g cm-3 at 80 degrees.

Perturbation of lecithin bilayer structure by globoside.

The ultrastructure of aggregates formed by mixtures of pig erythrocyte lecithin, cholesterol and globoside in aqueous systems was studied by electron microscopy and X-ray diffraction. Globoside and lecithin in up to equimolar amounts formed a lamellar mesophase, although the structure of the lamellae was perturbed. Mixtures containing excess globoside formed complex tubular or reticular aggregates. Cholesterol appeared to promote mixing of lecithin and globoside. The flexibility gradient of the hydrocarbon (hc) region of the lipid bilayers was studied using electron spin resonance (esr) spectroscopy of various nitroxide-labelled stearic acid probes. Globoside in equimolar amounts greatly perturbed the order parameters of lecithin bilayers, reducing the fluidity of the hc region and flattening the flexibility gradient near the polar (p) surface. The effect of globoside on lecithin-cholesterol bilayers was not so pronounced, since the latter was already more ordered than lecithin bilayers. A phase transition of pure globoside at 55 degrees C, involving 'melting' of the hc chains was also detected using X-ray and esr spectroscopic techniques. The interbilayer spacing, dw, of equimolar lecithin-globoside lamellar phase increased by 42% from that of lecithin bilayers, indicating that the glycolipid p group may increase the net repulsive force between bilayers, as was previously predicted theoretically.

Lysolecithin-cholesterol interaction. A spin-resonance and electron-micrographic study.

Another publication (rand, R. P., Pangborn, W., Purdon, A. D., and Tinker, D. O.(1975) Can. j. Biochem. 53, 189-195) has established that lysolecithin and cholesterol interact to form an equimolar complex. We have investigated this complex using the techniques of electron spin resonance (e.s.r) and electronmicroscopy. By varying the cholesterol concentration with lysolecithin in both thin films and dispersions studied by these techniques, we have observed the interaction between lysolecithin and equimolar complex below 50 mol % cholesterol, and between crystalline cholesterol and equimolar complex above 50 mol % cholesterol. We have observed an interesting alteration in morphology by electron microscopy, and an isotropic to anisotropic spectral change using 3-dosylcholestane and 12-doxylstearic acid spin-labelled probes when the cholesterol concentration is increased from 20 to 33 mol %. The equimolar complex is stable in the presence of crystalline cholesterol, and exhibits no phase changes in the physiological temperature range. Implications for membrane structure are discussed.

Lysolecithin and cholesterol interact stoichiometrically forming bimolecular lamellar structures in the presence of excess water, of lysolecithin or cholesterol.

The structural interaction of egg lysolecithin, derived from egg lecithin, and cholesterol in aqueous solution has been investigated using X-ray diffraction. When mixed in any proportions, either suspended in excess buffer or up to 85% lipid by dry weight, a separate lamellar phase containing equimolar proportions of lysolecithin and cholesterol forms, separate from excess water, or lysolecithin or cholesterol. The cholesterol disorders the crystalline chains of the lysolecithin. The equimolar phase is stable up to 50 degrees C unlike lysolecithin alone, which forms micelles, Thes results show that lysolecithin and cholesterol combine stoichiometrically in a stable complex. We propose as a structural model, that cholesterol fills the space of the missing fatty acyl chain making the lysolecithin more cylindrical rather than wedge shaped. This interaction could reduce both the lytic action of lysolecithin on membranes and its induction of cell fusion. It suggest another role of cholesterol in cell membranes: namely, to act as a stabilizer of bilayer structure by being a mobile component that can fill free volume in the hydrocarbon interior. Lysolecithin-cholesterol interaction may also be important in the early events of atherosclerosis where lysolecithin levels in vessel walls increase fivefold.

A model of lateral diffusion in phosphatide bilayers and natural membranes.

Lateral diffusion of phosphatide molecules in liquid crystalline bilayers has been analysed as a case of co-operative lattice diffusion. The potential energy of interaction between two molecules is assumed to arise from Van der Waals interactions of the hydrocarbon chains, and to have the form suggested by Salem [6]. From the observed values of the self-diffusion constant (of the order of 10-8 cm2sec-1) the depth of the potential "well" for two molecules at the equilibrium separation was estimated to have a lower limit of 1.95 kcal per mole, and the energy barrier to lateral motion was estimated to have an upper limit of 7.21 kcal per mole.