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G M Helmkamp

Publications and source records attributed to G M Helmkamp.

At least 37 records · Page 2Linked to original sources
PubMed1988

General kinetic model for protein-mediated phospholipid transfer between membranes.

Phospholipid transfer protein catalyzes the transfer of phospholipids between bilayer membranes. A general model is developed for describing the kinetics of this process. While previous models derive detailed expressions only for the initial rate of transfer from donor to acceptor membranes, this model takes into account donor-to-donor, acceptor-to-acceptor, and acceptor-to-donor transfers, in addition to the usual donor-to-acceptor transfer. The apparent rate of transfer along any of these specific routes is given as the product of the total rate of transfer (the sum of the rates of transfer along all four routes) and a probability function uniquely defined for each route. The model explains adequately the effects of membrane concentration on phospholipid transfer activity as well as the consequences of varying membrane surface charge and size. Using bovine liver phosphatidylcholine transfer protein, the model is applied to the kinetic analysis of phosphatidylcholine transfer between two populations of small unilamellar vesicles. Rates of protein-catalyzed phosphatidylcholine transfer between vesicles with identical phosphatidic acid content (2 or 6 mol%) are determined experimentally as a function of total vesicle concentration to calculate apparent dissociation constants and maximum rates of transfer; apparent rates of transfer between various combinations of vesicles containing 2 or 6 mol% phosphatidic acid are then deduced from the derived velocity expression. Reasonably good agreement is seen between theoretical apparent rate-vesicle concentration relationships and those measured experimentally. The results support the general treatment of the kinetics of protein-mediated phospholipid transfer and permit an estimation of useful kinetic parameters.

Animals↗
PubMed1988

Design, synthesis, and characterization of bis-phosphatidylcholine, a mechanistic probe of phosphatidylcholine transfer protein catalytic activity.

The design, synthesis, and characterization of 1-(17,18-dithiatetratriacontandioyl)-bis(2-hexadecanoyl-sn-glycero -3- phosphocholine) is described. Bis-phosphatidylcholine is a dimeric phospholipid comprised of two glycerophosphocholine groups linked together by a disulfide bond at the distal ends of the sn-1 fatty acyl chains. Electron microscopy and [14C]glucose trapping studies indicate that hydrated dispersions of bis-phosphatidylcholine form closed, spherical structures which have diameters in the range of 125-500 nm. Sensitivity to phospholipase hydrolysis suggests that this bipolar lipid is organized in a membrane such that the two polar head groups of the molecular are oriented at the same surface of the membrane. Using conditions in which bovine liver phosphatidylcholine transfer protein transfers both unsaturated and saturated diacyl phosphatidylcholines between fluid phosphatidylcholine vesicles, no transfer of the bipolar phospholipid is observed. The lack of activity toward bis-phosphatidylcholine suggests that this molecule may be a useful tool for elucidating the role of membrane phosphatidylcholine in the catalytic mechanism of the phosphatidylcholine transfer protein.

Androgen-Binding Protein↗
PubMed1988

Effect of acceptor membrane phosphatidylcholine on the catalytic activity of bovine liver phosphatidylcholine transfer protein.

Protein-mediated transfer of phosphatidylcholine (PC) by bovine liver phosphatidylcholine transfer protein (PC-TP) was examined using a vesicle-vesicle assay system. Donor and acceptor membranes were prepared from Escherichia coli phospholipids and limiting amounts of egg yolk PC. PC transfer between vesicles of E. coli lipid/egg PC was markedly higher than transfer of PC from vesicles of E. coli lipid/egg PC to vesicles of E. coli lipid. Kinetic parameters of the interaction between PC-TP and E. coli lipid vesicles with or without PC was investigated. The apparent dissociation constants of the complex formed between PC-TP and these vesicles were determined kinetically and from double-reciprocal plots of intrinsic PC-TP fluorescence intensity increase versus vesicle concentration. The magnitude of the dissociation constant decreased as the PC content of the vesicles increased from 0 to 5 mol%. In addition, kinetic analysis revealed that the presence of PC in acceptor vesicles increased both the association and dissociation of PC-TP from vesicles. The effect of membrane PC molecules on transfer rates was examined using bis-phosphatidylcholine, a dimeric PC molecule which is not transferred by PC-TP. Rates of PC transfer to acceptor vesicles comprised of E. coli lipid/bis-PC were virtually identical to rates observed with acceptors vesicles prepared from E. coli lipid. The results suggest that transfer of PC by PC-TP is enhanced only when insertion of protein-bound PC occurs concurrently with the extraction of a molecule of membrane PC, i.e., a concerted, one-step catalytic mechanism for phospholipid exchange.

Androgen-Binding Protein↗
PubMed1988

Effects of dietary fish oil on platelet function and plasma lipids in hyperlipoproteinemic and normal subjects.

We studied the effects of dietary supplementation with an encapsulated fish oil concentrate (Maxepa) on platelet function, fibrinolysis, and plasma lipids and lipoproteins in 9 normal subjects, 10 patients with type IV hyperlipoproteinemia, and 6 with type IIB hyperlipoproteinemia. After a baseline period, the subjects crossed over randomly between treatment periods with Maxepa (providing 3.24 g eicosapentaenoic acid and 2.16 g docosahexaenoic acid per day) and safflower oil (used as a control), given for 6 weeks each. Administration of Maxepa led to a slight prolongation of the bleeding time in all groups and to modest inhibition of platelet aggregation in the type IV hyperlipoproteinemics and normal subjects, with partial (41%) inhibition of thromboxane synthesis from baseline levels noted in the normal group. Plasma total fibrinolytic actively did not change significantly in any group. Maxepa treatment resulted in a marked decrease in triglyceride and VLDL-cholesterol and a slight increase in HDL-cholesterol was noted after Maxepa in the type IV hyperlipoproteinemics (4.11 +/- 0.13 mmol/l vs. 3.10 +/- 0.16 mmol/l, Maxepa vs. safflower oil). We conclude that dietary supplementation with fish oil results in a relatively minor degree of inhibition of platelet function in normal and hyperlipoproteinemic subjects, and a potentially adverse increase in LDL-cholesterol in type IV hyperlipoproteinemics.

Adult↗
PubMed1986

Phospholipid transfer proteins: mechanism of action.

Phospholipid transfer proteins are generally localized in the cytosolic fraction of cells and are capable of catalyzing the flux of phospholipid molecules among membranes. Artificial membranes also participate in protein-catalyzed phospholipid movements. In this review the major phospholipid transfer proteins are discussed with respect to their phospholipid substrate specificity and the contributions of membrane physical properties to this process. The phenomenon of net transfer of phospholipids is described. The use of various kinetic approaches to the study of these catalysts is reviewed. A detailed consideration of the distinct phospholipid binding and membrane interaction domains of one phospholipid transfer protein is presented. Finally, some recent applications of phospholipid transfer proteins to the examination of membrane structure and function and further directions for the continued research activity with this class of proteins are summarized.

Animals↗
PubMed1985

Purification and characterization of a phosphatidylinositol transfer protein from human platelets.

We report the purification of a phospholipid transfer protein from human platelets. This protein preferentially transfers phosphatidylinositol, with phosphatidylcholine and phosphatidylglycerol being transferred to a lesser extent. Phosphatidylethanolamine is not transferred. Transfer activity is detected by measuring the transfer of radiolabeled phospholipids between two populations of small unilamellar vesicles. The protein was purified approximately 1000-fold over the platelet cytosol by chromatography on Sephadex G-75, sulfooxyethyl cellulose, and hydroxylapatite. The molecular weight of this protein appears to be 28 000 as determined by gel filtration chromatography. When the purified protein is analyzed on sodium dodecyl sulfate-polyacrylamide gels, two major components and several minor ones are observed. The molecular weight of the two major bands are 28 600 and 29 200. Isoelectric focusing of the platelet cytosol yielded phosphatidylinositol and phosphatidylcholine transfer activity at pH 5.6 and 5.9. The platelet phospholipid transfer protein is able to catalyze the transfer of phosphatidylinositol and phosphatidylcholine between vesicles and human platelet plasma membranes. One possible physiological role for this transfer protein is an involvement in the rapid turnover of inositol-containing lipids which occurs upon exposure of platelets to various stimuli.

Blood Platelets↗
PubMed1985

Phosphatidylinositol transfer proteins: structure, catalytic activity, and physiological function.

Among the diverse lipid transfer proteins which are found in tissues and biological fluids are those which exhibit a specificity toward phosphatidylinositol and phosphatidylcholine, with a preference for the former. Phosphatidylinositol transfer proteins (PI-TPs) have been purified from several eukaryotic sources; those present in bovine brain and heart have been extensively studied. This review examines the tissue distribution of PI-TPs and the means by which transfer activity is measured using natural and artificial membranes. The interaction of these proteins with lipid monolayers and bilayers is discussed in terms of phospholipid fatty acyl and polar head group compositions. The inhibition of transfer activity by sulfhydryl agents and amphiphilic amines is summarized. The metabolism of the phosphoinositides is considered and a role for PI-TPs is proposed.

Animals↗
PubMed1984

Partition of amphiphilic molecules into phospholipid vesicles and human erythrocyte ghosts: measurements by ultraviolet difference spectroscopy.

Molar partition coefficients for chlorpromazine and methochlorpromazine between phospholipid vesicles or human erythrocyte ghosts and buffer are determined by ultraviolet difference spectroscopy. The partition coefficients between small unilamellar egg phosphatidylcholine vesicles and buffer at pH 7.4 are 4.4 X 10(5) for chlorpromazine and 0.8 X 10(5) for methochlorpromazine, determined with 10 microM amphiphile. An increase in the partition of chlorpromazine into vesicles is seen as the pH is increased to the pKa of chlorpromazine at 9.2. Chlorpromazine also partitions preferentially into fluid-phase phospholipid compared to solid-phase phospholipid. Molar partition coefficients between unsealed human erythrocyte ghosts and buffer at pH 8.0 with 10 microM amphiphile are determined to be 6.5 X 10(5) for chlorpromazine and 2.5 X 10(5) for methochlorpromazine. Difference spectroscopy is an equilibrium technique that does not require separation of bound from free amphiphile, as do many other methods of determining membrane-buffer partition coefficients. This method is useful for any amphiphile that has an appreciable absorbance below its critical micelle concentration and whose absorbance is sensitive to environment.

Chemical Phenomena↗
PubMed1984

Acyl chain specificity of phosphatidylcholine transfer protein from bovine liver.

The specificity of bovine liver phosphatidylcholine transfer protein for various phosphatidylcholine (PC) molecular species was examined at 37 degrees C. The amount of transfer between donor and acceptor vesicles of defined phospholipid composition was determined. Protein-mediated transfer between vesicles of long chain, fluid phase PCs (di-16:1 PC, di-17:1 PC, di-18:1 PC, di-18:2 PC, or egg PC) was markedly higher than transfer between vesicles of solid phase or short chain, fluid phase PCs (di-18:0 PC, di-16:0 PC, or di-14:0 PC). When di-14:0 PC and di-18:1 PC were present in the same vesicle, protein-mediated transfer of di-18:1 PC was still higher, indicating that the protein's specificity toward long chain, fluid phase PCs is based on true acyl chain structure preference rather than a bulk phase physical property of the longer chain PCs. The effect of adding a third type of vesicle to a system which consisted of transfer protein, donor vesicles, and acceptor vesicles was investigated. Addition of solid phase PC vesicles does not affect transfer of the well transferred species, while addition of the poorly transferred short chain, fluid phase PCs does inhibit transfer. These results suggest that the transfer protein has the ability to bind to any fluid phase PC vesicle, although it preferentially extracts and transfers long chain, fluid phase PCs.

Androgen-Binding Protein↗
PubMed1984

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity.

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.

Animals↗
PubMed1984

Bovine brain phosphatidylinositol transfer protein. Selective inhibition by chlorpromazine and other amphiphilic amines.

Cationic amphiphilic amines of varied pharmacological activity were evaluated as modulators of the protein-catalyzed, intermembrane transfers of phosphatidylinositol and phosphatidylcholine. The catalytic agent was brain phosphatidylinositol transfer protein; the membrane system consisted of two populations of single bilayer phospholipid vesicles. The majority of the amines tested caused decreases in phospholipid transfer activity with the relative potencies in the following order: chlorpromazine greater than dibucaine greater than propranolol much greater than tripelennamine approximately chloroquine greater than dipyridamole. Concentrations required for 50% inhibition of phosphatidylinositol transfer were 0.24 mM chlorpromazine, 0.46 mM dibucaine, and 0.78 mM propranolol. The phosphatidylcholine transfer activity of this protein was somewhat less sensitive to these compounds. Comparison of chlorpromazine and its quaternary amine analogue, methochlorpromazine, at different pH values indicated that the observed inhibition can be attributed in large part to the charged forms of the amphiphiles. Direct association of methochlorpromazine with egg phosphatidylcholine bilayers was demonstrated by molecular sieve chromatography; no such association of the amphiphile with phosphatidylinositol transfer protein was apparent. Anionic agents, such as indomethacin, phenylbutazone, and tolmetin, were without significant effect on protein-catalyzed phospholipid transfers. Electrostatic interaction between the cationic amines and anionic or zwitterionic phospholipids, forming ion pairs in the lipid bilayers, is suggested as a possible molecular mechanism for the observed inhibition.

Amines↗
PubMed1983

The inactivity of brain phospholipid transfer protein toward phosphatidylinositol 4-phosphate.

Bovine brain phosphatidylinositol transfer protein catalyzes the transport of phosphatidylinositol and, to a lesser extent, phosphatidylcholine between model phospholipid membranes. To investigate the transport of phosphorylated phosphatidylinositol derivatives, 32P-labeled lipids were isolated from guinea pig brain, separated by chromatography on a column of neomycin-coated glass beads, and incorporated into single bilayer vesicles. Under conditions where significant transfer of phosphatidylinositol was observed, no protein-catalyzed transport of phosphatidylinositol 4-phosphate was detected. Thus, the substrate specificity of the brain phospholipid transfer protein cannot be extended to phosphatidylinositol 4-phosphate.

Animals↗
PubMed1982

Modulation of phospholipid transfer protein activity. Inhibition by local anesthetics.

The transfer of phospholipid molecules between biological and synthetic membranes is facilitated by the presence of soluble catalytic proteins, such as those isolated from bovine brain which interacts with phosphatidylinositol and phosphatidylcholine and from bovine liver which is specific for phosphatidylcholine. A series of tertiary amine local anesthetics decreases the rates of protein-catalyzed phospholipid transfer. The potency of inhibition is dibucaine greater than tetracaine greater than lidocaine greater than procaine, an order which is compared with and identical to those for a wide variety of anesthetic-dependent membrane phenomena. Half-maximal inhibition of phosphatidylinositol transfer by dibucaine occurs at a concentration of 0.18 mM, significantly lower than the concentration of 1.9 mM required for half-maximal inhibition of phosphatidylcholine transfer activity of the brain protein. Comparable inhibition of liver protein phosphatidylcholine transfer activity is observed at 1.6 mM dibucaine. For activity measurements performed at different pH, dibucaine is more potent at the lower pH values which favor the equilibrium toward the charged molecular species. With membranes containing increasing molar proportions of phosphatidate, dibucaine is increasingly more potent. No effect of Ca2+ on the control transfer activity or the inhibitory action of dibucaine is noted. These results are discussed in terms of the formation of specific phosphatidylinositol or phosphatidylcholine complexes with the amphiphilic anesthetics in the membrane bilayer.

Anesthetics, Local↗
PubMed1981

Intermembrane phospholipid fluxes catalyzed by bovine brain phospholipid exchange protein.

Bovine brain phospholipid exchange protein catalyzes the transfer of phosphatidylinositol and phosphatidylcholine between two populations of single bilayer vesicles. The inclusion of lactosylceramide in one of the vesicle populations and the ability to precipitate those vesicles in the presence of Ricinus communis agglutinin assures the quantitative separation of donor and acceptor vesicles following incubation with exchange protein. When both vesicle populations contain phosphatidylinositol and phosphatidylcholine and transfers are monitored in both directions, the flux of phosphatidylinositol (or phosphatidylcholine) in the forward direction equals that in the reverse. When one of the vesicle populations initially lacks phosphatidylinositol, a net unidirectional transfer of that phospholipid occurs. Concurrently, a compensatory flux of phosphatidylcholine takes place in the opposite direction, such that the bidirectional fluxes of total phospholipid are equal. A net transfer of phosphatidylcholine is also demonstrated. A mechanism of true molecular exchange between vesicles, rather than net transfer, is proposed for the bovine brain phospholipid exchange protein.

Animals↗
PubMed1981

Protein-catalyzed phospholipid exchange between gel and liquid-crystalline phospholipid vesicles.

Bovine liver phospholipid exchange protein catalyzes the transfer of phosphatidylcholine between two populations of single bilayer phospholipid vesicles. Donor vesicles are prepared from egg phosphatidylcholine--phosphatidic acid--lactosylceramide (90:2:8) mol %); acceptor vesicles are prepared from phosphatidylcholine--phosphatidic acid (98:2 mol %). Activity is determined from the rate of transfer of 3H-labeled egg phosphatidylcholine from donor to acceptor vesicles in the presence of phospholipid exchange protein. Donor vesicles are quantitatively precipitated by Ricinus communis agglutinin, while acceptor vesicles remain in the supernate. When egg phosphatidylcholine acceptor vesicles over the temperature range 11--45 degrees C are used, a linear Arrhenius plot is obtained, in keeping with the observation that these membranes exist only in the liquid-crystalline state. When dimyristoylphosphatidylcholine acceptor vesicles under the same conditions are used, however, a biphasic plot is seen with decreasing transfer activity at lower temperatures. The discontinuity occurs at 31 degrees C and corresponds with the onset of the liquid-crystalline to gel phase transition. The incorporation of cholesterol into dimyristoylphosphatidylcholine vesicles at a concentration sufficient to abolish the thermotropic phase transition yields a monophasic Arrhenius plot of transfer activity. The results indicate that bovine liver phospholipid exchange protein interacts catalytically with phospholipid bilayer vesicles composed of saturated or unsaturated phosphatidylcholines but preferentially with liquid-crystalline membranes.

Animals↗
PubMed1980

Studies of Fc gamma receptors of human B lymphocytes: phospholipase A2 activity of Fc gamma receptors.

The presence of phospholipase A2 activity within human B cell Fc gamma receptors was investigated. Lysate produced by detergent treatment of chronic lymphocytic leukemia cells that had 1% of the cells surface radioiodinated was subjected to affinity chromatography by using either rac-1-(9-carboxynonyl)-2-hexadecylglycero-3-phosphorylcholine-Sepharose (PC-Sepharose) or heat-aggregated human IgG-Sepharose 4B conjugate (IgG-Sepharose). The materials eluted from both adsorbants by ethylenediaminetetraacetate- or urea-containing buffer were further purified by gel filtration and isoelectric focusing in the presence of 6 M urea. Both isolated PC- and IgG-binding materials were homogeneous, when judged by gel filtration and isoelectric focusing, and had identical isoelectric points (pI = 6.5), peptide maps, and amino acid compositions. Furthermore, both preparations catalyzed equally the hydrolysis of phosphatidylcholine to release fatty acid from the 2 position. Optimal enzymatic activity depended on the presence of Ca2+, was maximal at pH 9.5, and was augmented by Fc gamma fragments. Both preparations specifically bound to the Fc portion of IgG and inhibited human antibody-coated erythrocyte rosette formation by peripheral mononuclear cells. Our data thus demonstrate the identity of PC- and IgG-binding materials and suggest that a functional activity of the human B cell Fc gamma receptor is the generation of phospholipase A2 activity within the plasma membrane.

Amino Acids↗