George M. Helmkamp

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.

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.

Tissue distribution, purification and characterization of rat phosphatidylinositol transfer protein

Phosphatidylinositol transfer activity is measured in cytosol fractions prepared from 13 rat tissues; specific activity is highest in brain and lowest in adipose and skeletal muscle. Based upon electrophoretic analysis phosphatidylinositol transfer protein is purified to homogeneity from whole rat brain. The protein has a molecular weight of 36,000 and exists as a mixture of species having isoelectric points of 4.9 and 5.3. In a vesicle-vesicle assay system, the intermembrane transfer rate is greatest for phosphatidylinositol and less by a factor of 2 for phosphatidylcholine; transfer of phosphatidylethanolamine, phosphatidylserine or sphingomyelin is not observed. Using a polyclonal rabbit antibody against bovine phosphatidylinositol transfer protein, immunologic cross-reactivity is noted between the rat protein and other mammalian phosphatidylinositol transfer proteins. A strong correlation is established between a tissues capacity for phosphatidylinositol transfer and the amount of immunoreactive transfer protein seen in that tissue. Purified phosphatidylinositol transfer protein is capable of transporting newly synthesized phosphatidylinositol molecules from rat brain microsomes to small unilamellar phospholipid vesicles. The results are discussed within the context of cellular phosphoinositide metabolism and the maintenance of the metabolically responsive pool of phosphatidylinositol in the plasma membrane.

Interaction of bovine brain phospholipid exchange protein with liposomes of different lipid composition

The major phospholipid exchange protein from bovine brain catalyzes the transfer of phosphatidylinositol and phosphatidylcholine between rat liver microsomes and sonicated liposomes. The effect of liposomal lipid composition on the transfer of these phospholipids has been investigated. Standard liposomes contained phosphatidylcholine-phosphatidic acid (98 : 2, mol%); in general, phosphatidylcholine was substituted by various positively charged, negatively charged, or zwitterionic lipids. The transfer of phosphatidylinositol was essentially unaffected by the incorporation into liposomes of phosphatidic acid, phosphatidylserine, or phosphatidylglycerol (5--20 mol%) but strongly depressed by the incorporation of stearylamine (10--40 mol%). Marked stimulation (2--4-fold) of transfer activity was observed into liposomes containing phosphatidylethanolamine (2--40 mol%). The inclusion of sphingomyelin in the acceptor liposomes gave mixed results: stimulation at low levels (2--10 mol%) and inhibition at higher levels (up to 40 mol%). Cholesterol slightly diminished transfer activity at a liposome cholesterol/phospholipid molar ratio of 0.81. Similar effects were noted for the transfer to phospholipidcholine from microsomes to these various liposomes. Compared to standard liposomes, the magnitude of Km tended to increase for liposomes which depressed phospholipid transfer and to decrease for those which stimulated; little change was observed in the values of V. Single phospholipid liposomes of phosphatidylinositol were inhibitory when added to standard liposomes. Because bovine brain phospholipid exchange protein is able to distinguish among a wide spectrum of membrane interfaces, taking into account variations in the polar head groups as well as the fatty acyl moieties of the liposomal phospholipids, it may be considered a reasonable model system for protein-lipid and protein-membrane interactions.

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.

Concerning the mechanism of action of bovine liver phospholipid exchange protein: Exchange or net transfer

Abstract Bovine liver phospholipid exchange protein catalyzes the transfer of phosphatidylcholine between donor and acceptor populations of single bilayer phospholipid vesicles. In comparing egg and dimyristoylphosphatidylcholine vesicles, larger transfer rates are found for the unsaturated phospholipid. The bidirectional transfer rates measured from donor to acceptor and from acceptor to donor, are equivalent, suggesting that the protein facilitates an exchange rather than a net transfer of phosphatidylcholine.

Transport and Metabolism of Phosphatidylinositol in Eukaryotic Cells

Phospholipids are the major lipid component of eukaryotic cell membranes. A diversity of phospholipid structures is reflected in distributions that are unique to specific intracellular membranes, as well as the cytoplasmic and extracytoplasmic surfaces of the membrane bilayer. Matched to the spectrum of phospholipids within the cell are cytosolic phospholipid transfer proteins. As a general class of catalyst, these proteins facilitate the transport of monomeric phospholipid molecules between membrane domains (Kader et al., 1982; Wirtz, 1982; Helmkamp, 1986). Although a number of interesting and often highly suggestive catalytic activities have been demonstrated in vitro for phospholipid transfer proteins, it has proved frustratingly difficult to define any in vivo function.

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.

Developmental patterns in rat brain of phosphatidylinositol synthetic enzymes and phosphatidylinositol transfer protein

Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum. Specific activities of CTP:phosphatidate cytidylyltransferase and CDPdiacylglycerol:inositol phosphatidyltransferase were found to have similar developmental patterns. Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60. Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60. Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60. Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7-10 days compared to the whole brain. Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28. The developmental pattern of CTP:phosphatidate cytidylyltransferase in rat brain is reported here for the first time.

Transport of phosphatidylinositol to rat hepatocyte plasma membrane catalyzed by phosphatidylinositol transfer protein

Plasma membrane sheets were isolated from fresh rat liver and characterized by electron microscopy and marker enzyme activities. Plasma membrane sheets were used as the acceptor membrane in the measure of transport of phosphatidyl[3H]inositol from small unilamellar phospholipid vesicles or rough endoplasmic reticulum donor membranes. Catalysis of this transport was achieved with phosphatidylinositol transfer protein purified from rat or bovine brain. Assays were designed to separate donor and acceptor membranes by density gradient centrifugation. Rates of transfer were directly proportional to incubation time and the amounts of transfer protein and plasma membrane sheet added. These results are discussed in terms of cellular phosphatidylinositol metabolism, membrane phospholipid composition, and vesicle trafficking in rat hepatocytes.