Addition Of Phosphatidylethanolamine Research Articles

Membrane Phospholipid Composition Governs Pah1 Phosphatidate Phosphatase Activity

In yeast and higher eukaryotes, all membrane phospholipids and the storage lipid triacylglycerol, are derived from phosphatidate (PA), which is also implicated as an activator of cell growth and proliferation, vesicular trafficking, secretion, and endocytosis. PA also regulates expression of lipid synthesis genes via the Henry (Opi1/Ino2‐Ino4) regulatory circuit, where the key step is the PA‐mediated translocation of the Opi1 repressor between the nuclear/ER membrane and the nucleus. Data indicate that several enzymes play important roles in the PA‐mediated regulation of lipid synthesis that occurs at the nuclear/ER membrane. Of the enzymes that metabolize PA, the PA phosphatase enzyme, which is encoded by the PAH1 gene, has emerged as key regulator of PA homeostasis. Indeed, cells lacking this enzyme exhibit several phenotypes that include elevated levels of PA, derepression of phospholipid synthesis genes, increased synthesis of membrane phospholipids, and the abnormal expansion of the nuclear/ER membrane. These mutant cells also exhibit a drastic reduction in TAG and lipid droplets. The PA phosphatase, which catalyzes the conversion of PA to diacylglycerol, must translocate from the cytoplasm to the ER membrane where its substrate PA resides to function in lipid synthesis. Thus, studies to examine mechanisms for the enzyme reaction at the membrane surface are warranted. In this work, we implemented defined unilamellar vesicles with a diameter of 100 nm composed of the major phospholipids within the ER membrane. Vesicles composed of phosphatidylcholine (PC) and 10 mol% PA supported PA phosphatase activity. The addition of phosphatidylethanolamine (PE) or phosphatidylinositol (PI) stimulated the activity, but phosphatidylserine (PS) had little effect. In more complex vesicle compositions, the maximum activity was observed with vesicles containing PC/PE/PS/PA or PC/PE/PI/PA. Yet, the activity was reduced when vesicles contained PC/PE/PI/PS/PA. The PA phosphatase activity was dependent on vesicle number as well as the surface concentration of PA within the vesicles. This vesicle system is being used to assess the roles of phosphorylation/dephosphorylation of Pah1 on membrane interaction and PA phosphatase activity.Support or Funding InformationSupported by NIH grant GM028140

Interaction of the Water-Soluble Protein Aprotinin with Liposomes: Gel-Filtration, Turbidity Studies, and 31P NMR Studies

The interactions of a water-soluble nonmembrane protein aprotinin with multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) from soybean phospholipids were studied using Sephadex G-75 gel chromatography combined with different methods of the analysis of the eluate fractions (fluorescence, light-scattering, turbidity; 31P NMR spectroscopy). The composition of the liposomes mainly containing soybean phosphatidylcholine (PC) was varied by the addition of phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). To evaluate the lipid-protein interactions, the amount of aprotinin in the MLV–aprotinin complexes was determined. Lipid–protein interactions were found to strongly depend on the liposome composition, medium pH and ionic strength. These dependencies point to the electrostatic nature of the aprotinin-lipid interactions. 31P NMR spectroscopy of the MLV–aprotinin complexes indicated that aprotinin influences the phospholipid structure in MLV at pH 3.0. In the case of PC:PE:PI and PC:PE:PI:lyso-PC vesicles, aprotinin induced liposome aggregation and a lamellar-to-isotropic phase transition of the phospholipids.

Generation of receptor-active, globotriaosyl ceramide/cholesterol lipid 'rafts' in vitro : A new assay to define factors affecting glycosphingolipid receptor activity.

Purified renal globotriaosyl ceramide (Gb3)/cholesterol mixtures sonicated heated in a Triton-containing buffer placed below a discontinuous sucrose gradient form glycosphingolipid (GSL)-containing dense lipid structures at the 30/5% sucrose interface after centrifugation. Inclusion of fluorescein-labeled verotoxin 1 B subunit (FITC-VT1 B) within the most dense sucrose layer results in the fluorescent labeling of this Gb3-containing raft structure. Alternatively inclusion of I-labeled VT1 fractionation allows quantitation of binding. FITC-VT1 B effectively competes for I-VT1/Gb3 raft binding. This assay will allow the definition of the optimal raft composition for VT1 (or any other ligand) binding. The effect of several potential cellular raft components are reported. Increased cholesterol content increased VT1 binding. Addition of phosphatidylethanolamine had minimal effect while phosphatidylserine was inhibitory. Although inclusion of sphingomyelin increased the Gb3 content of the "raft" reduced VT1 binding was seen. Inclusion of other glycolipids can also be inhibitory. The addition of globotetraosyl ceramide had no effect; however addition of sulfogalactosyl ceramide but not sulfogalactoglycerolipid inhibited VT1/Gb3 raft binding. These results suggest that certain GSLs can disfavor the formation of the appropriate 'raft' structure for ligand binding that this is dependent on both their carbohydrate lipid structure. Such "deceptor" GSLs may provide an as yet unappreciated mechanism for the regulation of cellular GSL receptor activity. This model is an effective tool to approach the dynamics ligand-binding specificity of GSL/cholesterol-containing lipid microdomains.

Serratia marcescens hemolysin (ShlA) binds artificial membranes and forms pores in a receptor-independent manner.

Both the inactive and active conformations of the hemolysin/cytolysin of Serratia marcescens (ShlA) binds membranes of erythrocytes, but only active ShlA is able to form pores. ShlA is unable to lyse prokaryotic membranes. To determine the receptors of the binding and pore-forming domains of active cytolysin on eukaryotic membranes, artificial large unilamellar vesicles (LUVs) of various membrane compositions were examined. In the current study, it is shown that significant pore formation and lysis was achieved with binary phosphatidylcholine/phosphatidylserine (PS) liposomes. No proteinaceous receptor was needed for either binding or pore formation by ShlA. Membrane integration and pore-forming activity were enhanced by addition of phosphatidylethanolamine. Phosphatidylserine is negatively charged at physiologic pH and is almost absent in prokaryotic membranes. Hence, membrane binding and insertion of ShlA are highly dependent on phosphatidylserine, which targets the toxic activity to eukaryotic cell membranes without any need of a proteinaceous receptor. This may explain why prokaryotic membranes were found to be resistant against ShlA in a previous study.

Ｔｈｉｏｂａｃｉｌｌｕｓ ｆｅｒｒｏｏｘｉｄａｎｓによる黄銅鉱のバクテリアリーチングに及ぼすｐｈｏｓｐｈａｔｉｄｙｌｅｔｈａｎｏｌａｍｉｎｅの影響

Phospholipids such as phosphatidylethanolamine and phosphatidylglycerol are detected as extracellular substances in cultures of Thiobacillus ferrooxidans and Thiobacillus thiooxidans, but their effect on bacterial leaching is not well understood. In this paper, basic experiments on bacterial leaching of chalcopyrite by Thiobacillus ferrooxidans were carried out in the presence of phosphatidylethanolamine.The copper extraction in chemical leaching of chalcopyrite with ferric sulfate increased slightly by the addition of phosphatidylethanolamine. In the medium containing ferrous sulfate as substrate, the ferrous oxidation by Thiobacillus ferrooxidans and its growth were not affected by the addition of phosphatidylethanolamine. In bacterial leaching, the addition of phosphatidylethanolamine had no effect on the copper extraction and bacteria adsorption on the mineral surfaces. These results were different from the results with phosphatidylglycerol.

The modulation of protein kinase C activity by membrane lipid bilayer structure.

The hypothesis that protein kinase C (PKC) activity is sensitive to phospholipid head group interactions was tested using lipid bilayers of defined composition with PKC purified from rat brain. The head group interactions were modulated by varying phosphatidylcholine cis-unsaturation, vesicle curvature, and by the addition of phosphatidylethanolamine and cholesterol. With unilamellar vesicles (including 20 mol% brain phosphatidylserine), increased phosphatidylcholine unsaturation potentiated basal and phorbol ester stimulated PKC activity. By contrast, in the presence of phosphatidylethanolamine, the activity decreased with increasing phosphatidylcholine unsaturation. Weakening phospholipid head group interactions spaces the head group region and increases interstitial water, and this effect was assessed from its effect on the fluorescence intensity of the phospholipid-labeled fluorophore 1-palmitoyl-2-N-(4-nitrobenzo-2-oxa-1,3-diazole)aminohexanoylphosphat idylcholin e (C6-NBD-PC). When the PKC activities with vesicles of varying phosphatidylcholine unsaturation, with and without phosphatidylethanolamine, were plotted as a function of the fluorescence intensity of C6-NBD-PC-labeled vesicles, a biphasic profile was obtained, which had an optimum value of intensity, relating to head group spacing, that corresponded to a maximal enzyme activity. A similar biphasic curve was also found when PKC activities were plotted as a function of published bilayer intrinsic curvature x-ray diffraction data, a parameter closely related to head group spacing. By contrast, no simple relationship was evident between PKC activity and 1,6-diphenyl-1,3,5-hexatriene anisotropy, taken as a measure of lipid order or fluidity. Therefore, increasing the level of phosphatidylcholine unsaturation, phosphatidylethanolamine, or cholesterol either potentiates or attenuates PKC activity, dependent on whether the initial condition is above or below its optimum.

Specific lipids enhance the activity of UDP-GlcNAc: Dolichol phosphate GlcNAc-1-phosphate transferase in rat liver endoplasmic reticulum membrane vesicles

The rate of the reaction catalyzed by UDP- N-acetylglucosamine (GlcNAc):dolichol phosphate GlcNAc-1-phosphate transferase in rat liver endoplasmic reticulum vesicles was shown to be influenced by particular lipids. Utilizing in vitro assay conditions where the membrane vesicles retained latency of glucose-6-phosphatase activity, the addition of phosphatidylethanolamine, cardiolipin, or monogalactosyldiglyceride resulted in severalfold increases in the rate of dolichol pyrophosphate N-acetylglucosamine synthesis. Other phospholipids were not stimulatory. These rates were dependent on the concentrations of the exogenous lipids and of the substrate dolichol phosphate. In the presence of cardiolipin, the membrane-bound enzyme became more susceptible to inactivation by protease K and to inhibition by tunicamycin. Titration of cardiolipin-containing endoplasmic reticulum vesicles with adriamycin indicated that the majority of the cardiolipin was exposed on the outer surface. These results suggest that the particular lipids altered membrane structure in a way that allowed further access of the enzyme to substrate, inhibitor, and other molecules. Lipids observed in these studies to be stimulatory are known to exist in the macromolecular hexagonal phase and may therefore be affecting the GlcNAc-1-phosphate transferase by locally disrupting the bilayer structure of the membrane. As other dolichol-utilizing enzymes have been previously observed by other investigators to be similarly influenced by such lipids, the effects may be common to enzymes of the dolichol cycle.

Effect of phospholipids on purified lipoamidase

Phospholipids were added to purified lipoamidase from porcine brain microsomal membranes, and changes in lipoamidase activity were examined. Approximately twofold activation of lipoamidase activity occurred upon the addition of phosphatidylethanolamine. On the other hand, phosphatidylserine, cardiolipin, and phosphatidic acid reduced the enzyme activity by approximately 80%. This pattern of the activation of lipoamidase by phosphatidylethanolamine and its inhibition by phosphatidylserine is similar to the pattern for adenylate cyclase, and contrasts with the pattern for ATPase.

Susceptibility of UDP-Glucose:(1,3)-β-Glucan Synthase to Inactivation by Phospholipases and Trypsin

UDP-glucose:(1,3)-beta-glucan synthase from Beta vulgaris L. was rapidly inactivated by treatment with phospholipases C, D, and A(2). Enzyme activity could not be restored to the phospholipase-treated enzyme by the addition of phosphatidylethanolamine or other phospholipids. Membrane-bound and solubilized glucan synthase were also trypsin-labile with inactivation rates equal in the presence or absence of divalent cations or chelators. Gradual activity declines were observed in membranes incubated with divalent cations, but not with chelators.

Phosphatases and phosphodiesterases interfere with 1,3-beta-D-glucan synthase activity in pea epicotyl membrane preparations.

Soluble and membrane-bound phosphatase and phosphodiesterase activities are present in preparations of 1,3-beta-D-glucan synthase from pea epicotyls. UDP-glucose phosphodiesterase and non-specific alkaline phosphatase could be partially inhibited by N-ethylmaleimide or iodoacetamide and partially removed from membranes by washing. Such treatments helped to prolong 1,3-beta-glucan synthase activity. Nevertheless, the 1,3-beta-D-glucan synthase activity in washed membranes still gradually decreased during incubation in buffer at 30 degrees C. The rate of decay was reduced by adding more specific phosphatase inhibitors, e.g. molybdate, vanadate or fluoride, or by addition of nucleotides, and much of the loss of 1,3-beta-D-glucan synthase activity during preincubation could be restored by addition of phosphatidylethanolamine to the assay mixtures. It is concluded that membrane phospholipid is an essential part of the environment of 1,3-beta-glucan synthase and must be maintained intact in order for the enzyme to remain fully active.

Effects of changed lipid composition on responses of liposomes to various odorants: possible mechanism of odor discrimination.

In a previous paper [Nomura, T., & Kurihara, K. (1987) Biochemistry (preceding paper in this issue)], we showed that azolectin liposomes are depolarized by various odorants and there is a good correlation between the responses in the liposomes and the frog or porcine olfactory responses. In this study, we examined effects of changed lipid composition on responses of liposomes to various odorants. The membrane potential changes in response to odorants were monitored with the fluorescent dye 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. Egg phosphatidylcholine (PC) liposomes showed depolarizing responses to nine odorants among ten odorants tested. The magnitudes of depolarization by alcohols were similar to those in azolectin liposomes, but those by other odorants were much less than those in azolectin liposomes. Addition of sphingomyelin (SM) to PC led to an increase in the magnitude of depolarization by most odorants. Addition of phosphatidylethanolamine (PE) to PC (PE/PC = 0.25) led to depolarizing responses to four odorants among six odorants tested, and a further increase in PE content (PE/PC = 0.54) led to depolarizing responses only to two odorants. Addition of SM to the lipids of this composition of PC and PE [SM/(PC + PE) = 0.22] led to depolarizing responses to four odorants again. Liposomes made of a mixture of SM, PE, and PC exhibited depolarizing responses to four odorants tested, and addition of cholesterol to the lipids [cholesterol/(PC + PE + SM) = 0.05 and 0.11] led to depolarizing responses only to two and one odorant, respectively. Thus, changes in lipid composition of liposomes led to great changes in specificity of the responses to odorants.(ABSTRACT TRUNCATED AT 250 WORDS)

Ganglioside-mediated modulation of cell growth. Specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor.

Glycosphingolipids added exogenously to 3T3 cells in culture were shown to inhibit cell growth, alter the membrane affinity to platelet-derived growth factor binding, and reduce platelet-derived growth factor-stimulated membrane phosphorylation (Bremer, E., Hakomori, S., Bowen-Pope, D. F., Raines, E., and Ross, R. (1984) J. Biol. Chem. 259, 6818-6825). This approach has been extended to the epidermal growth factor (EGF) receptor of human epidermoid carcinoma cell lines KB and A431. GM3 and GM1 gangliosides inhibited both KB cell and A431 cell growth, although GM3 was a much stronger inhibitor of both KB and A431 cell growth. Neither GM3 nor GM1 had any affect on the binding of 125I-EGF to its cell surface receptor. However, GM3 and, to a much lower extent, GM1 were capable of inhibiting EGF-stimulated phosphorylation of the EGF receptor in membrane preparations of both KB and A431 cells. Further characterization of GM3-sensitive receptor phosphorylation was performed in A431 cells, which had a higher content of the EGF receptor. The following results were of particular interest. (i) EGF-dependent tyrosine phosphorylation of the EGF receptor and its inhibition by GM3 were also demonstrated on isolated EGF receptor after adsorption on the anti-receptor antibody-Sepharose complex, and the receptor phosphorylation was enhanced on addition of phosphatidylethanolamine. (ii) Phosphoamino acid analysis of the EGF receptor indicated that the reduction of phosphorylation induced by GM3 was entirely in the phosphotyrosine and not in the phosphoserine nor phosphothreonine content. (iii) The inhibitory effect of GM3 on EGF-dependent receptor phosphorylation could be reproduced in membranes isolated from A431 cells that had been cultured in medium containing 50 nmol/ml GM3 to effect cell growth inhibition. The membrane fraction isolated from such growth-arrested cells was found to be less responsive to EGF-stimulated receptor phosphorylation. These results suggest that membrane lipids, especially GM3, can modulate EGF receptor phosphorylation in vitro as well as in situ.

Effects of solute hydrophobicity and phospholipid composition on permeability of composite membranes containing phospholipids

Permeabilities of several solutes through the composite membranes containing phospholipids have been measured. They were inversely proportional to the content of the phospholipids in the membrane. Both the permeability of solutes and the degree of permeability change around the phase transition temperature of the phospholipids for the hydrophobic solutes such as n-butanol and salicylamide were larger than those for the hydrophilic solutes such as amino acids and pyridoxine. These results suggest that the permeation path of hydrophobic solutes is different from that of hydrophilic ones. The addition of phosphatidyl ethanolamine, phosphatidyl serine, or phosphatidic acid to the composite membrane influenced the solute permeability due to the introduced negative charge and/or the change in the molecular packing of phospholipid.

The sodium channel from rat brain. Reconstitution of voltage-dependent scorpion toxin binding in vesicles of defined lipid composition.

Purified sodium channels incorporated into phosphatidylcholine (PC) vesicles mediate neurotoxin-activated 22Na+ influx but do not bind the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx) with high affinity. Addition of phosphatidylethanolamine (PE) or phosphatidylserine to the reconstitution mixture restores high affinity LqTx binding with KD = 1.9 nM for PC/PE vesicles at -90 mV and 36 degrees C in sucrose-substituted medium. Other lipids tested were markedly less effective. The binding of LqTx in vesicles of PC/PE (65:35) is sensitive to both the membrane potential formed by sodium gradients across the reconstituted vesicle membrane and the cation concentration in the extravesicular medium. Binding of LqTx is reduced 3- to 4-fold upon depolarization to 0 mV from -50 to -60 mV in experiments in which [Na+]out/[Na+]in is varied by changing [Na+]in or [Na+]out at constant extravesicular ionic strength. It is concluded that the purified sodium channel contains the receptor site for LqTx in functional form and that restoration of high affinity, voltage-dependent binding of LqTx by the purified sodium channel requires an appropriate ratio of PC to PE and/or phosphatidylserine in the vesicle membrane.

UDP-Glucose: Sterol Glucosyltransferase and a Steryl Glucoside Acyltransferase Activity in Amyloplast Membranes from Potato Tubers

Envelope membranes were isolated from potato tuber amyloplast by a discontinuous sucrose density gradient and high speed centrifugation. These membranes catalyzed the transfer of [14C]glucose from UDP-[14C]glucose to endogenous sterol acceptors and, in turn, catalyzed the esterification of steryl glucosides with fatty acids from an endogenous acyl donor. The synthesis of steryl glucosides was stimulated in the presence of Triton X-100, while formation of acyl steryl glucosides was inhibited by the detergent. However, in the presence of an added sterol acceptor and Triton X-100, the inhibition of acyl steryl glucoside synthesis was overcome by the addition of phosphatidylethanolamine. The enzyme involved in steryl glucoside formation was solubilized by treatment of the envelope membranes with 0-3% Triton X-100. The solubilized enzyme had an almost absolute requirement for sterol acceptors.

Effect of liposome composition on the activity of detergent-solubilized acylcoenzyme A: cholesterol acyltransferase.

Acylcoenzyme A:cholesterol acyltransferase (ACAT) was solubilized from Ehrlich ascites cell microsomes with Triton X-100. After removal of the detergent, ACAT activity per mg protein was reduced by 50 to 65% as compared with untreated microsomes. When this microsomal extract was combined with liposomes composed of cholesterol and egg phosphatidylcholine, the ACAT activity increased 5.4- to 6.7-fold. Under these conditions sucrose density gradient centrifugation indicated that more than 50% of the added lipid was incorporated into vesicles having the same density as the ACAT activity, suggesting the formation of a complex. ACAT activity increased 2.9-fold when the phosphatidylcholine content of the liposomes was raised from 0.5 to 5.0 mumol/mg microsomal protein. By contrast, the ACAT activity increased only 42% when the cholesterol content of the liposomes was raised from 0.17 to 0.57 mumol/mg microsomal protein. Addition of phosphatidylethanolamine to the liposomes produced little change in ACAT activity, whereas the activity was reduced by 25 and 50%, respectively, when sphingomyelin or phosphatidylserine was added. ACAT activity was five times higher when the liposomes were prepared from dioleoylphosphatidylcholine than from saturated phosphatidylcholines, including hydrogenated egg yolk, dimyristoyl or dipalmitoyl phosphatidylcholine. Likewise, the ACAT activity with liposomes made from soybean or egg yolk phosphatidylcholine was almost 3.5-fold greater than with those prepared from the saturated phosphatidylcholines. These results are consistent with the view that the activity of ACAT can be modified by changes in the composition of the membrane lipids with which the enzyme is associated.

(Na+ + K+)-stimulated ATPase inhibition by cesalin and macromomycin.

(Na+ + K+)-stimulated ATPase was purified from dog kidney and its catalytic and glycoprotein subunits were separated. The enzymatic activity of the ATPase was totally inhibited by ouabain and partially inhibited by cesalin, maromomycin, concanavalin A, and wheat germ agglutinin. The inhibitions by cesalin or macromomycin can be reversed by the addition of phosphatidylethanolamine or phosphatidylserine, but not phosphatidylcholine. The specific binding of 125I-cesalin to the enzyme was inhibited by concanavalin A, wheat germ agglutinin, macromomycin, and the antibodies prepared in rabbits against the holoenzyme and the glycoprotein subunit, but not by ouabain, simple sugars, or the antibody against the catalytic subunit. It is proposed that the antitumor activities of cesalin and macromomycin are initiated in part by their binding to the glycoprotein subunit of (Na+ + K+)-ATPase in the plasma membrane.

Oxidative Dimerization of Tocopherols during the Course of Thermal Oxidation of Saturated and Unsaturated Triglycerides. I.

The effect of phospholipids on the oxidative decomposition of d-γ-tocopherol (γ-Toc) and the formation of its dimers during the thermal oxidation of trilaurin (Tri-La) and of trilinolein (Tri-Li) was examined. After triglycerides, in which γ-Toc and additives (phospholipids or their model compounds) dissolved, were oxidized with air at 180 for 10 h, residual γ-Toc and formed dimers were determined by HPLC. On the oxidation in Tri-La, little change of γ-Toc consumption was observed and the amount of formed 5- (γ-tocopheroxy) -γ-tocopherol (γ-TED) decreased by the addition of phospholipids (Table-1). When a small amount of phosphatidyl choline was added to Tri-Li, γ-Toc consumption greatly decreased, and a similar effect was observed also by the addition of phosphatidyl ethanolamine (Fig.-1). From this fact, it was assumed that nitrogenous base of phospholipid molecule is independent of the inhibitory effect of phospholipids on the thermal oxidation of γ-Toc. For clarifying the mechanism of the suppresssion of the thermal oxidation of γ-Toc with phospholipids, the following experiments were carried out : the oxidation of γ-Toc in Tri-Li containing tri-n-octylamine (TOA) as a model compound of nitrogenous base, or tri-n-butyl phosphate (TBP) as a model compound of phosphate ester. A large amount of TOA depressed the oxidation, but a small amount has little influence. On the other hand, γ-Toc consumption was remarkably reduced by the addition of a small amount of TBP (Fig.-4). The action of TBP is very similar to that of phospholipids. From the above results, it is considered that the functional group which suppresses the thermal oxidation of γ-Toc in Tri-Li is phosphate ester linkage. The dimers which formed during the thermal oxidation of γ-Toc in the presence of phospholipids were qualitatively the same as those in the absence of phospholipids. The amount of γ-TED increased to some extent, and the amounts of 5- (γ-tocopherol-5-yl) -γ-tocopherol and its atropisomer [γ-TBD (H) and γ-TBD (L)] were not influenced by the addition of phospholipids (Figs.-2 and 3). When TOA or TBP was added to Tri-Li, a little increase of the amount of γ-TED formed was observed, but the change of the amounts of γ-TBD (H) and γ-TBD (L) was less than that of γ-TED (Figs.-5 and 6). All of the examined additives depressed the thermal oxidation of Tri-Li (Fig.-1).

UDPG:Sterol glucosyltransferase in etiolated pea seedlings

Sterol UDPglucose glucosyltransferase was located predominantly in the axis tissue of etiolated pea seedlings. During the first 11 days of growth the activity reached a peak in the axis tissue after seven days. Centrifugation of tissue homogenates showed the cell fraction sedimenting between 13000 nd 25000 g to have the highest specific activity and also the bulk of the total activity. Sitosterol is the major free sterol of this fraction and cholesterol is a trace component. The composition of the aglycones of the isolated steryl glycosides shows cholesterol to be the major sterol. Although exhibiting no metal ion requirement, the enzyme is stimulated by Ca 2+ and Mg 2+, partially inhibited by EDTA and EGTA and completely inhibited by Zn 2+. The membranous nature of the enzyme is manifested by its stimulation by the addition of phosphatidyl -ethanolamine, -choline and -serine. After brief treatment with phospholipases A, C and D, enzyme activity is partially lost. After phospholipase A treatment the activity may be completely restored by the addition of phosphatidyl ethanolamine but phosphatidyl-choline and -serine are without effect. After phospholipase C and D treatment, each phospholipid brings about a partial recovery of activity but phosphatidyl ethanolamine is again superior.

Determinants of the Formation and Activity of Factor V-phospholipid Complexes II. Molecular Properties of the Complexes

The stability, buoyancy, and intrinsic activity for factor V-phospholipid complexes were investigated. The decay of factor V activity in the absence of phospholipid followed first order kinetics; however, in the presence of several phospholipids a biphasic decay curve was observed. The addition of phosphatidylethanolamine to factor V produced only a small loss of activity in the first 2 minutes but decreased the subsequent rate of inactivation fourfold. A PE-factor V complex with a low bouyant density was separated from uncomplexed factor V by sucrose density ultracentrifugation. The association constant for this complex was 5 X 10(6) M-1 with approximately 2 moles of factor V bound per mole of lipid micelle. The isolated complex was capable of increasing prothrombin conversion 10-fold without additional phospholipid. A still lighter complex increased the rate of prothrombin conversion 18-fold. Phosphatidyl serine produced a concentration-dependent loss of up to 95% of the factor V activity in the first 2 minutes. After ultracentrifugation on a sucrose density gradient, a PS-factor V complex of increased density was detected. This complex failed to accelerate prothrombin conversion in the intrinsic two-stage assay. Except at very high concentrations, phosphatidylcholine did not alter the kinetics of inactivation of factor V. A factor V-phosphatidylcholine complex could not be detected after ultracentrifugation. When added to factor V, cardiolipin (200 mug/ml), produced a rapid 50% decline in activity with a subsequent three-fold increase in the rate of inactivation. No activity was recovered after ultracentrifugation of factor V in the presence of cardiolipin. Saturated phosphatidylethanolamine produced a concentration dependent initial loss of activity, but only a minimal increase in the subsequent rate of inactivation. At 200 mug/ml almost no light complex was detected after ultracentrifugation, but at 800 mug/ml a light complex was observed. This behavior corresponds to the ability of saturated phosphatidylethanolamine to accelerate prothrombin conversion only at very high concentrations. Thus, phospholipids combine with factor V to form complexes which differ in their ability to accelerate prothrombin conversion. The most active species are stable lipoprotein complexes of lower buoyant density than factor V.