Phospholipids with a short acyl chain modulate phospholipase and acyltransferase activities.

1-Acyl lysophosphatidylcholine prepared from egg yolk has been chemically reacylated to form decanoyl, dodecanoyl, myristoyl and palmitoyl derivatives of phosphatidylcholine. The liposomes formed by these semi-synthetic phospholipids have been characterized by calorimetry, X-ray diffraction and fluorescence probe methods. Asymmetric phosphatidylcholines tend to promote formation of excimers of a codispersed fluorescent phospholipid (1-palmitoyl-sn-2-(1-pyrenedecanoyl)-L-alpha-phosphatidic acid) (2 mol%). Excimer formation is correlated with the rate of hydrolysis of the fluorescent anionic phospholipid by Crotalus venom phospholipase A2. Codispersion with the semi-synthetic phosphatidylcholine of cholesterol or unsaturated fluid lecithin modulated both excimer formation and the susceptibility of the fluorescent probe to hydrolysis by venom phospholipase A2 at 22 degrees C. Similar results were obtained with hydrolysis of a radiolabelled substrate, 1-palmitoyl-sn-2-[1-14C]linoleoylphosphatidylethanolamine, codispersed with the semi-synthetic phosphatidylcholine. Enrichment of rat hepatocyte plasma membranes with semi-synthetic asymmetric phosphatidylcholines was mediated by incubation of membranes with phospholipid dispersions in the presence of a phospholipid exchange protein. Enrichment of the membranes with semi-synthetic phosphatidylcholines of between 30 and 60% of the membrane phosphatidylcholine was achieved. The resulting alteration of the biomembrane is associated with a decreased activity of endogenous membrane phospholipase A2 acting on extramembranous radiolabelled substrate vesicles. By contrast, the activity of acyl-CoA:lysophospholipid acyltransferase is increased in membranes enriched with highly asymmetric phospholipids.

Acyl-CoA synthetase activity depends on the phospholipid composition of rat liver plasma membranes.

The dependence of acyl-CoA synthetase on the lipid composition of rat liver plasma membranes has been investigated. For this purpose the composition of the membranes was modified by incorporation of different phospholipids in the presence of partially purified lipid transfer proteins. Another approach to the modification of the membrane phospholipid composition was treatment with exogenous phospholipase C and subsequent enrichment with different phospholipids. The experiments performed in vitro indicated that the presence of certain phospholipids such as phosphatidylnositol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine was essential for the activation of long chain fatty acids by acyl-CoA synthetase. However, some differences were observed when oleate and palmitate were used as substrates. Sphingomyelin was found to inhibit this activity especially when oleic acid served as substrate. In addition, we tried to modify in vivo the membrane lipid composition by treatment with D-galactosamine, which is known to induce acute hepatitis and cause biochemical and biophysical alterations in liver membranes. The results thus obtained confirmed the idea that the augmentation of the membrane lipids and especially of PI, PE and PG was accompanied by acyl-CoA synthetase activation. The presence of two different enzymes, activating the saturated and unsaturated fatty acids is discussed.

Phospholipase C activities in rat liver plasma membranes depend on the phospholipid composition.

Investigations were carried out on the influence of rat liver plasma membranes phospholipid composition on phospholipase C activity using PIP, PIP2, PC and PE as substrates. The membrane phospholipids were modified by incorporation of definite phospholipids with the aid of lipid transfer proteins or after partial delipidation with exogenous phospholipases A2 and C. The results indicated that sphingomyelin inhibited all phospholipase C activities. The incorporation of two different molecular species of phosphatidylcholine did not alter significantly the investigated phospholipase C activities, indicating that membrane fluidity was not essential in this case. Phosphatidylglycerol, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine served as specific activators of plasma membrane-bound phospholipase C when PIP, PIP2 and PC were used as substrates. However, these four phospholipids inhibited phospholipase C activity towards PE. The role of phosphoinositide-specific phospholipase C in the production of second messengers as well as the eventual biological significance of PC and PE as substrates for phospholipase C is discussed.

Effect of membrane phospholipid composition and fluidity on rat liver plasma membrane tyrosine kinase activity.

1. The effect of membrane phospholipid composition and fluidity on tyrosine kinase activity was investigated in rat liver plasma membranes. 2. The phospholipid composition has been modified by in vitro enrichment of plasma membranes with different phospholipids in the presence of lipid transfer proteins and by partial delipidation with exogenous phospholipases A2, C and D and subsequent enrichment with phosphatidylglycerol. 3. Phosphatidylglycerol and dioleoylglycerophosphocholine caused dramatic elevation of this activity, while phosphatidylserine and phosphatidylethanolamine were less effective. Enrichment with dipalmitoylglycerophosphocholine and sphingomyeline reduced tyrosine kinase activity.

Alterations in microsomal and plasma membranes during liver regeneration.

Investigations have been carried out on the alterations of membrane lipids and some enzyme activities during liver regeneration. The results indicated that 32 h after partial hepatectomy the membrane phospholipids per mg protein were augmented. The cholesterol esters were also increased in both microsomal and plasma membranes. The specific radioactivity of the separate phospholipid fractions, estimated by incorporation of 14C-palmitate into the phospholipid molecules, was higher in membranes from partially hepatectomized rats, compared to sham-operated ones, indicating an enhanced phospholipid synthesis. The content and specific radioactivity of diacylglycerols and triacylglycerols was enhanced in both types of membranes from regenerating liver. Moreover, we observed a fluidization of these membranes, which is illustrated by the decrease of the structural order parameter (SDPH) of the lipid bilayer as well as by the elevation of the excimer to monomer fluorescent ratio (IE/IM). 1,6-Diphenyl-1,3,5-hexatriene and pyrene were used as fluorescent probes for determination of the membranes physical state. Palmitoyl-CoA and oleoyl-CoA synthetase, acyl-CoA: lysophosphocholine and acyl-CoA:lysophosphoethanolamine acyltransferase as well as phospholipase C activities were augmented in membranes from partially hepatectomized rats. The biological significance of these alterations in the process of liver regeneration is discussed.

Phospholipid dependence of rat liver microsomal acyl:CoA synthetase and acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase.

Investigations were performed on the influence of the phospholipid composition and physicochemical properties of the rat liver microsomal membranes on acyl-CoA synthetase and acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase activities. The phospholipid composition of the membranes was modified by incubation with different phospholipids in the presence of lipid transfer proteins or by partial delipidation with exogenous phospholipase C and subsequent enrichment with phospholipids. The results indicated that the incorporation of phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine induced a marked activation of acyl-CoA synthetase for both substrates used--palmitic and oleic acids. Sphingomyelin occurred as specific inhibitor for this activity especially for palmitic acid. Palmitoyl-CoA: and oleoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase activities were found to depend on the physical state of the membrane lipids. The alterations in the membrane physical state were estimated using two different fluorescent probes--1,6-diphenyl-1,3,5-hexatriene and pyrene. In all cases of membrane fluidization this activity was elevated. On the contrary, in more rigid membranes obtained by incorporation of sphingomyelin and dipalmitoylphosphatidylcholine, acyltransferase activity was reduced for both palmitoyl-CoA and oleoyl-CoA. We suggest a certain similarity in the way of regulation of membrane-bound acyltransferase and phospholipase A2 which both participate in the deacylation-reacylation cycle.

Phospholipid-dependence of rat liver plasma membrane protein kinase activities--a new approach.

The influence of the phospholipid composition and fluidity on protein kinase A and protein kinase C activities in rat liver plasma membranes was studied. We observed that enrichment of membranes with phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine and dioleoylphosphatidylcholine caused activation of both protein kinases. Phosphatidylglycerol was found to be most effective activator. The enrichment of plasma membranes with dipalmitoylphosphatidylcholine and sphingomyelin led to decrease in protein kinase A and C activities. The stimulatory effect of phosphatidylglycerol was confirmed in plasma membranes pretreated with exogenous phospholipases A2, C and D, and subsequently enriched with phosphatidylglycerol. We suggest that besides the specific presence of definite phospholipids protein kinases A and C require a more fluid membrane lipid bilayer to display an optimal activity.

Phospholipid modifications influence acyl-CoA:1-acyl-glycero-3-phosphocholine O-acyltransferase in rat liver plasma membranes.

The influence of the membrane lipid composition and physical state on the activity of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase in rat liver plasma membranes has been investigated. The membrane's lipid composition has been modified either by lipid transfer proteins or by partial delipidation with exogenous phospholipases. The results indicate that membrane fluidity is of particular importance for membrane-bound palmitoyl-CoA: and oleoyl-CoA:1-acyl-glycero-3-phosphocholine acyltransferase. The incorporation of phospholipids that induce membrane fluidization such as dioleoylphosphatidylcholine, egg yolk phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine was accompanied by an elevation of acyltransferase activity. On the contrary, the phospholipids causing augmentation of membrane rigidity induced a decrease of this activity. A suggestion is made concerning the possible role of the membrane physical state for the deacylation-reacylation cycle in rat liver plasma membranes.

Phospholipid dependence of phospholipase C in rat liver plasma membranes.

Investigations have been carried out on the lipid dependence of membrane-bound phosphatidylinositol-specific phospholipase C in rat liver plasma membranes. For this purpose the phospholipid composition of rat liver plasma membranes has been modified in two different ways. The first method included enrichment of plasma membranes with different phospholipids in the presence of lipid transfer proteins, and the second a partial delipidation by means of exogenous phospholipases A2 and C and selective enrichment with different phospholipids. The results indicated that almost all used phospholipids induced activation of phosphatidylinositol-specific phospholipase C except sphingomyelin. Phosphatidylethanolamine and egg yolk phosphatidylcholine were observed to be most effective in phospholipase C activation.

Acyl-CoA: 1-acyl-glycero-3-phosphoethanolamine O-acyltransferase and liver plasma membrane fluidity.

Investigations have been carried out on the influence of membrane lipid composition and physical state on acyl-CoA: 1-acyl-glycerol-3-phosphoethanolamine O-acyltransferase activity in rat liver plasma membranes. The lipid composition of the membranes was modified either by way of lipid transfer proteins or by partial delipidation with exogenous phospholipases and subsequent enrichment of the membranes with different phospholipids. The results indicated that membrane rigidification by enrichment of the membranes with DPPC or SM reduced the transfer of oleic and palmitic acid to lysophosphatidylethanolamine, whereas all phospholipids inducing membrane fluidization lead to acyltransferase activation. The eventual role of membrane fluidity in the deacylation-reacylation cycle is discussed.

Effect of probucol on the lipid composition of blood plasma, erythrocyte ghosts and liver membranes in mice.

1. Probucol treatment of mice (0.6 g/kg) induced a decrease of cholesterol (CH) and total phospholipids (PLs) in blood plasma, erythrocyte ghosts, liver plasma and microsomal membranes. 2. The incorporation of [14C]acetate in the microsomal lipids of probucol-treated mice was lowered by 23% compared to controls. 3. Probucol administration induced a reduced specific activity of PLs, CH and CH esters, whereas in triacylglycerols it was augmented. 4. Phospholipase A2 and neutral sphingomyelinase activities were not enhanced, indicating that the catabolism of the membrane PL was not elevated.

Age-related changes in rat liver phospholipid transfer activity.

The age-induced changes in the liver cytosol phospholipid transfer activity of male and female rats have been investigated. These changes were found to be closely related to the age-induced alterations in the two major microsomal phospholipids--phosphatidylcholine and phosphatidylethanolamine. Regression analysis indicated a linear correlation between the phospholipid transfer activity and the level of phosphatidylcholine (positive) and phosphatidylethanolamine (negative) in liver microsomes of both male and female rats.

Age-related changes in rat liver plasma membrane sphingomyelinase activity.

The age-induced changes of some phospholipid fractions, membrane fluidity and neutral membrane-bound sphingomyelinase (EC 3.1.4.12) activity in rat liver plasma membranes have been investigated. Alterations in the percentage participation of phosphatidylcholine and sphingomyelin with aging have been established. Regression analysis indicated a positive linear correlation (r = 0.927) between the membrane-bound neutral sphingomyelinase activity and the phosphatidylcholine percent in the total plasma membrane phospholipids, as well as a negative linear correlation (r = -0.937) between the enzyme activity and the sphingomyelin/phosphatidylcholine ratio.

D-galactosamine induced changes in rat liver plasma membranes lipid composition and some enzyme activities.

The influence of D-galactosamine administration on rat liver plasma membranes lipid composition, fluidity and some enzyme activities was investigated. D-Galactosamine was found to induce an increase of the total phospholipids, the cholesterol level and membrane rigidity. In liver plasma membranes of D-galactosamine-treated rats the exogenous phospholipase A2 activity was enhanced about 2 fold, whereas the endogenous activity was slightly decreased. No alteration of the neutral sphingomyelinase activity was observed.