Biomedical subjects

M Waite

Publications and source records attributed to M Waite.

At least 19 recordsLinked to original sources

PubMed1999

Transacylase formation of bis(monoacylglycerol)phosphate.

Recent work within our laboratory has focused on the enzymes we hypothesize are involved in the biosynthesis of bis(monoacylglycerol)phosphate from phosphatidylglycerol. Here we describe a transacylase, active at acidic pH values, isolated from a macrophage-like cell line, RAW 264.7. This enzyme acylates the head group glycerol of sn-3:sn-1' lysophosphatidylglycerol to form sn-3:sn-1' bis(monoacylglycerol)phosphate. Here we demonstrate that this enzyme uses two lysophosphatidylglycerol molecules, one as an acyl donor and another as an acyl acceptor, and that the acyl contributions from all other lipids tested are comparatively minor. This enzyme prefers saturated acyl chains to monounsaturates, 16 and 18 carbon fatty acids over 14 carbon fatty acids, and saturated acyl chains at the sn-1 position to monounsaturated acyl chains on the sn-2 carbon of lysophosphatidylglycerol. We present data which show the transacylase activity depends on the presence of a lipid-water interface and the lipid polymorphic state.

Acyltransferases↗

PubMed1999

A novel phosphatidylglycerol-selective phospholipase A2 from macrophages.

In our recent studies on the synthesis of bis(monoacylglycero)phosphate (BMP), we postulated that the first step involved a PLA2 that cleaved the 2-acyl group from phosphatidylglycerol (PG). In the present study, a novel lysosomal PLA2 was partially purified and characterized from RAW 264.7, macrophage like cells. Cells were homogenized and delipidated, and the PLA2 activity in the soluble fraction was purified by Sephacryl S100 and DEAE Sephacel. Further purification was performed using Con-A Sepharose, Phenyl Sepharose, DEAE Sephacel, and Superdex 75 FPLC. The enzyme at this stage of purification showed a dominant band around 45 kDa plus several minor bands on SDS-PAGE. The molecular mass determined by Superdex 75 column FPLC was about 45 kDa. The highly purified fraction hydrolyzed at the sn-1 position, implying that this PLA2 also has some intrinsic PLA1 activity. This enzyme preferentially hydrolyzed PG, has an acidic pH optima, and does not require divalent metal ions. Comparison using PG with various acyl chains on the sn-2 position showed that oleate and linoleate were preferred relative to arachidonate. MAFP, a known cytosolic PLA2 inhibitor, strongly inhibited this PLA2 activity. MJ33, AACOCF3, DENP, and Amiodarone also gave moderate inhibition. The characteristics of this enzyme showed this to be a new type of PLA, and the overwhelming preference for PG as substrate suggests its physiological role is in the biosynthesis of BMP.

Acrylic Resins↗

PubMed1999

Lipid exchange between mixed micelles of phospholipid and triton X-100.

If phospholipase catalyzed hydrolysis of phospholipid dissolved in a detergent mixed micelle is limited to the phospholipid carried by a single micelle, then hydrolysis ceases upon exhaustion of that pool. However, if the rate of phospholipid exchange between micelles exceeds the catalytic rate then all of the phospholipid is available for hydrolysis. To determine phospholipid availability we studied the exchange of 1,2-dioleoyl-sn-glycero-3-phosphocholine between mixed micelles of phospholipid and non-ionic Triton detergents by both stopped-flow fluorescence-recovery and nuclear magnetic resonance-relaxation techniques. Stopped-flow analysis was performed by combining mixed micelles of Triton and phospholipid with mixed micelles that contained the fluorescent phospholipid 1-palmitoyl-2-(12-[{7-nitro-2-1, 3-benzoxadiazo-4-yl}amino]dodecanoyl)-sn-glycero-3-phosphocholine (P-2-NBD-PC). The concentration dependence of fluorescence recovery suggested a second-order exchange mechanism that was saturable. The true second-order rate constant depends on the specific mechanism for exchange, which was not determined in this study, but the rate constant will be on the order of 106 to 107 M-1s-1. Incorporation of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine into micelles increased the rate of proton relaxation and gave a limiting relaxation time of 1.3 ms. The results demonstrate that phospholipid exchange was rapid and that the phospholipid content of a single micelle did not limit the rate of phospholipid hydrolysis by phospholipases.

Fluorescence↗

PubMed1998

Hydrolysis of surfactant-associated phosphatidylcholine by mammalian secretory phospholipases A2.

Hydrolysis of surfactant-associated phospholipids by secretory phospholipases A2 is an important potential mechanism for surfactant dysfunction in inflammatory lung diseases. In these conditions, airway secretory phospholipase A2 (sPLA2) activity is increased, but the type of sPLA2 and its impact on surfactant function are not well understood. We examined in vitro the effect of multiple secretory phospholipases A2 on surfactant, including their ability to 1) release free fatty acids, 2) release lysophospholipids, and 3) increase the minimum surface tension (gammamin) on a pulsating bubble surfactometer. Natural porcine surfactant and Survanta were exposed to mammalian group I (recombinant porcine pancreatic) and group II (recombinant human) secretory phospholipases A2. Our results demonstrate that mammalian group I sPLA2 hydrolyzes phosphatidylcholine (PC), producing free fatty acids and lysophosphatidylcholine, and increases gammamin. In contrast, mammalian group II sPLA2 demonstrates limited hydrolysis of PC and does not increase gammamin. Group I and group II secretory phospholipases A2 from snake venom hydrolyze PC and inhibit surfactant function. In summary, mammalian secretory phospholipases A2 from groups I and II differ significantly from each other and from snake venom in their ability to hydrolyze surfactant-associated PC.

Animals↗

PubMed1997

Hydrolysis of phosphatidylcholine by hepatic lipase in discoidal and spheroidal recombinant high-density lipoprotein.

Hepatic lipase (HL) hydrolysis of phosphatidylcholine (PC) was studied in recombinant high-density lipoprotein particles (r-HDL). r-HDL were made from cholate mixed micelles that contained PC, apo AI, and, in some cases, unesterified cholesterol. r-HDL were characterized using chemical composition, nondenaturing gradient gel electrophoresis, transmission electron microscopy, and dynamic light scattering. The r-HDL were found to be discoidal and in the size range of native HDL. Upon treatment of cholesterol-containing r-HDL with lecithin-cholesterol acyltransferase (LCAT), to form cholesteryl ester, the discoidal r-HDL became spheroidal. The effects of r-HDL morphology and size on HL activity were studied on r-HDL made of palmitoyloleoyl-PC, unesterified cholesterol, cholesteryl ester, and apolipoprotein AI. Spheroidal r-HDL were hydrolyzed at a faster rate than discoidal r-HDL. Protein-poor r-HDL were hydrolyzed by HL at a faster rate than protein rich r-HDL. Unesterified cholesterol had no apparent effect on particle PC hydrolysis. The hydrolysis of different species of PC [dipalmitoyl (DPPC), dioleoyl(DOPC), palmitoylarachidonoyl (PAPC), and palmitoyloleoyl (POPC)] in r-HDL was also investigated. In discoidal r-HDL, we found that POPC >/= DOPC = PAPC/DPPC. However, in LCAT-treated spheroidal r-HDL, POPC = DOPC > PAPC/DPPC. In both discoidal and spheroidal rHDL, DPPC containing r-HDL were not hydrolyzed to a significant extent. Collectively, these studies demonstrate that the physico-chemical properties of particles (such as phospholipid packing and phospholipid acyl composition) play a significant role in hydrolysis of HDL phospholipid by HL and, therefore, in reverse cholesterol transport.

Apolipoprotein A-I↗

PubMed1996

Transacylase and phospholipases in the synthesis of bis(monoacylglycero)phosphate.

Bis(monoacylglycero)phosphate (monoacyl-sn-glycero-1-phospho-1'-monoacyl-sn-glycerol) is a unique lipid that represents greater than 15% of the total phospholipid of the resident alveolar macrophage. Bis(monoacylglycero)phosphate is not synthesized de novo but rather is derived from phosphatidylglycerol of the lung surfactant. There are two enantiomers of bis(monoacylglycero)phosphate synthesized, but only the sn-1, and not the sn-3, enantiomer accumulates in vivo. We recently published a scheme in which the sn-3 enantiomer was an intermediate in the synthesis of the final sn-1 bis(monoacylglycero)-phosphate. Here we further expand the understanding of the biosynthesis of bis(monoacylglycero)phosphate by examining the proposed first two steps of the pathway. A phospholipase A and a transacylase activity are partially separated by gel permeation chromatography. Both are optimally active in the acid pH range that supports the concept that they function in the lysosome-endosome compartment of the cell. Independently, these two enzyme systems are incapable of converting phosphatidylglycerol into sn-3 bis(monoacylglycero)phosphate. However, combination of the two partially purified enzymes reestablishes the synthesis of sn-3 bis(monoacylglycero)phosphate from phosphatidylglycerol. The results presented here support our hypothesis that the phospholipase and transacylase are separate enzymes essential to the synthesis of bis(monoacylglycero)phosphate.

Acyltransferases↗

PubMed1995

Stereospecific labeling of the glycerol moiety: synthesis of 1,2-dioleoyl-sn-[3-3H]glycero-3-phospho(1-rac-glycerol).

1,2-Dioleoyl-sn-[3-3H]glycero-3-phospho(1-rac-glycerol) was synthesized from 1,2-dioleoyl-sn-glycerol using a new radiosynthetic procedure. 1,2-Dioleoyl-sn-glycerol was oxidized to the corresponding aldehyde using pyridinium dichromate and pyridine. The aldehyde was reduced to the radiolabeled alcohol using tritiated sodium borohydride and crown ether. This material was then converted to the phosphocholine derivative using 2-chloro-2-oxo-1,3,2-dioxaphospholane, followed by displacement with trimethylamine. In the last step, the 1,2-dioleoyl-sn-[3-3H]glycero-3-phosphocholine was converted to 1,2-dioleoyl-sn-[3-3H]glycero-3-phospho-(1-rac-glycerol) via a classic transphosphatidylation reaction using glycerol and cabbage phospholipase D. A theoretical explanation of unusual chemical behavior of the primary alcohol of diglycerides is also given, based on semi-empirical calculations.

Diglycerides↗

PubMed1995

Biosynthetic conversion of phosphatidylglycerol to sn-1:sn-1' bis(monoacylglycerol) phosphate in a macrophage-like cell line.

Bis(monoacylglycerol) phosphate has a unique stereoconfiguration of sn-glycero-1-phospho-1'-sn-glycerol and is synthesized from exogenous phosphatidylglycerol by macrophages. Previous work by our laboratory showed that the macrophage-like cell line RAW 264.7 synthesizes sn-glycero-1-phospho-1'-sn-glycerol bis(monoacylglycerol) phosphate. Here we describe studies using RAW 264.7 cells that examine the biosynthetic pathway by which bis(monoacylglycerol) phosphate is formed. Experiments were conducted using precursors that were specifically radiolabeled on the glycerol backbone in order to examine the stereoconfiguration of the intermediates and products formed in intact RAW 264.7 cells. The results of our studies indicate that a complex series of reactions are involved in the synthesis of bis(monoacylglycerol) phosphate. In this proposed pathway phosphatidylglycerol is hydrolyzed to form 1-acyllysophosphatidylglycerol which is then acylated on the headgroup glycerol to form the sn-glycero-1-phospho-1'-sn-glycerol enantiomer of bis(monoacylglycerol) phosphate. The sn-glycero-1-phospho-1'-sn-glycerol enantiomer of bis(monoacylglycerol) phosphate is then thought to undergo a stereoconversion that proceeds via the required removal of the acyl group at the sn-1 position. The resulting sn-glycero-1-phospho-1'-sn-glycerol enantiomer of lysophosphatidylglycerol with the acyl moiety on the original headgroup glycerol is then acylated to form sn-glycero-1-phospho-1'-sn-glycerol bis(monoacylglycerol) phosphate.

Animals↗

PubMed1994

Lipoprotein lipase domain function.

Human lipoprotein lipase (LPL) monomer consists of two domains, a larger NH2-terminal domain that contains catalytic residues and a smaller COOH-terminal domain that modulates substrate specificity and is a major determinant of heparin binding. Analyses of NH2-terminal domain function were performed after site-directed mutagenesis of the putative active-site serine residue, while COOH-terminal domain function was assessed following reaction with a monoclonal antibody. The native enzyme and mutant LPL in which serine 132 was replaced with alanine, cysteine, or glycine were transiently expressed in COS-7 cells. Mutant proteins were synthesized and secreted at levels comparable to native LPL; however, none of the mutants retained enzymatic activity. The mutant with alanine replacing serine 132 was purified and shown to be inactive with both esterase and lipase substrates; however, binding to a 1,2-didodecanoyl-sn-glycero-3-phosphatidylcholine monolayer was comparable to native LPL. These results are consistent with a catalytic, and not a lipid binding, role for serine 132. To investigate the function of the smaller COOH-terminal domain, LPL lipolytic and esterolytic activities as well as heparin binding properties were determined after reaction with a monoclonal antibody specific for this domain. Lipolytic activity was inhibited by the monoclonal antibody, whereas esterolytic activity was only marginally affected, indicating that the LPL COOH-terminal domain is required for lipolysis, perhaps by promoting interaction with insoluble substrates. Also, the affinity of antibody-reacted LPL for heparin was not significantly different from that of LPL alone, suggesting that (i) the heparin-binding site is physically distinct from the COOH-terminal domain region required for lipolysis and (ii) binding of antibody did not cause dimer dissociation. A model is proposed for the two LPL domains fulfilling different roles in the lipolytic process.

Animals↗

PubMed1994

Ventricular entry during resection of malignant gliomas: effect on intracranial cerebrospinal fluid tumor dissemination.

The ventricular system is not infrequently entered during the course of maximum cytoreductive surgery for high-grade supratentorial gliomas. It is unclear if ventricular entry during surgery and/or proximity of the tumor to the ventricular system affects cerebrospinal fluid (CSF) tumor dissemination or the patients' overall survival rate. The authors retrospectively reviewed hospital records and neuroradiological studies of 51 patients operated on at the University of Washington between 1987 and 1991. Inclusion in this study necessitated a pathological diagnosis of malignant glioma and the availability of preoperative and postoperative computerized tomography scans or magnetic resonance images. Patients were excluded from the study if they had radiographic evidence of ventricular entry or CSF tumor dissemination prior to referral to the authors' institution. The index operation was defined as the first operation at the University of Washington or (in those patients with ventricular entry) the operation in which the ventricle was entered. Patients were followed until time of death or, in the case of survivors, until February, 1992. The effect of both ventricular entry and the proximity of the tumor to the ventricular system on CSF tumor dissemination and survival rate was assessed using statistical survival methodology. There was no significant difference in time from diagnosis to the index operation between groups compared (Mann-Whitney U-test, p > 0.40). Cerebrospinal fluid dissemination was radiographically documented in 18 patients (35%) following the index operation. This occurrence was not significantly influenced by either ventricular entry during surgery (Mantel-Cox test, p = 0.13), the proximity of the tumor to the ventricular system (p = 0.63), or these two variables combined (p = 0.28). Survival rate following the index operation was not significantly affected by ventricular entry (p = 0.66), proximity of the tumor to the ventricular system (p = 0.61), or these two variable considered in combination (p = 0.44). However, survival rate was significantly decreased once CSF tumor dissemination had occurred (Cox model, p = 0.03).

Adult↗

PubMed1994

Antioxidant status in controlled and uncontrolled hypertension and its relationship to endothelial damage.

Hypertension is associated with an increased risk of atherosclerosis. Free radical oxidative damage has been implicated in the atherogenic process. We measured levels of the antioxidants uric acid, thiols, vitamins C, A and E as well as the total antioxidant capacity in 21 normotensive controls, 22 patients whose hypertension was controlled on drugs and 30 patients with uncontrolled hypertension. Mean BPs in the groups were 125/76, 132/80 and 181/98 mmHg, respectively. When compared with controls, both hypertensive groups had significantly lower serum ascorbic acid (54 +/- 5 vs. 37 +/- 6 vs. 38 +/- 5 mumol/l, P < 0.05) and albumin-corrected thiol levels (9.91 +/- 0.18 vs. 8.69 +/- 0.20 vs. 8.92 +/- 0.19 mumol/g, P < 0.05). The levels of the other antioxidants did not differ significantly between the groups. Levels of von Willebrand factor, a marker of endothelial damage, were correlated with SBP but not with antioxidant status. We conclude that hypertensive subjects have lower levels of the antioxidants vitamin C and thiols and this may reflect greater oxidative consumption. The implications for atherogenesis and endothelial function and integrity in hypertension are discussed.

Adult↗

PubMed1993

Regulation of rat hepatic lipase by the composition of monomolecular films of lipid.

The regulation of hepatic lipase (HL) by the lipid composition of monomolecular substrate films was examined using a monolayer technique at constant surface pressure. HL-catalyzed hydrolysis of triacylglycerol, a poor substrate for HL in pure monomolecular films, was activated by diradylglycerol and its phosphorylated derivatives in mixed films containing 10 mol % triacylglycerol. When triacylglycerol was progressively diluted with dialkylglycerol, triacylglycerol hydrolysis by HL was maximal between 90 and 98 mol % dialkylglycerol. The best activators, dialkylphosphatidic acid and dialkylphosphatidylethanolamine, increased triacylglycerol hydrolysis 13-14-fold, and the enhancement of HL-catalyzed triacylglycerol hydrolysis by the activator lipids was inversely related to the average mean molecular area of the mixed films. The hydrolysis of 5 mol % triacylglycerol in mixed films that also contained phosphatidylcholine and 0-20 mol % cholesterol was inhibited approximately 80% when the concentration of cholesterol was 10-13 mol %. Interestingly, between 15 and 17 mol % cholesterol the hydrolysis rate was restored to about 50% of the uninhibited rate, but at 20 mol % cholesterol this value decreased back to 80% inhibition of hydrolysis. The hydrolysis of phosphatidylethanolamine in mixed films with 0-20 mol % cholesterol decreased approximately 30% in films containing 10-12 mol % cholesterol. However, at 15 mol % cholesterol the hydrolysis rate was restored to the same level observed for a pure phosphatidylethanolamine film. This enhancement of HL activity occurred at about the same cholesterol concentration as the restoration of triacylglycerol hydrolysis observed for the triacylglycerol/phosphatidylcholine/cholesterol films.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Free radicals, antioxidants, and endothelial cell damage after percutaneous transluminal coronary angioplasty.

BACKGROUND: Oxygen-derived free radicals (ODFRs) and subsequent lipid peroxidation may be responsible for myocardial damage associated with ischaemia/reperfusion after percutaneous transluminal coronary angioplasty (PTCA). These potentially cytotoxic ODFRs are likely to be generated in the plasma, where the primary target organ or cell may be the endothelium. Some of these toxic effects may be limited by the action of antioxidant enzymes such as glutathione peroxidase (GPx). The aim of this study was to determine whether there was evidence of ODFR-mediated damage to the endothelium after PTCA and to examine the roles of lipid peroxides and GPx. METHODS: Serial samples of plasma were obtained at the time of catheter insertion, at the time of balloon inflation, 10 min after inflation, and 60 min after inflation, from 16 patients undergoing PTCA. We measured levels of von Willebrand factor (vWf, a specific product of the endothelium and a marker of damage), thiobarbituric acid reactive substances (TBARS, an indirect measure of the activity of ODFRs in peroxidizing lipoproteins), and the antioxidant GPx. RESULTS: There was a simultaneous peak in the levels of TBARS (P = 0.0096) with a fall in the levels of GPx (P = 0.004) 10 min after balloon deflation. Sixty min after balloon deflation, GPx levels were still reduced (P = 0.005) but there was a rise in levels of vWf (P = 0.038). CONCLUSIONS: Our interpretation of these data is that an early peak in ODFR activity associated with PTCA causes an endothelial injury 1 h after inflation. This may be related to the reduced ability of GPx to scavenge ODFRs.

Adult↗

PubMed1993

von Willebrand factor and endothelial damage in essential hypertension.

The relationship between von Willebrand factor antigen (vWFAg, a specific endothelial cell product) and hypertension was examined. Circulating vWFAg levels were measured in serum from patients attending a hypertension clinic and in normotensive controls. The vWFAg was higher in those patients with uncontrolled hypertension at 131 +/- 34 IU/dl than in controls (90 +/- 30 IU/dl) and in those whose hypertension was controlled 104 +/- 29 IU/dl (P < 0.001). Levels of vWFAg correlated with both SBP (r = 0.42, P < 0.0002) and DBP (r = 0.25, P < 0.05), but serum from neither group of patients was more cytotoxic to cultured endothelial cells in vitro than was serum from controls. Neither symptoms of cardiovascular or peripheral vascular disease or two of its risk factors (hypercholesterolaemia or smoking, alone or in combination) appeared to further increase vWFAg in patients with uncontrolled hypertension. However, vascular disease and the same risk factors did increase vWFAg to 133 +/- 36 IU/dl in those patients with controlled hypertension (P < 0.001). These data indicate a relationship between vWFAg and hypertension and suggest that endothelial damage may indeed be important in the vascular complications of hypertension.

Adult↗

PubMed1992

Action of lysosomal phospholipase A1 on bis(monoacylglycerol)phosphate.

Bis(monoacylglycerol)phosphate (BMP) in macrophages is known to rapidly turn over its acyl moiety(s) located at primary positions of the glycerols, yet the glycerols and phosphate remain stable within the BMP molecule. Here we examine whether the phospholipase A1 isolated from rat-liver lysosomes is capable of deacylating BMP. By comparison with the precursor of BMP, phosphatidylglycerol, BMP is a very poor substrate for the phospholipase A1. We conclude, therefore, that a direct deacylation of the acyl groups at the primary alcohol level of the glycerol probably does not occur, but postulate that transacylations may occur to account for the removal of the acyl moiety.

Animals↗

PubMed1992

Role of apolipoprotein E in hepatic lipase catalyzed hydrolysis of phospholipid in high-density lipoproteins.

We reported earlier that hepatic lipase (HL)-catalyzed hydrolysis of phospholipid monolayers is activated by apolipoprotein (apo) E [Thuren et al. (1991b) J. Biol. Chem. 266, 4853-4861]. On the basis of these studies, it was postulated that apoE-rich high-density lipoproteins (HDL) were preferred substrates for HL. In the present study, we tested this hypothesis, as well as further characterizing the activation of HL hydrolysis of phospholipid by apoE. The apoE-rich HDL, referred to as HDL-I, were isolated by heparin-Sepharose chromatography, and the phospholipid hydrolysis by HL was compared to an apoE-poor HDL, designated HDL-II. The hydrolysis of HDL-I phosphatidylcholine was approximately 3-fold higher than HDL-II, supporting the hypothesis that HL preferably hydrolyzes the phospholipids in apoE-rich HDL. In order to gain additional insight into the nature of the activation, we used phospholipid monolayers as model systems. Comparison of the ability of the two thrombolytic fragments of apoE (22 kDa, residues 1-191; 12 kDa, residues 192-299) revealed that only the 12-kDa fragment was capable of activating the hydrolysis of phospholipid by HL (1.75-fold). However, activation was less than with the intact protein (2.8-fold for apoE3), suggesting that the intact protein was required for full activation. The fact that the 12-kDa fragment, which represents a major lipid region of the protein, did activate HL suggests that activation occurs at the lipid-water interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗

PubMed1992

Phosphatidylinositol hydrolysis by phospholipase A2 and C activities in human peripheral blood neutrophils.

We describe here and partially characterize a Ca(2+)-independent phospholipase A2 that acts on phosphatidylinositol in normal human peripheral blood neutrophils. Neutrophils incubated with myo-[3H]inositol to form [3H]phosphatidylinositol and then stimulated with the calcium ionophore A23187 produced [3H]lysophosphatidylinositol. This deacylation was further characterized in cell sonicates by the specific release of [3H]arachidonic acid from exogenous [1-14C]stearoyl-2-[3H]arachidonyl-phosphatidylinositol. This phospholipase A2 is Ca2+ independent, retaining full activity in the presence of 10 mM EDTA, and is optimally active at alkaline pH (pH 9). A phosphatidylinositol-hydrolyzing phospholipase C activity was characterized by the production of [3H]-/[14C]-diglycerides. This phospholipase C activity is dependent on the presence of exogenous Ca2+ and is optimally active at neutral pH (pH 7.5). The lipoxygenase/cyclooxygenase inhibitors eicosatetraenoic acid and nordihydroguaiaretic acid and the calmodulin antagonist trifluoperazine were the only compounds tested that showed significant inhibition of phospholipase A2 activity. However, none of these phosphatidylinositol-hydrolyzing phospholipase A2 inhibitory compounds resulted in the accumulation of any radiolabeled diglyceride, monoglyceride, or phosphatidic acid intermediates. Following subcellular fractionation on sucrose density gradients, it was found that the plasma membrane-enriched fractions contained the highest specific activity for phospholipase A2; however, the cytosolic fraction contained a large part of the total phospholipase A2 activity. Furthermore, when neutrophils were first exposed to several agents, including lipopolysaccharide, phorbol myristate acetate, or N-formyl-methionyl-leucyl- phenylalanine, and then subfractionated, there was a significant translocation of the enzyme activity from the cytosolic fraction to the membrane-enriched fractions. These data suggest that this Ca(2+)-independent, phosphatidylinositol-hydrolyzing phospholipase A2 may play an important role in early cell activation, providing free arachidonic acid for subsequent metabolism into biologically active eicosanoids.

Arachidonic Acid↗

PubMed1991

Activation of hepatic lipase catalyzed phosphatidylcholine hydrolysis by apolipoprotein E.

The effect of apolipoproteins A-I, A-II, C-II, C-III and E on the hydrolysis of phosphatidylcholine and triacylglycerol by hepatic lipase was studied. Hepatic lipase catalyzed phospholipid hydrolysis was 1.8-fold activated by apolipoprotein E while the other apolipoproteins did not affect the hydrolysis by this enzyme. Triacylglycerol hydrolysis by hepatic lipase was 1.5-fold activated by apolipoprotein E while the other apolipoproteins inhibited hepatic lipase. These results suggest that lipoproteins containing apolipoprotein E may be preferred substrates for hepatic lipase.

Animals↗