Studies on phospholipase A2 in human seminal plasma.

1. Human seminal plasma and posterior lobe of prostate was found to have phospholipase A2 (PLA2) activity hydrolysing phosphatidylethanolamine with 14C-labelled linoleic and arachidonic acid. 2. A negative relationship was between sperm count and PLA2 activity in human seminal plasma. 3. The purified PLA2 from human seminal plasma showed high affinity to heparin, sensitivity toward p-bromophenacyl bromide, Pb2+, dithioerythritol and EDTA and it was activated by Ca2+ and Mn2+. 4. The purified PLA2 had alkaline pH optimum (7.5-10.0) and pI-value of 5.3. In SDS-PAGE enzyme preparation resulted in two bands with mol. wt of 14,000 and 16,000.     

Further studies of plasma protease inhibitors in the hedgehog, Erinaceus europaeus; collagenase, papain and plasmin inhibitors

Hedgehog plasma was separated by gel filtration on Sephacryl S-200, the fractions resolved by electrophoresis and the electrophoretograms characterized for collagenase, papain and plasmin inhibiting activities with the high mol. wt substrate casein. The three inhibitors previously identified as alpha 2-, alpha 2-beta- and beta-macroglobulins were found to inhibit all three proteases. These were the only collagenase inhibitors found in plasma. Hedgehog alpha 2-chymotrypsin inhibitor and beta-protease inhibitor were both found to also inhibit papain. Three new inhibitors specific for papain (gamma-, alpha 2- and alpha 1-cysteine protease inhibitors) and one for plasmin (alpha 2-antiplasmin) were also found, bringing the number of protease inhibitors in hedgehog plasma to 14. Immunological cross-reactivity as studied by immunoelectrophoresis showed homology between hedgehog alpha 2-macroglobulin and rat murinoglobulin I and between hedgehog alpha 2-antithrombin and rat antithrombin III.     

Hydrolysis and protection from hydrolysis of enkephalins in human plasma

Seven groups of enkephalin-degrading enzymes and three groups of inhibitors active on these enzymes were separated from human plasma. The activity of the enzymes in hydrolyzing enkephalins and of the inhibitors in protecting enkephalins from proteolysis was measured. Results obtained with the endogenous inhibitors were compared to those relative to synthetic inhibitors. Data obtained indicate that all enkephalin-degrading enzymes found in plasma are significantly inhibited by the endogenous substances present in this tissue. The inhibition of the different classes of plasma enzymes by two of the three groups of endogenous substances is quite uniform, while one group of inhibitors appears specific to dipeptidylpeptidases. Results obtained are discussed in terms of the functional role of the inhibitory substances and of the possible pharmacological implication of their presence in human plasma.     

Mechanism of activation of inactive renin in human plasma by puff adder venom

Venom of the puff adder (Bitis arietans) contains a potent, basic, Mr 24,000 metalloproteinase activity that can destroy all detectable trypsin and chymotrypsin inhibitory activity, when venom is incubated with human plasma. We have found that during such incubation, concomitant activation of inactive renin occurs. In an examination of the mechanism involved we now report the activation, in addition, of plasma prekallikrein and serine proteinase activity, but not plasminogen, when human plasma is incubated with venom. Furthermore, venom was not able to release active trypsin from its complex with alpha 1-proteinase inhibitor and human renin was not inhibited by alpha 1-proteinase inhibitor. The activities in venom and venom/plasma mixtures were analysed using Sephacryl S-200 gel filtration and the effect of 10 mM EDTA and 5 mM phenylmethanesulphonyl fluoride on activities in column fractions was tested. The inactive-renin-activating, plasma prekallikrein-activating and serine proteinase-activating activities could be accounted for to a large extent by a venom metalloproteinase which was estimated to have a Mr of 24,000 by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. This enzyme activity appeared to complex with alpha 2-macroglobulin when venom was mixed with plasma. Since both EDTA and phenylmethanesulphonyl fluoride could inhibit the activation of inactive renin by this metalloproteinase, it is suggested that the enzyme activates serine proteinase(s), which then activate inactive renin. Plasma kallikrein may have a role in this process. Additional peaks of inactive-renin-activating activity eluted from Sephacryl S-200 at Mr 30,000 and 80,000 (minor) and an additional, minor peak of caseinolytic activity eluted at Mr 60,000. The Mr 24,000 metalloproteinase in venom may have considerable utility in activating inactive renin at physiological pH owing to its ability to destroy plasma proteinase inhibitors at the same time.     

Acid-stable trypsin-plasmin inhibitors formed enzymatically from plasma precursor protein

Enzymatic formation of acid-stable trypsin-plasmin inhibitors (ASTPIs) in human plasma with several proteinases, particularly SH-proteinases, was demonstrated. The maximal activity obtained with bromelain was 40 U/ml plasma, which corresponded to about a 10-fold increase as compared to the untreated control plasma (4.2 U/ml). Gel filtration revealed at least two ASTPI activity peaks of molecular weight 16,000 (main peak) and 8000 (minor peak). The main ASTPI was further purified by trypsin-Sepharose affinity chromatography, isoelectric focusing and gel filtration on Sephadex G-75 superfine. The purified inhibitor was found to be identical to the active fragment of plasma ASTPI or urinary trypsin inhibitor (UTI) formed by bromelain treatment. It had an isoelectric point (pI) of 3.7, a molecular weight of 16,000 by SDS-polyacrylamide gel electrophoresis and was a glycine- and glutamic acid-rich protein lacking histidine. The NH2-terminal amino acid sequence was H2N-(Lys)-Glu-Asp-Ser-X-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-X-Met-Gly-Met-Th r-X-Arg - Tyr-Phe-Tyr-... COOH, which was homologous to the Lys22-Met36 part (or Glu23-Met36 part; 30% of the total) of the plasma ASTPI or UTI molecule (molecular weight 70,000-80,000 by gel filtration). The purified ASTPI displayed the same antigenicity as UTI and exerted strong inhibitory effects on trypsin, chymotrypsin and plasmin amidolysis, but had a much lesser effect on plasmin fibrinolysis. It also strongly inhibited non-plasmic fibrinolysis with human leukocyte proteinase and earthworm proteinase.     

Measurement of endogenous Na+,K+-ATPase inhibitors in human plasma and urine using high-performance liquid chromatography

This study was undertaken to assess endogenous Na+,K+-ATPase inhibitors in both plasma and urine in the same subjects. Samples were chromatographed on reverse-phase HPLC using an acetonitrile gradient and the eluent screened using Na+,K+-ATPase inhibition and cross-reaction with anti-digoxin antibodies. The donors were divided into inhibiting and non-inhibiting subjects using a previously described method, plasma action on ouabain binding and on Na+,K+-ATPase activity. Three Na+,K+-ATPase inhibitors (1P, 2P and 3P) were detectable in plasma; the antibodies cross-reaction of the peaks 2P and 3P were larger than that of peak 1P. The peaks 2P and 3P were significantly higher in inhibiting subjects as compared to non-inhibiting subjects. The 24-h urine is resolved into two peaks inhibiting Na+,K+-ATPase activity (1U and 2U). Peak 2U cross-reacted with anti-digoxin antibodies to a greater extent than peak 1U and is significantly larger in inhibiting subjects in terms of Na+,K+-ATPase inhibition. These data support the heterogeneity of human Na+,K+-ATPase inhibitor in both plasma and urine.     

Effect of phospholipase A2 inhibitors on the release of arachidonic acid and cell viability in cold-stored hepatocytes

We investigated the effect of phospholipase A(2) (PLA(2)) inhibitors on PLA(2) activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [(3)H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA(2) inhibitors. PLA(2) activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m-chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA(2) activity was also suppressed (58% of control) by trifluoperazine (50 microM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 microM) suppressed PLA(2) activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA(2) activity. Thus, the inhibitory activity of these agents appears unrelated to Ca-calmodulin-phospholipid interaction but to have an inhibitory effect on PLA(2) activity. To study the effects of PLA(2) inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs-Henseleit buffer for 2 h at 37 degrees C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA(2) inhibitors blocked PLA(2) activity, there was no suppression of LDH release. PLA(2) may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes.     

Inhibitors of reverse serotonin uptake and of specific imipramine binding in human blood plasma

Human blood plasma contains low-molecular substances that inhibit in a dose-dependent manner both high-affinity specific binding of imipramine and reverse serotonin uptake by platelets. Incubation of human blood plasma with alumina was made use of to extract and study these imipramine-like inhibitors. The extract obtained from human blood plasma inhibited imipramine binding and reverse uptake of serotonin with median inhibitory concentrations of 0.18 +/- 0.1 and 0.36 +/- 0.15 mg/ml, respectively. After gel chromatography on Biogel P-2 the elution profile of the extract showed 2 major peaks of reverse serotonin uptake and imipramine binding inhibition and 3 additional peaks of reverse serotonin uptake inhibition, which did not have any considerable effect on imipramine specific binding. It is assumed that endogenous inhibitors of imipramine binding and reverse serotonin uptake are involved in the development of affective disorders.     

Effect of plasma biotinyl-peptides on biotinidase activity

Purified biotinidase (enriched 24,000-fold) from fresh human plasma exhibited reduced catalytic activity when incubated with heat-inactivated dialyzed plasma. The polypeptide fractions separated from the heat-inactivated dialyzed plasma using streptavidin-Sepharose resin showed the same effect on purified biotinidase. These inhibitory effects on biotinidase were partial (25-45%) rather than complete. The polypeptide fraction from streptavidin-Sepharose resin was analyzed by SDS-PAGE in the Laemmli system and by various types of HPLC. Analyses by ion-exchange and reversed-phase HPLC revealed the existence of three relatively small mol. wt polypeptides. Each of these peak fractions exhibited similar inhibitory effects on biotinidase activity. SDS-PAGE analysis indicated that the streptavidin affinity resin fraction was composed of four major polypeptides whose mol. wts were 120,000, 76,000, 53,000 and 27,000. The two bands of 120,000 and 76,000 corresponded to the mol. wts of the biotinyl subunit of pyruvate carboxylase, beta-methyl-crotonyl-CoA and/or propionyl-CoA carboxylase respectively. However, the polypeptides of mol. wts 53,000 and 27,000 were found to be two unique biotinyl-peptides present in human plasma. These bands on the gels were transblotted and exhibited a fluorescent activity after incubated with a FITC-avidin. These findings strongly suggest the existence of circulating plasma biotinyl-polypeptides as inhibitory factor(s) on human plasma biotinidase.     

Guinea pig plasma trypsin inhibitors. Purification and characterization of two functionally distinct proteinase inhibitors.

Two trypsin inhibitors (TI-1, TI-2) were isolated from guinea pig plasma and purified to homogeneity. In amino-acid composition as well as molecular masses, TI-1 (Mr 58,000) and TI-2 (Mr 57,000) are similar to each other and to human and mouse alpha 1-proteinase inhibitors, and mouse con-trapsin. The two inhibitors form equimolar complexes with proteinases. The effectiveness of the inhibitors was characterized by association rate constants under second-order rate conditions. The inhibitory action of TI-1 was rapid for bovine trypsin, porcine pancreatic elastase and guinea pig plasma kallikrein, but slow for bovine thrombin and guinea pig plasmin and not detectable for bovine chymotrypsin and porcine pancreatic kallikrein. The inhibitory action of TI-2 was rapid for trypsin and chymotrypsin, but slow for guinea pig plasma kallikrein and not detectable for other proteinases. These results show that TI-1 and TI-2 are physicochemically similar but functionally distinct from each other and from human alpha 1-proteinase inhibitor that inhibits trypsin, chymotrypsin and elastase.     

Isolation, characterization and molecular cloning of AnMIP, a new alpha-type phospholipase A2 myotoxin inhibitor from the plasma of the snake Atropoides nummifer (Viperidae: Crotalinae)

A new phospholipase A(2) (PLA(2))-inhibitory protein was isolated from the plasma of Atropoides nummifer, a crotaline snake from Central America. This inhibitor was named AnMIP, given its ability to neutralize the activity of basic PLA(2) myotoxins of its own and related venoms. The cDNA of AnMIP was cloned and sequenced, showing that it belongs to the alpha group of phospholipase A(2) inhibitors (PLIs). AnMIP appears as a homotrimer in the native state, held together by non-covalent forces, with a subunit molecular mass of 22,247-22,301 and an isoelectric point of 4.1-4.7. This trimeric structure is the first observed in a PLIalpha from American crotaline snakes, previously reported only in Asian species. Sequencing, mass spectrometry, and analytical isoelectrofocusing indicated the existence of isoforms, as reported for other PLIalphas isolated from snake plasma. The inhibitory profile of AnMIP showed specificity towards group II PLA(2)s, either belonging to the catalytically-active (D49) or -inactive (K49) subtypes, exemplified in this study by Bothrops asper myotoxin I and A. nummifer myotoxin II, respectively. By phylogenetic analysis it was shown that AnMIP is closely related to CgMIP-II, previously isolated from the plasma of Cerrophidion godmani, showing 93% amino acid sequence identity.     

Competitive inhibition of lipolytic enzymes. VI. Inhibition of two human phospholipases A2 by acylamino phospholipid analogues.

The competitive inhibition of human pancreatic and a mutant human platelet phospholipase A2 (PLA2) was investigated using acylamino phospholipid analogues, which are potent competitive inhibitors of porcine pancreatic PLA2 [De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257]. Both the mutant platelet PLA2 and the human pancreatic PLA2 are effectively inhibited by these compounds. The enzyme from platelets is most strongly inhibited by compounds with a negatively charged phosphoglycol headgroup. Compounds with a neutral phosphocholine headgroup are only weak inhibitors, whereas an inhibitor with a phosphoethanolamine headgroup shows an intermediate inhibitory capacity. The platelet PLA2 is most effectively inhibited by negatively charged inhibitors having a relatively short (four or more carbon atoms) alkylchain on position one and a acylamino chain of 14 carbon atoms on position two. For the pancreatic enzyme an inhibitor with a phosphoethanolamine headgroup was more effective than inhibitors with either a phosphocholine or a phosphoglycol headgroup. The chainlength preference of the pancreatic enzyme resembles that of the platelet PLA2. The largest discrimination in inhibition between the human platelet and the human pancreatic PLA2 is obtained with inhibitors with a negatively charged phosphoglycol headgroup, an alkyl chain of four carbon atoms on position one and a long acylamino chain of 14-16 carbon atoms on position two. Because the platelet PLA2 is thought to have several biological functions, specific inhibitors of this enzyme could have important implications in the design of pharmaceutically interesting compounds.     

Inhibition of human factor Xa by various plasma protease inhibitors

The inhibitory effects of the plasma protease inhibitors antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin on the activity of human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide susbtrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithromin III, alpha 2-macroglobulin and alpha 1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin were 5.8 . 10(4), 4.00 . 10(4) and 1.36 . 10(4) M -1 . min -1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as alpha 1-antitrypsin greater than antithrombin III greater than alpha 2-macroglobulin in the ratio 4.64: 2.08: 1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.     

Inhibition of the antioxidative activity of plasma by sodium azide

Powerful antioxidant activity of human plasma was demonstrated by measuring the thiobarbituric acid reaction and Fe+2-induced chemiluminescence. Inhibition of lipid peroxidation was shown both for plasma lipids and for the suspension of egg lipoproteins, which was taken as a model system. The inhibitory effect of plasma peroxidation was removed by azide Na taken in the concentration of 0.5 mg/ml, but caeroplasmin activity in the plasma was completely suppressed at NaN3 concentration equal to 0.1 mg/ml. A low correlation (r = 0.75) between caeruloplasmin activity in the blood plasma and extent of chemiluminescence activation obtained in the presence of NaN3 was found. The presented data led to an assumption that only a part of lipid peroxidation inhibitors in the plasma can be attributed with caeruloplasmin.     

Xenorhabdus nematophilus inhibits p-bromophenacyl bromide (BPB)-sensitive PLA2 of Spodoptera exigua

Xenorhabdus nematophilus is a Gram-negative symbiotic bacterium of the entomopathogenic nematode, Steinernema carpocapsae. The bacteria delivered into the insect hemocoel by the nematodes cause immunodepression of the target insects to protect host nematodes and themselves from the cellular immune reaction. Previous reports suggest that the immunodepression is caused by inhibition of the eicosanoid pathway that is known to be critically important to mediate cellular immunity. This study focused on the inhibitory effect of X. nematophilus on PLA2 activity of Spodoptera exigua. The PLA2 activity was functionally associated with the activation cascade of prophenoloxidase (pPO). Dexamethasone (DEX), a specific PLA2 inhibitor, inhibited pPO activation completely at the higher doses of approximately 2.4 muM in vitro condition. The inhibitory effect of DEX was reversed by the addition of arachidonic acid, the catalytic product of PLA2. By means of this in vitro PLA2 inhibitor assay system, two different PLA2 inhibitors were used to compare their inhibitory effects on the hemolymph PLA2 of S. exigua. p-Bromophenacyl bromide (BPB), a specific inhibitor of secretory PLA2 (sPLA2), significantly inhibited pPO activation, but methylarachidonyl fluorophosphates (MAFP), a specific inhibitor of cytosolic PLA2 (cPLA2), did not show any inhibitory effect. BPB also inhibited pPO activation of the plasma, though much higher PO activation and its inhibition by BPB was found in the hemocytes. Growth medium of X. nematophilus at the stationary phase had a PLA2 inhibitory effect. Via the in vitro PLA2 inhibitor assay, it was shown that the ethyl ether extract of the medium contained significant PLA2 inhibitor activity. These results indicate that X. nematophilus produces and secretes PLA2 inhibitor, which acts on BPB-susceptible PLA2 of S. exigua.     

Identification and some properties of a new fast-reacting plasmin inhibitor in human plasma.

Fresh plasma was seeded with trace amounts of highly purified biologically intact iodine-labelled plasminogen and the plasmin-inhibitor complexes formed after activation with streptokinase or urokinase separated by gel filtration. Two radioactive peaks were observed, the first one eluted in the void volume and the second one just before the 7-S globulin peak. In incompletely activated samples, the second peak was always predominant over the first one. Both components were purified with high yield by a combination of affinity chromatography on lysine-agarose and gel filtration, and investigated by dodecylsulphate-polyacrylamide gel electrophoresis and immunoelectrophoresis. Neither component reacted with antisera against alpha1-antitrypsin, antithrombin III, C1-esterase inhibitor, inter-alpha-trypsin inhibitor or alpha1-antichymotrypsin. The component of the first peak appeared to be a complex between plasmin and alpha2-macroglobulin which reacted with antisera against human plasminogen and against alpha2-macroglobulin. The component of the second peak had a molecular weight (Mr) of 120000-140000 by dodecyl-sulphate-polyacrylamide gel electrophoresis and lpon reduction displayed a doublet band with an Mr of 65000-70000 and a band with Mr 11000. It reacted with antisera against plasminogen and with antisera raised against this complex and absorbed with purified plasminogen. The latter antisera reacted with a single component in plasma which is different from the above-mentioned plasma protease inhibitors. Specific removal of this component from plasma by immuno-absorption resulted in disappearance of the fast-reacting antiplasmin activity whereas alpha2-macroglobulin was found to represent the slower-reacting plasmin-neutralizing activity. In the presence of normal plasma levels of these proteins, the specific removal or absence of alpha1-antitrypsin, antithrombin III or C1-esterase inhibitor did not alter the inactivation rate of plasmin when added to plasma in quimolar amounts to that of plasminogen. It is concluded that only two plasma proteins are important in the binding of plasmin generated by activation of the plasma plasminogen, namely a fast-reacting inhibitor which is different from the known plasma protease inhibitors and which we have provisionally named antiplasmin, and alpha2-macroglobulin, which reacts more slowly.     

Inhibitors of rabbit plasma prostaglandin A isomerase

1. The potent inhibitory activities of three groups of prostaglandin analogues on the prostaglandin A isomerase of rabbit plasma were demonstrated. 2. Six of the compounds were prepared by NaBH(4) reduction of the C-9 oxo groups of prostaglandin A(2) and prostaglandin C(2) and their C-15 epimers. The remaining four were racemates and were synthesized in another laboratory. Unknown configurations at C-9 and at C-15 were assigned. 3. All the compounds were found to be competitive inhibitors of the isomerase in vitro. K(m)/K(i) ratios were determined and it was found that both the 15(S) and 15(R) epimers have potent inhibitory activity. 4. One of the inhibitors was used to study the reversibility of the isomerase. 5. It is suggested that these compounds may be useful for determining the biological significance of prostaglandin A isomerase. In view of its weak biological activity and possibly extended half-life in vivo, the reduction product of 15-epiprostaglandin C(2) may be the most suitable agent for this purpose.     

Structural and functional analysis of BmjMIP,a phospholipase A2 myotoxin inhibitor protein from Bothrops moojeni snake plasma

A protein, which neutralizes the enzymatic, toxic, and pharmacological activities of various basic and acidic phospholipases A(2) from the venoms of Bothrops moojeni, Bothrops pirajai, and Bothrops jararacussu, was isolated from B. moojeni snake plasma by affinity chromatography using immobilized myotoxins on Sepharose gel. Biochemical characterization of this myotoxin inhibitor protein (BmjMIP) showed it to be an oligomeric glycoprotein with a M(r) of 23,000-25,000 for the monomeric subunit. BmjMIP was stable in the pH range from 4.0 to 12.0, between 4 and 80 degrees C, even after deglycosylation. The role of the carbohydrate moiety was investigated and found not to affect the in vitro function of the inhibitor. The corresponding 500bp cDNA obtained by RT-PCR from the liver of the snake encodes a mature protein of 166 amino acid residues including a 19 amino acid signal peptide. The primary structure of BmjMIP showed a high similarity with other snake phospholipase A(2) inhibitors (PLIs) in which the carbohydrate recognition domain (CRD) and the glycosylation site (Asn103) are conserved. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either the BmjMIP or the target protein occur upon their interaction. BmjMIP has a wide range of inhibitory properties against basic and acidic PLA(2)s from Bothrops venoms (anti-enzymatic, anti-myotoxic, anti-edema inducing, anti-cytotoxic, anti-bactericidal, and anti-lethal). However, the inhibitor showed a reduced ability to neutralize the biological activities of crotoxin B (CB), the PLA(2) homologue associated with crotapotin in Crotalus durissus terrificus snake venom. Finally, the purified PLA(2) inhibitor was shown to protect in vivo against the toxic and pharmacological effects of a homologous PLA(2) enzyme, suggesting that PLIs or a corresponding derived peptide may prove useful in the treatment of snakebite victims or, more importantly, in the treatment of the many human diseases in which these enzymes have been implicated.     

Purification and characterization of a 32-kDa phospholipase A2 inhibitory protein (lipocortin) from human peripheral blood mononuclear cells

A 32-kDa protein was isolated from human monocytes after calcium precipitation and chromatography. The protein activity was assessed by the inhibition of soluble phospholipase A2 (PLA2). This in vitro inhibitory effect on phospholipases A2 was found only with negatively charged phospholipids. The protein was also able to inhibit cellular PLA2 in mouse thymocytes. The biochemical properties and amino acid composition strongly suggest that the protein shares similarities with endonexin. Using a neutralizing monoclonal antibody against rat lipocortin, we found a cross-reactivity with the 32-kDa protein. According to the biochemical and immunological properties, we propose to relate this PLA2 inhibitory protein from human monocytes to lipocortin.     

Human cytomegalovirus carries a cell-derived phospholipase A2 required for infectivity

Human cytomegalovirus (HCMV) is known to carry host cell-derived proteins and mRNAs whose role in cell infection is not understood. We have identified a phospholipase A2 (PLA2) activity borne by HCMV by using an assay based on the hydrolysis of fluorescent phosphatidylcholine. This activity was found in all virus strains analyzed and in purified strains. It was calcium dependent and was sensitive to inhibitors of cytosolic PLA2 (cPLA2) but not to inhibitors of soluble PLA2 or calcium-independent PLA2. No other phospholipase activity was detected in the virus. Purified virus was found to contain human cellular cPLA2alpha, as detected by monoclonal antibody. No homology with PLA2 was found in the genome of HCMV, indicating that HCMV does not code for a PLA2. Decreased de novo expression of immediate-early proteins 1 and 2 (IE1 and IE2), tegument phosphoprotein pp65, and virus production was observed when HCMV was treated with inhibitors of cPLA2. Cell entry of HCMV was not altered by those inhibitors, suggesting the action of cPLA2 was postentry. Together, our results indicate a selective sorting of a cell-derived cPLA2 during HCMV maturation, which is further required for infectivity.