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The Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) displays both a phospholipase A2 and a lysophospholipase activity. Numerous hydrolases, including lipases, catalyze the hydrolysis of ester bonds by means of an active site triad of amino acids that includes a serine or a cysteine residue. We have examined whether human cPLA2 belongs to this class of enzymes by using site-directed mutagenesis. Although chemical inactivation of cPLA2 by the sulfhydryl reagent N-ethylmaleimide made it appear that cysteine(s) may be essential for catalysis, all 9 cysteine residues of cPLA2 proved dispensable, allowing near-normal enzyme activity when substituted by alanine. We noted that cPLA2 contains a 110-amino-acid region with sequence homology to phospholipase B (PLB) from Penicillium notatum. Interestingly, one of the conserved serines of cPLA2, Ser-228, within this domain aligns with the lipase consensus sequence Gly-X(Leu)-Ser(137)-X(Gly)-Gly of PLB. Replacement of Ser-228 by alanine (or threonine or cysteine) yielded catalytically inactive cPLA2, even though the native conformation was maintained as determined by CD spectroscopy. Likewise, the lysophospholipase activity was completely abolished by the Ser-228 mutations. In contrast, substitution by alanine of three different serines of cPLA2 (Ser-195, Ser-215, or Ser-577) that also aligned with the PLB sequence allowed for substantial enzymatic activity of cPLA2. Our findings provide evidence that 1) Ser-228 participates in the catalytic mechanism of cPLA2 and that 2) both the phospholipase A2 and the lysophospholipase activities of cPLA2 are catalyzed by the same active site residue(s).

Calcium-sensitive cytosolic phospholipase A2 (cPLA2) is responsible for receptor-mediated liberation of arachidonic acid, and thus plays an important role in the initiation of the inflammatory lipid-mediator cascade generating eicosanoids and platelet-activating factor. In this study we have investigated the cellular distribution of cPLA2 in brain using a monoclonal antibody raised against cPLA2 to immunostain tissue sections of human cerebral cortex. We have localized cPLA2 in astrocytes of the gray matter. Colocalization with glial fibrillary acidic protein (GFAP) confirmed that cPLA2 is associated predominantly with protoplasmic astrocytes. Astrocytes of the white matter, on the other hand, were not immunoreactive. In experiments using different human astrocytoma cell lines we found that cPLA2 can be immunochemically localized in UC-11 MG cells, but cannot be detected in U-373 MG cells. This finding is consistent with the observation that cPLA2 mRNA as well as cPLA2 enzymatic activity can be readily measured in UC-11 MG astrocytoma cells, yet cannot be detected in U-373 MG cells. Our data suggest that the astrocyte is a primary source of cPLA2 in the brain and provide further evidence for the importance of this cell type in inflammatory processes in the brain.

Receptor-mediated activation of human platelets by thrombin initiates a series of rapid biochemical events that include activation of phospholipase A2 to liberate arachidonic acid for further conversion to thromboxane A2. The identity of the phospholipase A2 involved has not been clear. Here we show by immunochemical analysis that human platelets contain significant amounts (60 ng/10(9) platelets) of the recently identified Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2). Metabolic labeling of human platelets with 33Pi revealed that the extent of phosphorylation of cPLA2 was greatly increased after thrombin treatment. Upon stimulation of platelets with thrombin, cPLA2 exhibits enhanced catalytic activity, as well as a change in its electrophoretic and chromatographic properties compared with cPLA2 in resting platelets. These alterations of cPLA2 are reversed by treatment with phosphatase, demonstrating that they are the consequence of thrombin-stimulated phosphorylation. Thrombin-induced phosphorylation and activation of cPLA2 is rapid (half-maximal by 1 min at 1 unit/10(9) platelets) and dose-dependent. Agonist-induced phosphorylation of cPLA2 is more sensitive to thrombin than the generation of thromboxane A2, suggesting that it may be an early event in the sequence of steps leading to the mobilization and further metabolism of arachidonic acid. By comparing the functional properties of cPLA2 from control versus thrombin-stimulated platelets, we found that while activated cPLA2 exhibits the same Ca2+ requirement and apparent substrate affinity (Km), its catalytic activity (Vmax) is increased compared with control cPLA2. We conclude that 1) cPLA2 is likely to play an important role in agonist-induced mobilization of arachidonic acid and 2) thrombin elicits rapid and full activation of cPLA2 not only by promoting a rise in cytosolic free Ca2+ but also by inducing phosphorylation of cPLA2 thereby improving its catalytic activity.

Through a series of synthetic model peptides, we have examined the structural requirements of the P2 and P3 residues in statine-based HIV protease (PR) inhibitors. Results agree with the general observations that, the more bulky the P3 aromatic hydrophobic side chain, the more potent is the inhibitor. At P2, an isopropyl side chain is critical in maintaining potency. Three-dimensional modeling demonstrates that the steric bulk of a leucyl residue or the unfavorable energy transfer, from water to enzyme, for a basic amino acid residue at P2 markedly compromises activity. A naphthylalaninyl-valyl P3-P2 substituted analogue inhibits PR with an IC50 value of 6 nM, and was also effective as an antiviral agent.

A critical step in the replicative cycle of the human immunodeficiency virus HIV-1 involves the proteolytic processing of the polyprotein products Prgag and Prgag-pol that are encoded by the gag and pol genes in the viral genome. Inhibitors of this processing step have the potential to be important therapeutic agents in the management of acquired immunodeficiency syndrome. Current assays for inhibitors of HIV-1 protease are slow, cumbersome, or susceptible to interference by test compounds. An approach to the generation of a rapid, sensitive assay for HIV-1 protease inhibitors that is devoid of interference problems is to use a capture system which allows for isolation of the products from the reaction mixture prior to signal quantitation. In this paper, we describe a novel method for the detection of HIV-1 protease inhibitors utilizing the concept of particle concentration fluorescence. Our approach involves the use of the HIV-1 protease peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val which has been modified to contain a biotin moiety on one side and a fluorescein reporter molecule on the other side of the scissile Tyr-Pro bond. This substrate is efficiently cleaved by the HIV-1 protease and the reaction can be readily quantitated. Known inhibitors of the protease were readily detected using this new assay. In addition, this approach is compatible with existing instrumentation in use for broad screening and is highly sensitive, accurate, and reproducible.

Synthetic peptides described as dog renin inhibitors were found to effectively inhibit the aspartyl protease of human immunodeficiency virus (HIV). The selection of oligopeptides for the HIV protease inhibition study was based on 1) the current strategy of inhibiting aspartyl proteases with transition state analogs, and 2) our previous observations regarding optimal structural differentiation at the P2 position among human, dog, and rat renin inhibitors. In an in vitro assay system consisting of recombinant HIV protease and a synthetic decapeptide substrate (at pH 5.5), results show that HIV protease was unaffected by statine-containing analogs carrying histidine at the P2 position whereas analogs containing valine at the same position yielded anti-protease IC50 values ranging from 50 to 500 nM. As anticipated, some analogs were also shown to inhibit processing of recombinant polyprotein substrate by HIV protease in vitro. The anti-viral activity of three inhibitors was studied in HIV-infected CEM and MT-2 cells. Results showed that one compound, Ac-Naphthylalanyl-Pro-Phe-Val-Statine-Leu-Phe-NH2 (antiprotease IC50 value = 0.4 microM), protected the infected cells effectively with IC50 values (0.73 microM for CEM cells and 0.88 microM for MT-2 cells). This antiviral effect is comparable to those obtained with AZT and ddC in parallel studies of MT-2 cells.