Substrate specificities of the human and rat kidney 85-kDa phospholipase A2 enzymes (hmw-PLA2) have been determined under conditions in which hydrolysis of substrate vesicles occurs without the desorption of enzyme from the interface (scooting mode catalysis). The rat kidney enzyme binds to vesicles of 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), which contain the substrate 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine (SAPC) and 10 mol% arachidonic acid (20:4) and 1-stearoyl-sn-glycero-3-phosphocholine (S-lyso-PC) as the hydrolysis reaction products, with a second-order rate constant k(on) approximately equal to 2 x 10(7) M-1 s-1. Upper limits of k(off) < or = 3 x 10(-4) s-1 and KD < = or 15 pM for the dissociation rate and equilibrium constants, respectively, are estimated from the vesicle binding measurements. The initial rates of hydrolysis of either radiolabeled 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphoserine (3H-SAPS), -phosphoethanolamine (3H-SAPE), -phosphoinositol (14C-SAPI), or -phosphate (3H-SAPA) and either 3H-SAPC or 14C-SAPC, which were incorporated into product-containing OPPC vesicles, were simultaneously measured with dual isotope radiometric assays. The plasmenylcholine 1-O-(Z-hexadec-1'-enyl)-2-arachidonyl-sn-glycero-3- phosphocholine (3H-PlasAPC) was also tested. Relative substrate specificity constants (Kcat/KM* values) were determined from the concentrations and initial rates of hydrolysis of the labeled substrates; the rank order of the values is SAPC approximately equal to SAPI approximately equal to PlasAPC > SAPE > SAPA approximately equal to SAPS. The maximal difference in specificity constants is 3.5-fold, indicating that the hmw-PLA2 does not significantly discriminate between phospholipids with different polar head groups. The diglyceride 1-stearoyl-2-arachidonyl-sn-glycerol is not a substrate for the human hmw-PLA2. Two mixtures of 1-stearoyl-2-acyl-sn-glycero-3-phosphocholine, which have different sn-2 acyl chains, were prepared and compared to SAPC as substrates. One mixture contained naturally-occurring unsaturated fatty acyl chains and the other contained a mixture of 20:4, all of its partially hydrogenated analogues (20:3, 20:2, and 20:1), and arachidic acid (20:0). The order of preference for the human hmw-PLA2 is sn-2-20:4 > sn-2-alpha-linolenoyl > sn-2-linoleoyl > sn-2-oleoyl > or = sn-2-palmitoyleoyl.(ABSTRACT TRUNCATED AT 400 WORDS)

The kinetics of hydrolysis of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine vesicles catalyzed by the high molecular weight phospholipase A2 from rat kidney show an anomalous behavior. The reaction progress lasts for several minutes and then stops after only 5-10% of the available substrate has been hydrolyzed. Addition of more enzyme but not more substrate leads to a new round of hydrolysis. Although this initially suggested that the enzyme becomes inactivated during the turnover, such a conclusion could not be substantiated. Addition of buffer containing 0.15 M NaCl and bovine serum albumin to the reaction after the progress ceased leads to the re-initiation of the lipolysis. The enzyme is not strongly inhibited by the reaction products. Although the enzyme does not bind irreversibly to vesicles composed of pure 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine, it does become irreversibly trapped on vesicles that contain a critical mole percentage of reaction products. This trapping is the most likely explanation for the cessation of the reaction progress. Both the binding of enzyme to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles and the hydrolysis of 1-stearoyl-2-[3H]arachidonyl-sn-glycerophosphocholine contained in these vesicles require the presence of products. Furthermore, the trapping of enzyme is independent of catalytic turnover. The trapping is sensitive to the structure of the fatty acid present in the vesicles and requires the presence of divalent metals (either Ca2+, Sr2+, Ba2+, or Mg2+). Since the concentrations of the metals needed for the enzymatic activity correlate with the amounts needed to promote the trapping, it is suggested that the role of the metal is only to promote the interfacial binding of the enzyme.

In admiration and memory of Prof. Eghert Hauinga (16. XII. 88) In a search for further synthetic routes to substituted [5.5.5.5]fenestranes, compound la, a derivative of 7-methoxyindane, was photolyzed. Two of the three photoproducts, oiz. the [3.5.5.5]fenestrane 3 a and the isomer 4 a, are formed according to the expected intramolecular meta-cycloaddition. A different mechanism is suggested for the formation of the major component 2a.

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