A novel Ca(2+)-independent phospholipase A2 (PLA2) has recently been purified from the murine macrophage-like cell line P388D1 (Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233). This enzyme is now shown to be inhibited by palmitoyl trifluoromethyl ketone (PACOCF3), arachidonyl trifluoromethyl ketone (AACOCF3), and a bromoenol lactone (BEL). Both PACOCF3 and AACOCF3 were found to inhibit the macrophage PLA2 in a concentration-dependent manner. PACOCF3 was found to be approximately 4-fold more potent than AACOCF3, with IC50 values of 3.8 microM (0.0075 mol fraction) and 15 microM (0.028 mol fraction), respectively. Reaction progress curves in the presence of either inhibitor were found to be linear, and the PACOCF3.PLA2 complex rapidly dissociated upon dilution. BEL was also found to inhibit the macrophage PLA2 in a concentration-dependent manner, with half-maximal inhibition observed at 60 nM after a 5-min preincubation at 40 degrees C. Inhibition was not reversed after extensive dilution of the enzyme into assay buffer. Treatment of the PLA2 with BEL resulted in a linear, time-dependent inactivation of activity, and the rate of this inactivation was diminished in the presence of PACOCF3. In addition, PLA2 treated with [3H]BEL resulted in the covalent labeling of a major band at M(r) 80,000. Inactivation of the PLA2 by 5,5'-dithiobis(2-nitrobenzoic acid) prior to treatment with [3H]BEL resulted in the near complete lack of labeling consistent with covalent irreversible suicide inhibition of the enzyme. The labeling of a M(r) 80,000 band rather than a M(r) 40,000 band upon treatment with [3H]BEL distinguishes the macrophage Ca(2+)-independent PLA2 from a previously identified myocardial Ca(2+)-independent PLA2 and provides strong evidence that the M(r) 80,000 protein is the catalytic subunit.

Cellular levels of free arachidonic acid (AA) are controlled by a deacylation/reacylation cycle whereby the fatty acid is liberated by phospholipases and reincorporated by acyltransferases. We have found that the esterification of AA into membrane phospholipids is a Ca2+-independent process and that it is blocked up to 60-70%o by a bromoenollactone (BEL) that is a selective inhibitor ofa newly discovered Ca2+-independent phospholipase A2 (PLA2) in macrophages.The observed inhibition correlates with a decreased steadystate level of lysophospholipids as well as with the inhibition of the Ca2+-independent PLA2 activity in these cells. This inhibition is specific for the Ca2 -independent PLA2 in that neither group IV PLA2, group II PLA2, arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with BEL. Moreover, two BEL analogs that are not inhibitors of the Ca2+-independent PIA2-namely a bromomethyl ketone and methyl-BEL-do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by BEL, indicating that de novo synthetic pathways are not inhibited by BEL. Collectively, the data suggest that the Ca2+-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction.

Large-scale mass spectrometry-based peptidomics for drug discovery is relatively unexplored because of challenges in peptide degradation and identification following tissue extraction. Here we present a streamlined analytical pipeline for large-scale peptidomics. We developed an optimized sample preparation protocol to achieve fast, reproducible and effective extraction of endogenous peptides from sub-dissected organs such as the brain, while diminishing unspecific protease activity. Each peptidome sample was analysed by high-resolution tandem mass spectrometry and the resulting data set was integrated with publically available databases. We developed and applied an algorithm that reduces the peptide complexity for identification of biologically relevant peptides. The developed pipeline was applied to rat hypothalamus and identifies thousands of neuropeptides and their post-translational modifications, which is combined in a resource format for visualization, qualitative and quantitative analyses.

Background: Cyclotides are useful scaffolds to stabilize bioactive peptides. Results: Four melanocortin analogues of kalata B1 were synthesized. One is a selective MC4R agonist. Conclusion:The analogues retain the native kalata B1 scaffold and introduce a designed pharmacological activity, validating cyclotides as protein engineering scaffolds. Significance: A novel type of melanocortin agonist has been developed, with potential as a drug lead for treating obesity.

The aggregation properties of different linear, single-chain alkyl phosphates and phosphonates in water were investigated at concentrations of up to 50 mM as a function of pH, focusing in particular on spontaneous vesicle formation. Under conditions where about half the molecules are monoionic and half the molecules are completely protonated (pH ≈ 2), n-dodecylphosphoric acid, n-decylphosphonic acid, and n-dodecylphosphonic acid spontaneously form vesicles at room temperature. For n-hexadecylphosphoric acid, stable vesicles only form above ∼40 °C. The presence of vesicles was evidenced by light and electron microscopy and in the case of n-dodecylphosphoric acid by entrapment experiments using as water soluble probes glucose, dextran, and pepsin. The phase-transition temperature of vesicles of n-dodecylphosphoric acid was 2.3 °C, as determined by differential scanning calorimetry. For n-hexadecylphosphoryladenosine evidence for micelle formation has been obtained with a cmc of 20−50 μM at 25 °C. In an experimental extension of the vesicle self-reproduction principles to phosphoamphiphiles, results are also presented on the alkaline hydrolysis of the water-insoluble di-n-decyl-4-nitrophenyl phosphate, which led to the formation of 4-nitrophenol and di-n-decyl phosphate, the latter being a known vesicle-forming amphiphile.

Studies are reported on two different types of activated ketones as inhibitors of two important intracellular phospholipase A 2 s (PLA 2 ): the group IV 85 kDa Ca 2+ -dependent phospholipase A 2 (cPLA 2 ) and the P388D 1 Ca 2+independent phospholipase A 2 (iPLA 2 ). In a mixed micelle assay, we observed that the reaction progress curve of cPLA 2 in the presence of a trifluoromethyl ketone (TFMK) is linear at pH 7.4, while at pH 9.0 it is nonlinear and slows with time. An investigation of this discrepancy demonstrated that the TFMKs are slow, tight-binding inhibitors of the cPLA 2 at both pH's, that the rate of dissociation of the enzyme-inhibitor complex is the same at both pH's, but that the rate of association of enzyme and inhibitor is slower at pH 7.4 than at pH 9.0. A novel group of activated ketone inhibitors has been synthesized that contain a fatty acyl tricarbonyl. These compounds also inhibit the cPLA 2 in the mixed micelle assay. The inhibition of cPLA 2 by the tricarbonyls is readily reversible upon dilution and does not involve slow binding. For both types of inhibitor, no preference for fatty acid chain was observed as the palmityl analogs inhibited as well as the arachidonoyl analogs, despite the fact that the cPLA 2 shows a strong preference for arachidonoyl-containing phospholipid substrates over palmitoyl-containing substrates. With the iPLA 2 , the inhibition by TFMKs is reversible and does not involve slow or tight binding. The tricarbonyls also inhibited the iPLA 2 , but were less potent than the TFMKs. Unlike the cPLA 2 , the iPLA 2 does exhibit a fatty acid preference as the palmityl analogs of both compounds inhibit better than the arachidonoyl analogs. The palmityl TFMK displayed a 10-fold lower IC 50 at pH 9.0 than at pH 7.5, whereas the potency of the tricarbonyl was unchanged in this range. Thus, the TFMKs inhibit both the cPLA 2 and the iPLA 2 , but the mechanism of inhibition of the two enzymes appears to be quite different. The tricarbonyls also inhibited both enzymes, but in both cases in a reversible manner and as such appear to be poorer inhibitors than the TFMKs.Ketones with enhanced electrophilic reactivity (hydrateforming ketones), such as trifluoromethyl ketones (TFMKs), R-diketones, R-keto esters, and R,β-dioxo esters or vicinal tricarbonyls, 1,2 are a well-established class of inhibitors for proteases, lipases, and other serine esterases. This type of inhibitor has now been used to study phospholipase A 2 s (PLA 2 ), a class of esterases which carry out a reaction similar to that of lipases. Of particular interest are two intracellular PLA 2 s, the Group IV, 85 kDa, Ca 2+ -dependent PLA 2 (cPLA 2 ) 3-5 and the murine P388D 1 80 kDa, Ca 2+ -independent PLA 2 (iPLA 2 ). 6 The mechanisms of action of these two PLA 2 s are not well established, although the cPLA 2 reaction is believed to proceed through an acyl-enzyme intermediate. 7,8 Sharp

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Activating an inactive bond: A new concept in synthetic peptide chemistry, backbone amide activation, proceeds through the selective conversion of a backbone amide into an imide, followed by nucleophilic acyl displacement (see scheme; Boc=tert‐butoxycarbonyl, Pg=protecting group). This methodology represents a new approach to solid‐phase synthesis of C‐terminal peptide thioesters, and may become a general tool for the synthesis of peptide thioesters.