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Research has been conducted on human group IIA secreted phospholipase A (sub)2. The preparation of phospholipase's H48Q and H48N mutants and the analysis of H48N and H48Q enzymes' activity are discussed.

The ability of fatty acid-binding proteins to interact with phospholipid interfaces that contain anionic phospholipids has been investigated. Results indicate that a complete loss of legand can be caused by the interaction with small phospholipid vesicles.

The mutation of the interfacial valine of human secreted phospholipase A2 (sPLA2) to a tryptophan was found to demonstrate the ability of this amphiphilic indole residue. The residue substantially improves the hydrolysis of zwitterionic interfaces found in the form of small unilamellar vesicles of phosphatidylcholine or cell membranes. Findings are in good agreement with the function of interfacial tryptophans of venom enzymes in permitting the penetration and hydrolysis of such condensed bilayers.

Human secretory group IIa phospholipase Asub2 (hIIa-PLAsub2) has high affinity for anionic surfaces such as anionic membranes. Site-directed mutagenesis of the protein was carried out to identify the cationic amino acid residues supporting binding to anionic membranes. Results suggest that electrostatic interactions between surface lysine and arginine residues and the interface contribute to interfacial hIIa-PLAsub2 binding to anionic vesicles.

The complex kinetic effects of gossypol and sulfated conjugates on phospholipid A2 activity were analyzed through quantitative determination of the primary rate and equilibrium constants for the catalytic turnover. Gossypol inactivated PLA2 through covalent modification of the alpha amino group at the amino terminal. Sulfated conjugates sequester the circulating PLA2 thereby altering the proteoglycan matrix which resulted in the loss of the regulatory role.

Research has been conducted on human group IIA phospholipase A (sub)2. Results demonstrate that the insertion of tryptophan produces mutants which can enhance human IIA enzyme activity against intact cell membranes and zwitterionic interfaces.

The conversion of desmosterol into cholesterol was earned out in the presence of either tritiated water or [4-³H2]NADPH. The side chain fragment consisting of carbon atoms 23-27 of cholesterol was obtained through the combination of biological and chemical techniques. Selective degradation of the fragment led to the conclusion that in the saturation of the 24,25-double bond of desmosterol a hydrogen atom from the medium is added to C-24 and another from the 4-position of NADPH to C-25. These results in conjunction with similar studies previously reported on the saturation of other C=C in steroid biosynthesis are interpreted in terms of a general mechanism. It is suggested that the first, crucial event, in the pyridine nucleotide linked reduction is the activation of the substrate through protonation to give an electron deficient species which in the next step is neutralized by the addition of a hydride from NAD(P)H to furnish the product. [ABSTRACT FROM AUTHOR]

3-Hydroxy-3-methyl-[3-14C]glutaryl-CoA was reduced to mevalonic acid in the presence of either [4A-³H]- or [4B-³H]NADPH and rat liver 3-hydroxy-3-methylglutaryl-CoA reductase. The resulting doubly labelled mevalonic acid incorporated tritium exclusively from the 4A (4R) position of NADPH. The distribution of tritium at C-5 of the mevalonic acid was determined by a biological degradation. It was concluded that during the reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid two hydrogen atoms from the 4A (4R) position of NADPH are transferred directly to mevalonic acid. [ABSTRACT FROM AUTHOR]

Liver FABP (fatty-acid-binding protein) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, lysophospholipids and bile acids. Liver FABP is also able to bind to anionic phospholipid vesicles under conditions of low ionic strength, and membrane binding results in the release of bound ligand. However, the molecular interactions involved in binding to the phospholipid interface and the mechanism of ligand release are not known. Ligand release could be due to a significant conformational change in the protein at the interface or interaction of a phospholipid molecule with the ligand-binding cavity of the protein resulting in ligand displacement. Two portal mutant proteins of liver FABP, L28W and M74W, have now been used to investigate the binding of liver FABP to anionic phospholipid vesicles, monitoring changes in fluorescence and also fluorescence quenching in the presence of brominated lipids. There is a large increase in fluorescence intensity when the L28W mutant protein binds to vesicles prepared from DOPG (dioleoyl-sn-phosphatidylglycerol), but a large decrease in fluorescence intensity when the M74W mutant binds to these vesicles. The Br(4)-phospholipid prepared by bromination of DOPG dramatically quenches both L28W and M74W, consistent with the close proximity of a fatty acyl chain to the tryptophan residues. The binding of liver FABP to DOPG vesicles is accompanied by only a minimal change in the CD spectrum. Overall, the results are consistent with a molecule of anionic phospholipid interacting with the central cavity of the liver FABP, possibly involving the phospholipid molecule in an extended conformation.