Biomaterial Database

Expression and characterization of human group V phospholipase A2. 1998

Y Chen, and E A Dennis

Department of Chemistry and Biochemistry, School of Medicine and Revelle College, University of California, San Diego, La Jolla, CA 92093-0601, USA.

Group V phospholipase A2 (GV-PLA2) has been shown to be involved in signal transduction and inflammatory processes in cellular studies, but the physical and biochemical properties of this important enzyme have been unclear. We report the over-expression and characterization of GV-PLA2. The GV-PLA2 cDNA was synthesized from human heart polyA+ mRNA by RT-PCR, and an expression construct containing the GV-PLA2 was established. After expression in Escherichia coli cells, the protein was solubilized and purified to homogeneity in a single step using nickel affinity chromatography. The purified GV-PLA2 protein was folded to form active enzyme. The recombinant GV-PLA2 has an absolute requirement for Ca2+ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with sonicated vesicles as substrate. GV-PLA2 preferentially hydrolyzes phosphatidylethanolamine (PE) vesicles compared to phosphatidylcholine (PC) vesicles. However, hydrolysis of PC and PE is equivalent in mixed vesicles of the phospholipids. The fatty acid preference of GV-PLA2 is linoleoyl>palmitoyl>arachidonyl with a PC head group and sonicated vesicles. 3-(3-Actamide-1-benzyl-2-ethylindolyl-5-oxy)propane phosphonic acid (LY311727), a potent inhibitor of human group IIA PLA2, strongly inhibits GV-PLA2 with an IC50 value of about 36 nM which is comparable to its inhibition of group IIA PLA2.

UI	MeSH Term	Description	Entries
D007211	Indoles	Benzopyrroles with the nitrogen at the number one carbon adjacent to the benzyl portion, in contrast to ISOINDOLES which have the nitrogen away from the six-membered ring.
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010741	Phospholipases A	Phospholipases that hydrolyze one of the acyl groups of phosphoglycerides or glycerophosphatidates.
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D006863	Hydrogen-Ion Concentration	The normality of a solution with respect to HYDROGEN ions; H+. It is related to acidity measurements in most cases by pH	pH,Concentration, Hydrogen-Ion,Concentrations, Hydrogen-Ion,Hydrogen Ion Concentration,Hydrogen-Ion Concentrations
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D013379	Substrate Specificity	A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts.	Specificities, Substrate,Specificity, Substrate,Substrate Specificities
D017510	Protein Folding	Processes involved in the formation of TERTIARY PROTEIN STRUCTURE.	Protein Folding, Globular,Folding, Globular Protein,Folding, Protein,Foldings, Globular Protein,Foldings, Protein,Globular Protein Folding,Globular Protein Foldings,Protein Foldings,Protein Foldings, Globular