CLONING OF THE CDNA CODING FOR 14 KDA GROUP-II PHOSPHOLIPASE-A2 FROM RAT-LIVER

The amino acid sequence of rat liver phospholipase A2 was partially el ucidated using peptide fragments generated by enzymatic or chemical cl eavage. Based on this sequence information, two oligonucleotide probes were constructed which were applied in a polymerase chain reaction on cDNA generated from rat liver total RNA. This resulted in cloning of the cDNA corresponding to the coding region of the mature phospholipas e A2. The deduced amino acid sequence showed the enzyme belongs to the group II phospholipases, and is almost completely identical to rat pl atelet and spleen membrane-associated phospholipase A2. However, in th e cDNA isolated one codon was different as compared to the platelet an d spleen enzymes, resulting in the substitution of Ala94 by Arg94 in t he liver enzyme. In Northern blot analyses the mRNA for rat group II p hospholipase A2 could not be detected in rat liver, neither in total R NA nor in poly(A)+ RNA. However, a polymerase chain reaction using tot al RNA originating from freshly isolated hepatocytes resulted in the a mplification of the described phospholipase A2 cDNA. This indicates th at group II PLA2 mRNA is present in these cells, but presumably at ver y low abundance. The observed increase in rat group II phospholipase A 2 secretion in rat mesangial cells upon stimulation with interleukin-1 beta (Pfeilschifter et al. (1989). Biochem. Biophys. Res. Commun. 159, 385-394) was shown to be accompanied by an increased transcription of the rat group II phospholipase A2 gene, indicating interleukin exerts its effect via increased phospholipase A2 mRNA synthesis. Based on No rthern blot analyses of stimulated rat mesangial cells, the size of th e mRNA for rat group II phospholipase A2 was determined to be 0.9 kb.