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 elucidated using peptide fragments generated by enzymatic or chemical cleavage. 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 phospholipase A2. The deduced amino acid sequence showed the enzyme belongs to the group II phospholipases, and is almost completely identical to rat platelet and spleen membrane-associated phospholipase A2. However, in the cDNA isolated one codon was different as compared to the platelet and spleen enzymes, resulting in the substitution of Ala94 by Arg94 in the liver enzyme. In Northern blot analyses the mRNA for rat group II phospholipase A2 could not be detected in rat liver, neither in total RNA nor in poly(A)+ RNA. However, a polymerase chain reaction using total RNA originating from freshly isolated hepatocytes resulted in the amplification of the described phospholipase A2 cDNA. This indicates that group II PLA2 mRNA is present in these cells, but presumably at very low abundance. The observed increase in rat group II phospholipase A2 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 Northern blot analyses of stimulated rat mesangial cells, the size of the mRNA for rat group II phospholipase A2 was determined to be 0.9 kb.
Van Schaik
R H
RH
Centre for Biomembranes and Lipid Enzymology, Utrecht University, The Netherlands.
Verhoeven
N M
NM
Neijs
F W
FW
Aarsman
A J
AJ
Van den Bosch
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000235:genetics*; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D051381:Rats; D016415:Sequence Alignment
D004247:DNA; D010741:Phospholipases A; D054467:Phospholipases A2