Purification of rabbit platelet secretory phospholipase A2 and its characteristics.
It was reported previously that rat platelets release phospholipase A2 upon in vitro stimulation by thrombin, ADP, or A23187 (Horigome, K., Hayakawa, M., Inoue, K., & Nojima, S. (1987) J. Biochem. 101, 53-61). Secretion of phospholipase A2 was also observed with rabbit platelets. Rabbit platelets seem to release phospholipase A2 upon stimulation in vivo, because the rabbit plasma taken immediately after intravenous injection of PAF contained an appreciable level of phospholipase A2 activity and fewer platelets. Rabbit platelet phospholipase A2 released in vitro was purified by column chromatography using Sepharose CL-4B conjugated with anti-rat platelet derived phospholipase A2 monoclonal antibody, followed by reversed-phase HPLC. The purified enzyme was subjected to structural analysis by HPLC peptide mapping and primary sequence determination of the separated peptides. Based on the homology with rat platelet secretory phospholipase A2 (Hayakawa, M., Kudo, I., Tomita, M., Nojima, S., & Inoue, K. (1988) J. Biochem. 104, 767-772), a partial primary structure (62 amino acid residues) of the rabbit enzyme was tentatively determined; the two sequences were highly homologous (72%). The rabbit sequence was also nearly identical to that of rabbit ascitic fluid phospholipase A2, which was determined by Forst et al. (Forst, S., Weiss, J., Elsbach, P., Maraganore, J.M., Reardon, I., & Heinrikson, R.L. (1986) Biochemistry 25, 8381-8385). Phospholipase A2 from the membrane fraction of rabbit platelets was also purified; it had the same characteristics and th same amino-terminal sequence as the purified secretory enzyme. Secretory and membrane-bound phospholipase A2 of rabbit platelets may in fact be identical.(ABSTRACT TRUNCATED AT 250 WORDS)
Mizushima
H
H
Faculty of Pharmaceutical Sciences, University of Tokyo.
Kudo
I
I
Horigome
K
K
Murakami
M
M
Hayakawa
M
M
Kim
D K
DK
Kondo
E
E
Tomita
M
M
Inoue
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001792:Blood Platelets / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010740:Phospholipases / Q000302:isolation & purification*; D010741:Phospholipases A / Q000097:blood / Q000276:immunology / Q000302:isolation & purification*; D054467:Phospholipases A2; D010972:Platelet Activating Factor / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D011817:Rabbits
D000911:Antibodies, Monoclonal; D010972:Platelet Activating Factor; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2