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Purification, characterization, and molecular cloning of group I phospholipases A 2 from the gills of the red sea bream, Pagrus major
Author(s) -
Iijima Noriaki,
Uchiyama Satoshi,
Fujikawa Yukichi,
Esaka Muneharu
Publication year - 2000
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-000-0653-4
Subject(s) - pagrus major , complementary dna , molecular mass , pagrus , biology , hepatopancreas , biochemistry , phospholipid , gill , phosphatidylcholine , microbiology and biotechnology , chromatography , chemistry , enzyme , fishery , gene , membrane , fish <actinopterygii>
Phospholipase A 2 (PLA 2 ) activity was investigated in various tissues of male and female red sea bream. In both male and female fishes, the specific activity of PLA 2 in the gills was 70 times higher than that in other tissues, such as the adipose tissue, intestine, and hepatopancreas. Therefore, we tried to purify PLA 2 from the gill filaments of red sea bream to near homogeneity by sequential chromatography on Q‐Sepharose Fast Flow, Butyl‐Cellulofine, and DEAE‐Sepharose Fast Flow columns, and by reversed‐phase high‐performance liquid chromatography. Two minor and one major PLA 2 , tentatively named G‐1, G‐2 and G‐3 PLA 2 , were purified, and all showed a single band with an apparent molecular mass of approximately 15 kDa by sodium dodecylsulfate‐polyacrylamide gel electrophoresis. The exact molecular mass values of G‐1, G‐2, and G‐3 PLA 2 were 14,040, 14,040 and 14,005 Da, respectively. G‐1, G‐2, and G‐3 PLA 2 had a Cys 11 and were all identical in N‐terminal amino acid sequences from Ala‐1 to Glu‐56. A full‐length cDNA encoding G‐3 PLA 2 was cloned by reverse transcriptase‐polymerase chain reaction and rapid amplification of cDNA ends methods, and G‐3 PLA 2 was found to be classified to group IB PLA 2 from the deduced amino acid sequence. G‐1, G‐2, and G‐3 PLA 2 had a pH optimum in an alkaline region at around pH 9–10 and required Ca 2+ essentially for enzyme activity, using a mixed‐micellar phosphatidylcholine substrate with sodium cholate. These results demonstrate that three group 1 PLA 2 , G‐1, G‐2, and G‐3 PLA 2 , are expressed in the gill filaments of red sea bream.