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Purification, characterization, and molecular cloning of group I phospholipases A2 from the gills of the red sea bream, Pagrus major

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Lipids

Abstract

Phospholipase A2 (PLA2) activity was investigated in various tissues of male and female red sea bream. In both male and female fishes, the specific activity of PLA2 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 PLA2 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 PLA2, tentatively named G-1, G-2 and G-3 PLA2, 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 PLA2 were 14,040, 14,040 and 14,005 Da, respectively. G-1, G-2, and G-3 PLA2 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 PLA2 was cloned by reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends methods, and G-3 PLA2 was found to be classified to group IB PLA2 from the deduced amino acid sequence. G-1, G-2, and G-3 PLA2 had a pH optimum in an alkaline region at around pH 9–10 and required Ca2+ essentially for enzyme activity, using a mixed-micellar phosphatidylcholine substrate with sodium cholate. These results demonstrate that three group 1 PLA2, G-1, G-2, and G-3 PLA2, are expressed in the gill filaments of red sea bream.

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Abbreviations

p-APMSF:

p-amidinophenylmethylsulfonyl fluoride

p-BPB:

p-bromophenacyl bromide

CHAPS:

3-[(3-cholamidopropyl) dimethylammoniol-1-propanesulfonate

CTAB:

cetyl trimethylammonium bromide

HPLC:

high-performance liquid chromatography

MALDI-TOF:

matrixassisted laser desorption ionization-time of flight

PAGE:

polyacrylamide gel electrophoresis

PCR:

polymerase chain reaction

PLA2 :

phospholipase(s) A2

POPC:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPE:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

POPG:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol

POPS:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine

RACE:

rapid amplification of cDNA ends

RP-HPLC:

reversed-phase-HPLC

RT:

reverse transcriptase

SDS:

sodium lauryl sulfate

TFA:

trifluoroacetic acid

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Authors and Affiliations

  1. Faculty of Applied Biological Science, Hiroshima University, 1-4-4 Kagamiyama, 739-8528, Higashihiroshima, Japan

    Noriaki Iijima, Satoshi Uchiyama, Yukichi Fujikawa & Muneharu Esaka

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  1. Noriaki Iijima
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  2. Satoshi Uchiyama
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  3. Yukichi Fujikawa
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  4. Muneharu Esaka
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Correspondence to Noriaki Iijima.

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Iijima, N., Uchiyama, S., Fujikawa, Y. et al. Purification, characterization, and molecular cloning of group I phospholipases A2 from the gills of the red sea bream, Pagrus major . Lipids 35, 1359–1371 (2000). https://doi.org/10.1007/s11745-000-0653-4

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  • DOI: https://doi.org/10.1007/s11745-000-0653-4

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