Purification, cDNA cloning, and regulation of lysophospholipase from rat liver.
A lysophospholipase was purified 506-fold from rat liver supernatant. The preparation gave a single 24-kDa protein band on SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzed lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, lysophosphatidylserine, and 1-oleoyl-2-acetyl-sn-glycero-3-phosphocholine at pH 6-8. The purified enzyme was used for the preparation of antibody and peptide sequencing. A cDNA clone was isolated by screening a rat liver lambda gt11 cDNA library with the antibody, followed by the selection of further extended clones from a lambda gt10 library. The isolated cDNA was 2,362 base pairs in length and contained an open reading frame encoding 230 amino acids with a Mr of 24,708. The peptide sequences determined were found in the reading frame. When the cDNA was expressed in Escherichia coli cells as the beta-galactosidase fusion, lysophosphatidylcholine-hydrolyzing activity was markedly increased. The deduced amino acid sequence showed significant similarity to Pseudomonas fluorescence esterase A and Spirulina platensis esterase. The three sequences contained the GXSXG consensus at similar positions. The transcript was found in various tissues with the following order of abundance: spleen, heart, kidney, brain, lung, stomach, and testis = liver. In contrast, the enzyme protein was abundant in the following order: testis, liver, kidney, heart, stomach, lung, brain, and spleen. Thus the mRNA abundance disagreed with the level of the enzyme protein in liver, testis, and spleen. When HL-60 cells were induced to differentiate into granulocytes with dimethyl sulfoxide, the 24-kDa lysophospholipase protein increased significantly, but the mRNA abundance remained essentially unchanged. Thus a posttranscriptional control mechanism is present for the regulation of 24-kDa lysophospholipase.
Sugimoto
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H
Department of Biochemistry, Gunma University School of Medicine, Maebashi, Japan.
Hayashi
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Yamashita
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry; D002454:Cell Differentiation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D016384:Consensus Sequence; D000458:Cyanobacteria / Q000201:enzymology; D018076:DNA, Complementary; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004926:Escherichia coli; D004950:Esterases / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015723:Gene Library; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008245:Lysophospholipase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D008297:Male; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009928:Organ Specificity; D011551:Pseudomonas fluorescens / Q000201:enzymology; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000096:biosynthesis
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004950:Esterases; C019724:arginine esterase; D002265:Carboxylic Ester Hydrolases; D008245:Lysophospholipase; D001616:beta-Galactosidase; D004121:Dimethyl Sulfoxide