Detection of hormone-sensitive lipase in various tissues. I. Expression of an HSL/bacterial fusion protein and generation of anti-HSL antibodies.
Hormone-sensitive lipase (HSL) is an intracellular neutral lipase found in a variety of tissues, but primarily in adipose and steroidogenic tissues, that hydrolyzes triglycerides and cholesteryl esters. In the present studies, a portion of rat HSL cDNA was subcloned into a pET expression system and the resulting recombinant fusion protein was over-expressed in E. coli. The approximately 26 kD HSL/fusion protein was used to generate polyclonal antibodies in rabbits that recognize intact HSL (84 kD) in rat adipose tissue, ovary, adrenal, testis, heart, and lung, as well as in human adipose tissue. In addition, there was an approximately 89 kD protein observed in all rat tissues expressing the 84 kD protein. Unique to testes, there was an immunoreactive protein of approximately 102 kD in sexually immature rats, and additional immunoreactive proteins of approximately 113 kD and approximately 127 kD in sexually mature rats. The anti-HSL/fusion protein antibodies could remove approximately 60-80% of total neutral cholesterol esterase activity from extracts of rat adipose tissue and immunoprecipitated a single 84 kD protein after labeling of adipocytes with [32P]orthophosphate. The incorporation of 32P into the 84 kD HSL protein was stimulated 4-fold by incubation of adipocytes with isoproterenol. The half life of [35S]methionine-labeled HSL was approximately 4 h in normal rat adipocytes. The production of an HSL/fusion protein and generation of antibodies that recognize native HSL should be valuable tools in exploring the mechanisms regulating the expression of HSL activity and the function and localization of its immunoreactive proteins.
Department of Veterans Affairs Medical Center, Palo Alto, CA 94304.
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Kraemer
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Patel
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Saedi
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Sztalryd
D000273:Adipose Tissue / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D008297:Male; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D002787:Sterol Esterase / Q000032:analysis* / Q000235:genetics / Q000276:immunology
D011993:Recombinant Fusion Proteins; D002787:Sterol Esterase