Expression of biologically active hormone-sensitive lipase in mammalian (COS) cells.
cDNAs encoding rat adipose tissue hormone-sensitive lipase were expressed in COS cells, under the control of the SV40 promoter to half the level in rat adipocytes, the richest native source of the enzyme. A cDNA lacking most of the long 5'-untranslated region of the full-length rat hormone-sensitive lipase cDNA was, with regard to the lipase activity, on the average 70% more efficiently expressed that the full-length cDNA. The recombinant protein was almost identical to hormone-sensitive lipase of rat adipose tissue with respect to specific activity, susceptibility to inhibitors, molecular size, phosphorylation and activation by cyclic AMP-dependent protein kinase. The described eukaryotic expression system will allow analysis of effects of amino acid substitutions introduced into the lipase molecule by site-directed mutagenesis.
Holm
C
C
Department of Medical and Physiological Chemistry 4, University of Lund, Sweden.
Davis
R C
RC
Fredrikson
G
G
Belfrage
P
P
Schotz
M C
MC
D000249:Adenosine Monophosphate / Q000378:metabolism; D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D015152:Blotting, Northern; D002461:Cell Line, Transformed; D015971:Gene Expression Regulation, Enzymologic; D007531:Isoflurophate / Q000494:pharmacology; D008628:Mercury / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013539:Simian virus 40 / Q000235:genetics; D002787:Sterol Esterase / Q000096:biosynthesis* / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D007531:Isoflurophate; D000249:Adenosine Monophosphate; D011494:Protein Kinases; D002787:Sterol Esterase; D008628:Mercury