Fb. Kraemer et al., DETECTION OF HORMONE-SENSITIVE LIPASE IN VARIOUS TISSUES .1. EXPRESSION OF AN HSL BACTERIAL FUSION PROTEIN AND GENERATION OF ANTI-HSL ANTIBODIES, Journal of lipid research, 34(4), 1993, pp. 663-671
Hormone-sensitive lipase (HSL) is an intracellular neutral lipase foun
d 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 ex
pression system and the resulting recombinant fusion protein was over-
expressed in E. coli. The approximately 26 kD HSL/fusion protein was u
sed 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 a
pproximately 89 kD protein observed in all rat tissues expressing the
84 kD protein. Unique to testes, there was an immunoreactive protein o
f approximately 102 kD in sexually immature rats, and additional immun
oreactive proteins of approximately 113 kD and approximately 127 kD in
sexually mature rats. The anti-HSL/fusion protein antibodies could re
move approximately 60-80% of total neutral cholesterol esterase activi
ty from extracts of rat adipose tissue and immunoprecipitated a single
84 kD protein after labeling of adipocytes with [P-32]orthophosphate.
The incorporation of P-32 into the 84 kD HSL protein was stimulated 4
-fold by incubation of adipocytes with isoproterenol. The half life of
[S-35]methionine-labeled HSL was approximately 4 h in normal rat adip
ocytes. The production of an HSL/fusion protein and generation of anti
bodies that recognize native HSL should be valuable tools in exploring
the mechanisms regulating the expression of HSL activity and the func
tion and localization of its immunoreactive proteins.