Rapid purification of detergent‐solubilized bovine hormone‐sensitive lipase by high performance hydrophobic interaction chromatography

Abstract

Hormone-sensitive lipase (HSL), the enzyme controlling the rate of adipose tissue lipolysis and also possibly involved in the regulation of steroidogenesis, has been purified from bovine omental adipose tissue. Partially detergent-solubilized, delipidated and purified HSL was obtained through step-elution at conventional DEAE ion-exchange chromatography, followed by concentration on hydroxylapatite. High performance hydrophobic interaction chromatography (HPHIC) on phenylsilica then resulted in an increase of HSL protein purity from 2% to more than 70%. Final purification of the enzyme to apparent homogeneity (greater than 95% protein purity), concentration and removal of most of the detergent was obtained by high performance cation exchange chromatography on Mono S. At least 0.5 mg of highly stable HSL was obtained from 5 kg of bovine omental fat within four working days. The purified lipase had a lower specific activity than previously reported for the corresponding rat enzyme but the preparations have proved very useful for enzyme structure studies and as an antigen.

Molecular Mechanisms Regulating Hormone-Sensitive Lipase and Lipolysis

“…This activity was given the name hormone-sensitive lipase in 1964 by Vaughan et al (173), who also made the distinction between HSL and MGL, based on differences in pH optimum and substrate specificity. HSL purified from adipose tissue is a hydrophobic and labile protein with an apparent M r of 84,000 (6, 42,[112][113][114]. Following the cDNA cloning of HSL (73), powerful expression systems were established based on baculovirus/insect cell technology, providing large amounts of homogenous protein for structural and functional studies (21, 120c).…”

Domain-structure analysis of recombinant rat hormone-sensitive lipase

“…After adding salt (0.8 M NaCl final concn.) the eluted protein was subjected to hydrophobic-interaction chromatography on phenyl-Sepharose CL-4B (Pharmacia), essentially as described for the purification of bovine adipose-tissue HSL on phenylsilica [11] : The pooled material was applied to a phenyl-Sepharose column (100 ml) that had been equilibrated with 20 vol. of 5 mM potassium phosphate (pH 7.4)\2 M NaCl\2 % C "$ E "# \1 mM dithioerythritol\10 % glycerol, followed by 10 vol.…”

“…Since the recombinant enzyme is intended for studies requiring a high degree of purity of the protein, such as crystallization studies, a purification scheme resulting in large amounts of homogenous enzyme was established. A modification of previous purification schemes for rat and bovine adipose-tissue HSL, utilizing HPLC and Mono Q [10] and phenyl-silica [11], was employed. The result of such a purification scheme starting with 12 litres of culture (approx.…”

“…HSL has been purified from adipose tissue of several species, and the limited amounts of pure protein obtained have been used for partial characterization [9][10][11][12]. HSL cDNAs have been isolated from rat, mouse and human adipose tissue, which, together with the biochemical characterization, have provided information about enzyme structure of this 84 kDa protein [5,[13][14][15].…”

Hormone-sensitive lipase protein expression and extent of phosphorylation in subcutaneous and retroperitoneal adipose tissues in the periparturient dairy cow

“…The initial step of lipolysis is the hydrolysis of triacylglycerols (Montague and O'Rahilly, 2000). In adipose tissue of cows, it is suggested that hydrolysis of triacylglycerols is mediated by hormone-sensitive lipase (HSL) under stimulation of catecholamines (Nilsson , 1989;Sumner and McNamara, 2007;Elkins and Spurlock, 2009). Yet, the exact physiological role of HSL in maintenance of energy homeostasis in dairy cows is not fully understood.…”