Avtor/Urednik | Osterlund, T; Danielsson, B; Degerman, E; Contreras, JA; Edgren, G; Davis, RC; Schotz, MC; Holm, C | |
Naslov | Domain-structure analysis of recombinant rat hormone-sensitive lipase | |
Tip | članek | |
Vir | Biochem J | |
Vol. in št. | Letnik 319 ( Pt 2) | |
Leto izdaje | 1996 | |
Obseg | str. 411-20 | |
Jezik | eng | |
Abstrakt | Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains. | |
Deskriptorji | AMINO ACID SEQUENCE CHOLESTEROL ESTERASE MOLECULAR SEQUENCE DATA RATS RECOMBINANT PROTEINS SEQUENCE ALIGNMENT SEQUENCE ANALYSIS |