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Cecilia Holm

Professor

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Domain-structure analysis of recombinant rat hormone-sensitive lipase

Author

  • Torben Österlund
  • Birgitta Danielsson
  • Eva Degerman
  • Juan Antonio Contreras
  • Gudrun Edgren
  • Richard C Davis
  • Michael C Schotz
  • Cecilia Holm

Summary, in English

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.

Department/s

  • Department of Translational Medicine
  • Molecular Endocrinology
  • Insulin Signal Transduction
  • Department of Experimental Medical Science

Publishing year

1996

Language

English

Pages

411-420

Publication/Series

Biochemical Journal

Volume

319

Issue

Pt 2

Document type

Journal article

Publisher

Portland Press

Topic

  • Biochemistry and Molecular Biology

Status

Published

Research group

  • Molecular Endocrinology
  • Insulin Signal Transduction

ISBN/ISSN/Other

  • ISSN: 0264-6021