Domain-structure analysis of recombinant rat hormone-sensitive lipase
Österlund, Torben LU ; Danielsson, Birgitta LU ; Degerman, Eva LU
; Contreras, Juan Antonio
LU
; Edgren, Gudrun
; Davis, Richard C
; Schotz, Michael C
and Holm, Cecilia
LU
(1996)
In Biochemical Journal
319(Pt 2).
p.411-420
- Abstract
- 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... (More)
- 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1109898
- author
- Österlund, Torben
LU
; Danielsson, Birgitta
LU
; Degerman, Eva
LU
; Contreras, Juan Antonio
LU
; Edgren, Gudrun
; Davis, Richard C
; Schotz, Michael C
and Holm, Cecilia
LU
- organization
- publishing date
- 1996
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Biochemical Journal
- volume
- 319
- issue
- Pt 2
- pages
- 411 - 420
- publisher
- Portland Press
- external identifiers
-
- pmid:8912675
- scopus:0029806454
- ISSN
- 0264-6021
- language
- English
- LU publication?
- yes
- additional info
- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Department of Experimental Medical Science (013210000), Insulin Signal Transduction (013212014), Experimental Clinical Chemistry (013016010), Molecular Endocrinology (013212018)
- id
- 3bb7f944-ec9c-440f-b8dd-c3658b1abb7d (old id 1109898)
- alternative location
- http://www.biochemj.org/bj/319/bj3190411.htm
- date added to LUP
- 2016-04-01 15:30:52
- date last changed
- 2022-01-28 05:43:36
@article{3bb7f944-ec9c-440f-b8dd-c3658b1abb7d,
abstract = {{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.}},
author = {{Österlund, Torben and Danielsson, Birgitta and Degerman, Eva and Contreras, Juan Antonio and Edgren, Gudrun and Davis, Richard C and Schotz, Michael C and Holm, Cecilia}},
issn = {{0264-6021}},
language = {{eng}},
number = {{Pt 2}},
pages = {{411--420}},
publisher = {{Portland Press}},
series = {{Biochemical Journal}},
title = {{Domain-structure analysis of recombinant rat hormone-sensitive lipase}},
url = {{http://www.biochemj.org/bj/319/bj3190411.htm}},
volume = {{319}},
year = {{1996}},
}