Resolution of hormone-sensitive triglyceride/diglyceride lipase from monoglyceride lipase of chicken adipose tissue

Lars Berglund, J. C. Khoo, D. Jensen, D. Steinberg

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Hormone-sensitive lipase isolated by conventional methods is a large lipid.protein complex showing four acylhydrolase activities (triglyceride, diglyceride, monoglyceride, and cholesterol ester hydrolases). The present studies show that Triton X-100 dissociates the complex and that, in the continuing presence of Triton X-100, significant purification can be obtained. Triglyceride and diglyceride lipase activies co-purified without change in relative activities throughout six steps of purification. Monoglyceride lipase activity, however, was resolved almost completely; the relative monoglyceride lipase activity in the most purified triglyceride/diglyceride lipase fraction was less than 2% that of the starting material. The monoglyceride lipase was purified 92-fold with a yield of 14%; it retained essentially no triglyceride lipase activity. Two-dimensional gel electrophoresis showed only a single predominant protein band (M(r) = 45,000) associated with the monoglyceride lipase activity. The triglyceride/diglyceride lipase was purified 300- to 500-fold. The predominant protein band in the final preparation showed a M(r) = 42,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and only this protein band accepted 32P from [γ-32P]ATP in the presence of cAMP-dependent protein kinase. Two-dimensional gel electrophoresis of a partially purified preparation showed two major protein bands of M(r)=42,000 and 45,000, respectively, the latter probably reflecting the presence of residual monoglyceride lipase. During the course of purification the capacity of triglyceride/diglyceride lipase to be activated by cAMP-dependent protein kinase fell progressively, as did the capacity to be stimulated by exposure to high ionic strength. Thus, the final preparation could be phosphorylated by protein kinase, yet it retained very little capacity to be activated. The results suggest that a protein of M(r) = 42,000 represents a component of the hormone-sensitive lipase but that other protein subunits (or lipid.protein complexes) dissociated during purification are essential to the activation process.

Original languageEnglish (US)
Pages (from-to)5420-5428
Number of pages9
JournalJournal of Biological Chemistry
Volume255
Issue number11
StatePublished - 1980

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Monoacylglycerol Lipases
Lipoprotein Lipase
Lipase
Adipose Tissue
Chickens
Triglycerides
Sterol Esterase
Purification
Hormones
Tissue
Electrophoresis
Connectin
Electrophoresis, Gel, Two-Dimensional
Octoxynol
Cyclic AMP-Dependent Protein Kinases
Proteins
Gels
Monoglycerides
Lipids
Diglycerides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Resolution of hormone-sensitive triglyceride/diglyceride lipase from monoglyceride lipase of chicken adipose tissue. / Berglund, Lars; Khoo, J. C.; Jensen, D.; Steinberg, D.

In: Journal of Biological Chemistry, Vol. 255, No. 11, 1980, p. 5420-5428.

Research output: Contribution to journalArticle

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abstract = "Hormone-sensitive lipase isolated by conventional methods is a large lipid.protein complex showing four acylhydrolase activities (triglyceride, diglyceride, monoglyceride, and cholesterol ester hydrolases). The present studies show that Triton X-100 dissociates the complex and that, in the continuing presence of Triton X-100, significant purification can be obtained. Triglyceride and diglyceride lipase activies co-purified without change in relative activities throughout six steps of purification. Monoglyceride lipase activity, however, was resolved almost completely; the relative monoglyceride lipase activity in the most purified triglyceride/diglyceride lipase fraction was less than 2{\%} that of the starting material. The monoglyceride lipase was purified 92-fold with a yield of 14{\%}; it retained essentially no triglyceride lipase activity. Two-dimensional gel electrophoresis showed only a single predominant protein band (M(r) = 45,000) associated with the monoglyceride lipase activity. The triglyceride/diglyceride lipase was purified 300- to 500-fold. The predominant protein band in the final preparation showed a M(r) = 42,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and only this protein band accepted 32P from [γ-32P]ATP in the presence of cAMP-dependent protein kinase. Two-dimensional gel electrophoresis of a partially purified preparation showed two major protein bands of M(r)=42,000 and 45,000, respectively, the latter probably reflecting the presence of residual monoglyceride lipase. During the course of purification the capacity of triglyceride/diglyceride lipase to be activated by cAMP-dependent protein kinase fell progressively, as did the capacity to be stimulated by exposure to high ionic strength. Thus, the final preparation could be phosphorylated by protein kinase, yet it retained very little capacity to be activated. The results suggest that a protein of M(r) = 42,000 represents a component of the hormone-sensitive lipase but that other protein subunits (or lipid.protein complexes) dissociated during purification are essential to the activation process.",
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