Arachidonoyl-Diacylglycerol Kinase from Bovine Testis: Purification and Properties

James P. Walsh, Rosa Suen, Rozenn N. Lemaitre, John A. Glomset

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

Previous work in our laboratory demonstrated the existence of a membrane-bound diacylglycerol kinase highly selective for diacylglycerols containing arachidonate as the sn-2 fatty acyl moiety (MacDonald, M. L., Mack K. F., Richardson, C. N., and Glomset, J. A. (1988) J. Biol. Chem. 263, 1575-1583). We now report the purification of arachidonoyl-diacylglycerol kinase 34,400-fold to apparent homogeneity from bovine testis. High concentrations of both salt and detergent were required to extract the enzyme from membranes and stabilize its activity, suggesting that in vivo the enzyme is part of a complex with other membrane or cytoskeletal proteins. Arachidonoyl-diacylglycerol kinase had an apparent Mr of 58,000 both on SDS-polyacrylamide gels and by size exclusion chromatography. The enzyme appeared to be an integral membrane protein. In a mixed micellar assay, arachidonoyl-diacylglycerol kinase followed surface dilution kinetics with respect to diacylglycerol. The purified enzyme retained the arachidonate selectivity observed previously in membranes. Kinetic analyses indicated a Km for sn-1-stearoyl-2-arachidonoylglycerol of 2.4 mol %, as compared to 43 mol % for sn-1-palmitoyl-2-oleoylglycerol. Calcium, an activator of some other diacylglycerol kinases, had no apparent effect on the arachidonate-specific enzyme. Guanosine triphosphate could effectively substitute for ATP as the phosphoryl donor and Mg2+ could be replaced by Mn2+ or Ca2+. Phosphatidylserine and, to a lesser extent, phosphatidylinositol inhibited the purified enzyme. Phosphatidylcholine and phosphatidylethanolamine had only small effects.

Original languageEnglish (US)
Pages (from-to)21155-21164
Number of pages10
JournalJournal of Biological Chemistry
Volume269
Issue number33
StatePublished - Aug 19 1994
Externally publishedYes

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Diacylglycerol Kinase
Purification
Testis
Enzymes
Diglycerides
Membranes
Membrane Proteins
Kinetics
Cytoskeletal Proteins
Size exclusion chromatography
Phosphatidylserines
Phosphatidylinositols
Guanosine Triphosphate
Phosphatidylcholines
Detergents
Dilution
Gel Chromatography
Assays
Salts
Adenosine Triphosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Walsh, J. P., Suen, R., Lemaitre, R. N., & Glomset, J. A. (1994). Arachidonoyl-Diacylglycerol Kinase from Bovine Testis: Purification and Properties. Journal of Biological Chemistry, 269(33), 21155-21164.

Arachidonoyl-Diacylglycerol Kinase from Bovine Testis : Purification and Properties. / Walsh, James P.; Suen, Rosa; Lemaitre, Rozenn N.; Glomset, John A.

In: Journal of Biological Chemistry, Vol. 269, No. 33, 19.08.1994, p. 21155-21164.

Research output: Contribution to journalArticle

Walsh, JP, Suen, R, Lemaitre, RN & Glomset, JA 1994, 'Arachidonoyl-Diacylglycerol Kinase from Bovine Testis: Purification and Properties', Journal of Biological Chemistry, vol. 269, no. 33, pp. 21155-21164.
Walsh JP, Suen R, Lemaitre RN, Glomset JA. Arachidonoyl-Diacylglycerol Kinase from Bovine Testis: Purification and Properties. Journal of Biological Chemistry. 1994 Aug 19;269(33):21155-21164.
Walsh, James P. ; Suen, Rosa ; Lemaitre, Rozenn N. ; Glomset, John A. / Arachidonoyl-Diacylglycerol Kinase from Bovine Testis : Purification and Properties. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 33. pp. 21155-21164.
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