J. Lipid Res.
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A more recent version of this article appeared on March 1, 2005

Papers In Press, published online ahead of print January 1, 2005
J. Lipid Res., doi:10.1194/jlr.D400021-JLR200
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Submitted on August 19, 2004
Revised on December 17, 2004
Accepted on December 17, 2004

Sensitive in vitro assay for hormone-sensitive lipase using a p-nitrobenzofurazan-labeled monoacylglycerol to detect low activities in rat adipocytes

Stefan Petry, Yassine Ben Ali, Henri Chahinian, Holger Jordan, Horst Kleine, Gunter Muller, Frederic Carriere, and Abdelkarim Abousalham

EIPL-UPR9025, CNRS, Marseille 13009

Corresponding Author: abousal{at}ibsm.cnrs-mrs.fr

The recent finding that p-nitrobenzofurazan (NBD)-fatty acid (NBD-FA) is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive fashion [G. Müller, H. Jordan, C. Jung, H. Kleine, S. Petry, Biochimie 85 (2003) 1245-1256], suggests that NBD-FA-labelled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing 12-(7-amino-benzo[1,2,3]oxadiazol-4-ylamino)-dodecanoic acid (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL in vitro. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes, and to a concomitant change of its spectrophotometric properties (a shift occurs from yellow to pink). The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics (a shift occurs from pink to yellow). The rate of lipolysis was monitored by measuring the increase in the OD at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. In order to assess the specific activity of recombinant human HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. With this novel quantitative assay, we established that the pH optimum of recombinant human HSL for cleavage of MAG was pH 6.0, and its specific activity was found to be 5 µmole.min-1.mg-1. This homogeneous and convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.


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