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Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobili-zation of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA)in vitro as well asin vivo, and Ser660 and Ser659 have been shown to be the activity-controlling sites in vitro. | Phosphorylation of hormone-sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area Christian Krintel1 2 Matthias Morgelin3 Derek T. Logan2 and Cecilia Holm1 1 Department of ExperimentalMedicalScience Division of Diabetes Metabolism and Endocrinology Lund University Sweden 2 Department of Molecular Biophysics Lund University Sweden 3 Department of ClinicalSciences Division of Infection Medicine Lund University Sweden Keywords cholesterol ester hydrolase electron microscopy fluorescence spectroscopy phospholipid vesicles Correspondence C. Holm Department of Experimental MedicalScience BMC C11 SE-221 84 Lund Sweden Fax 46 462224022 Tel 46 462228581 E-mail cecilia.holm@med.lu.se Received 10 March 2009 revised 17 May 2009 accepted 25 June 2009 doi 10.1111 j.1742-4658.2009.07172.x Hormone-sensitive lipase EC 3.1.1.79 HSL is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL Ser563 Ser659 and Ser660 are phosphorylated by protein kinase A PKA in vitro as well as in vivo and Ser660 and Ser659 have been shown to be the activity-controlling sites in vitro. The exact molecular events of PKA-mediated activation of HSL in vitro are yet to be determined but increases in both Vmax and S0.5 seem to be involved as recently shown for human HSL. In this study the hydrophobic fluorescent probe 4 4 -dianilino-1 1 -binaphthyl-5 5 -disulfonic acid bis-ANS was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL. The interaction of HSL with bis-ANS was found to have a Kd of 1 pM in binding assays. Upon PKA phosphorylation the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased. By negative stain transmission electron microscopy phosphorylated HSL was found to have a closer interaction with .
