[ effect of some detergents and stabilizing agents on plasma membrane phosphatidate phosphoydrolase of rat liver]

ABSTRACT

Phosphatidate phosphohydrolaze [PAP] catalyzes the dephosphorylation of phosphatidic acid to yield P[i] and diacylglycerol. Two different forms of PAP have been reported in rat hepatocyte PAP[1] that participates in the synthesis of phospholipids and triacylglyerols and PAP[2] which is involved in lipid signaling pathways. Two isoforms of PAP[2] are PAP[2a] and PAP[2b]. This study examines the effect of detergents such as Tween 80 lubrol, PX and CTAB as well as stabilizing factors including trehalose, sucrose, and albumin on the stability and activity of PAP[2b]. 14 Wistar rats weighing between 200-250 grams were used in the study. PAP[2b] was purified from liver plasma membrane by solubilizing with n-octyle glucoside and through several chromatography stages. Gel electrophoresis [SDS-PAGE] was performed in 10% gel slab in order to determine the purity level and to measure the molecular weight and number of the enzyme subunit. The effect of trehalose, sucrose, and albumin was examined on the stimulation of enzyme activity in different concentrations. The specific activity of purified enzyme was 7350 mU/mg protein. The purified enzyme showed a single band on SDS-PAGE with a MW of about 33.8 kDa. The enzyme was approximately activated 3 times by lubrol PX and Tween 80 both at 3 mM. the activation of CTAB occurred at 1 Mm. Trehalose, sucrose and albumin had the most stability effect on PAP[2b] in concentration of 3, 7 and 10 percent respectively. Tween 80, lubrol PX and CTAB have the ability to activate PAP[2b]. In case a nondetergent agent is required to stabilize PAP[2b] trehalose is preferred to sucrose and albumin. The lubrol PX has a higher potential stimulatory effect to activate PAP[2b] compared to other ionic and nonionic detergents