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

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

Evidence for histidine residues on plasma membrane phosphatidate phosphohydrolase from rat liver

Phosphatidate phosphohydrolase [PAP] catalyzes the dephosphorylation of phosphatidic acid to yield P[i], and diacylglycerol. Two different forms of PAP in rat hepatocyte have been reported. PAP[1] is located in cytosolic and microsomal fractions and participates in the synthesis of triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine, whereas the other form of phosphatidate phosphohydrolase [PAP[2]] is primarily involved in lipid signaling pathways. In rat liver, PAP2 has two isoforms; one PAP[2a] and another PAP2b- In this study, essential histidine residues were investigated in native form of rat purified PAP[2b] with diethylpyrocarbonate. PAP[2b] purified from rat liver plasma membrane by solubilizing with n-octyle glucoside and several chromatography steps. Gel electrophoresis [SDS-PAGE] performed on purified enzyme in order to evaluate its purity and to measure the molecular weight of the enzyme subunit. The enzyme inactivated with diethylpyrocarbonate [DEPC] and the number of moles of histidine residues modified per mol of enzyme determined. The specific activity of purified enzyme was 7350mU/mg protein and it showed only a single band on SDS-PAGE with a MW of about 33.8 kDa. The PAP[2b] inactivated by DEPC. The maximum 6 moles of histidine residues modified per mole of PAP[2b], when about 90% of enzyme activity is lost with DEPC. The data showed that the incubation of PAP[2b] by DEPC can inhibit enzyme activity. Our findings also, revealed the presence of essential histidines in the structure of PAP[2b] which involve in its activity. This enzyme is likely to have a similar hydrolysis catalytic mechanism as its super family through a phosphohistidine intermediate

relationship between cation-induced substrate configuration and enzymatic activity of phosphatidate phosphohydrolase from human liver

The mechanism by which bi-and trivalent cations affect human liver phosphatidate phosphohydrolase [PAP] activity was investigated. Bivalent cations up to 1 mM increased PAP activity whereas at higher concentrations the activity of the enzyme decreased. The stimulatory concentration for trivalent cations such as Al[3+] and Cr[3+], however, was much lower being 2 mM and 1 mM, respectively. All cations affecting PAP activity were also able to induce phase transition of phosphatidate from lamellar [La] to inverted hexagonal [HII] form. The rate of La-HII transition was different for each cation. At 100 mM concentration of Mg[2+] only 26% of the original phosphatidate remained in La form and for other cations tested ranged from 14.5% to 76%. The phase transition was blocked by EDTA. Magnesium from 0.8 to 1.5 mM concentration raised PAP activity [3-fold] with La form of substrate but not with the HII phase. Monovalent cations such as Na[+] and K[+] neither affected enzyme activity nor substrate configuration. These data suggest that cation-induced PAP activation is not as a result of cation-protein interaction, but is due to formation of a suitable substrate configuration for the enzyme catalysis during phosphatidate phase transition. It appears that the real substrate configuration for PAP activity is situated between La and HII phases

Epinepherine Inhibits the Activity of Phosphatidate Phosphohydrolase of Isolated Human Hepatocytes

The effect of epinephrine on phosphatidate phosphohydrolase [PAP] activity of isolated human hepatocytes was studied. Epineprine inhibited the enzyme activity progressively at concentrations above 0.1 micro M, reaching a maximum inhibition of 64.5% at 100 micro concentration. Inclusion of alprenolol, a Beta- receptor blocker, in the incubation mixture abolished the inhibitory effect of epinephrine on PAP, whereas the alpha-receptor antagonist phentolamine, or agonist phenylephrine, did not significantly change the hormone's effect. Addition of dibutyryl-cAMP or aminophylline [a cAMP phosphodiesterase inhibitor] to the incubation mixture together with epinephrine caused further enzyme inhibitism reaching 65.6% and 63.7%, respectively, compared to 49% inhibition caused by epinephrine alone under the same conditions. Dibutyryl-cAMP alone also inhibited PAP activity [51%]. The results suggested that epinephrine affects human hepatocyte PAP activity through beta- adrenoceptor activation and cAMP is involved in the mechanism by which PAP activity is altered

effects of adrenalectomy, orchiectomy, and selected hormones on glucose 6-phosphate dehydrogenase of rat liver

The effects of adrenalectomy, orchidectomy, and several selected hormones on rat liver G6PD were studied. Adrenalectomy decreased the enzyme activity by 56% within 12 days. Orchidectomy, however, caused significant increase in G6PD activity [123%] during the first four days after which the enzyme activity declined but remained above control levels on day 12. The enzyme activity increased by injecting estradiol [101%], progesterone [87%], and epinephrine [64%] in female rats but decreased by testosterone in both male [25%] and female [48%] animals. The data suggest that both adrenal and sex hormones may be involved in regulating G6PD activity