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:: Volume 12, Issue 2 (Quaterly 2008) ::
Feyz 2008, 12(2): 1-8 Back to browse issues page
Identification of essential histidine residues at the active site of rat liver plasma membrane phosphatidate phosphohydrolase
Esfandiar Heidarian *, Bahram Haghighi
, heidarian46@ yahoo.com
Abstract:   (7974 Views)

Background: Phosphatidate phosphohydrolase enzyme (PAP) catalyzes the transformation of phosphatidic acid to Pi and diacylglycerol. Two different forms of PAP have been reported in rat hepatocytes PAP1 which participates in the synthesis of phospholipids and triacylglycerols, and PAP2 which involved in lipid signaling transduction. PAP2 has two isoforms namely PAP2a and PAP2b. In this study the role of essential histidine residues in PAP2b was assessed.

Material and Methods: The rat liver plasma membrane was solved using n-octyle glucoside and PAP2b purified during several chromatography steps. The kinetics parameters were assessed based on surface kinetic dilution model. Discontinuous gel electrophoresis (SDS-PAGE) was performed on purified enzyme in order to evaluate its purity and to measure the molecular weight of the enzyme subunit in gel 10%. The number of mol histidine residues modified per mol enzyme was determined based on the formation of N-carbetoxy histidine.

Results: The specific activity of purified enzyme was 7350mU/mg protein. PAP2b showed only a single band on SDS-PAGE with a mass weight of 33.8 kDa by electrophoresis. The PAP2b was inactivated by DEPC. The inhibition was competitive with respect to phosphatidate and the maximum 6 moles of histidine residues were modified per mole PAP2b.

Conclusion: The data have shown that the incubation of PAP2b by DEPC plus phosphatidate can prevent the inhibitory effect of DEPC on enzyme activity. Our findings also revealed the role of histidine in the active site of PAP2b .This enzyme is likely to have a similar hydrolysis catalytic mechanism as its super family through a phosphohistidine intermediate.

Keywords: Diethylpyrocarbonate, Histidine, Phosphatidic acid, Phosphatidate phosphohydrolase
Full-Text [PDF 366 kb]   (3080 Downloads)    
Type of Study: Research | Subject: General
Received: 2008/12/22 | Published: 2008/07/15
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Heidarian E, Haghighi B. Identification of essential histidine residues at the active site of rat liver plasma membrane phosphatidate phosphohydrolase. Feyz. 2008; 12 (2) :1-8
URL: http://feyz.kaums.ac.ir/article-1-631-en.html

Volume 12, Issue 2 (Quaterly 2008) Back to browse issues page
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