Purification and kinetic characterization of chicken liver inorganic pyrophosphatase.
Soluble inorganic pyrophosphatase (EC 3.6.1.1) was isolated from chicken liver, RIR breed, to apparent homogeneity. The enzyme showed a molecular mass of 100 kDa as estimated by gel filtration and a subunit mass of 49 kDa on SDS-PAGE. The enzyme was very specific for pyrophosphate (PPi) and magnesium, and there was no measurable activity on replacing Mg2+ with Zn2+. At optimal conditions of assay, 50% of the enzyme activity was inhibited at 42 microM Ca2+, 70 microM fluoride and 0.91 mM Cd2+. There was a 50% inactivation of enzyme activity at 0.1 M guanidine hydrochloride (GuHCl). Kinetic analysis of GuHCl inactivation revealed 2 essential binding sites for this ligand. The enzyme showed allosteric behaviour with the substrate PPi and Mg2+. The apparent Hill coefficient of 1.47 and 1.48 for PPi and Mg2+, respectively indicate positive cooperatively. Hill plots also gave [S]0.5 of 0.177 mM and 2.5 microM for Mg2+ and PPi, respectively.
Syiem
D K
DK
Department of Biochemistry, North Eastern Hill University, Umshing, Shillong, India.
Sharma
R
R
D000818:Animals; D002412:Cations / Q000494:pharmacology; D002645:Chickens; D004795:Enzyme Stability; D066298:In Vitro Techniques; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D011755:Pyrophosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity
D002412:Cations; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium