Purification and characterization of thylakoid membrane-bound inorganic pyrophosphatase from Spinacia oleracia L.
An inorganic pyrophosphatase (PPase) was purified from thylakoid membrane of spinach leaves to electrophoretic purity by methods including detergent solubilization, ammonium sulfate fractionation, and successive chromatographic techniques. Current protocol yielded about 10% recovery of total activity with a 30-fold purification. The specific activity of the purified enzyme was approximately 400 micromol PPi consumed/mg protein x h. This enzyme is a monomer with a molecular mass of 55 kDa. Several properties, including subunit composition, substrate specificity, ion requirements, inhibitor sensitivities, and amino acid composition, have been studied. Mg2+ is an essential cofactor for the thylakoid PPase. The preferred substrate for the hydrolytic reaction of PPase appears to be dimagnesium pyrophosphate. K+ could not stimulate the enzymatic activity of thylakoid PPase, while F- was a potent inhibitor. Group-specific modification of the thylakoid PPase demonstrates possible involvement of carboxylate residues in the enzymatic activity. Furthermore, antibodies raised against thylakoid PPase in a rabbit could inactivate the PPi hydrolysis of thylakoid and the purified enzyme, but not that of vacuolar H+-PPase, indicating both PPi hydrolases are structurally distinct.
Jiang
S S
SS
Institute of Radiation Biology, College of Nuclear Science, National Tsing Hua University, Hsin Chu, Taiwan, Republic of China.
Fan
L L
LL
Yang
S J
SJ
Kuo
S Y
SY
Pan
R L
RL
D000596:Amino Acids / Q000032:analysis; D002413:Cations, Divalent / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D002736:Chloroplasts / Q000201:enzymology*; D011756:Diphosphates / Q000378:metabolism; D043564:Inorganic Pyrophosphatase; D007425:Intracellular Membranes / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D009711:Nucleotides / Q000378:metabolism; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D002413:Cations, Divalent; D002414:Cations, Monovalent; D011756:Diphosphates; D009711:Nucleotides; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium