Purification, physicochemical properties, and subcellular location of alkaline inorganic pyrophosphatase from sesame (Sesamum indicum L.) cotyledons.
Abstract: Changes in the levels of inorganic pyrophosphatases (PPases) were monitored in germinating sesame seeds at regular intervals. Activities of acid and alkaline PPases increased markedly in cotyledons up to day 4, remained at the peak level up to day 7, and then showed a considerable decline thereafter. An alkaline PPase was isolated and purified from 5-day-old sesame cotyledons following acetone precipitation, ammonium sulfate fractionation, and chromatography on DEAE-Sephadex. Current protocol yielded about 20% recovery of total activity with a 6.4-fold purification. The enzyme was a monomer with a molecular mass of 20.8 kDa. Some of the properties of alkaline PPase including stability, substrate specificity, ion requirement, and amino acid composition were studied. Alkaline PPase showed maximum activity at pH 8.6 in the presence of Mg2+ and at 50 degrees C. However, the metal ion could not protect the enzyme against thermal denaturation. Alkaline PPase was highly specific for inorganic pyrophoaphate (PP) as substrate and the Km value was 0.7677 +/- 0.0528 mM. Full activation of the enzyme was achieved with a Mg2+/PPi ratio of 2. Divalent metal ions such as Ca2+, Cu2+, and Zn2+ inhibited PPase activity. Mg2+, partially relieved the inhibition caused by adenosine 5'-triphosphate. Studies related to the localization of alkaline PPase in microbodies revealed that the enzyme was distributed between glyoxysomes and mitochondria, with the former containing more of it.
Hemalatha
K P J
KP
Department of Biochemistry, Andhra University, Andhra Pradesh, India.
Prasad
D Siva
DS
D000227:Adenine Nucleotides / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D018548:Cotyledon / Q000201:enzymology / Q000502:physiology; D003576:Electron Transport Complex IV / Q000378:metabolism; D004795:Enzyme Stability; D018525:Germination; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase; D007425:Intracellular Membranes / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology; D015388:Organelles / Q000201:enzymology; D011489:Protein Denaturation; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000201:enzymology / Q000502:physiology; D020539:Sequence Analysis, Protein; D031685:Sesamum / Q000201:enzymology*; D013379:Substrate Specificity
D000227:Adenine Nucleotides; D000596:Amino Acids; D000255:Adenosine Triphosphate; D003576:Electron Transport Complex IV; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium