Purification of apyrase from yellow lupin cotyledons after extraction with perchloric acid.
Neutralized 1 M perchloric acid (PCA) extracts of yellow lupin (Lupinus luteus) seedling cotyledons contain considerable amounts of apyrase (EC 3.6.1.5). Investigators who use PCA extraction for the estimation of nucleotide levels, particularly in plant tissues, should be aware of this danger. Only when the material is treated with 1.8-2 M PCA are the extracts obtained free of apyrase activity. Chromatography of neutralized 1 M extracts obtained from 7-day-old seedling cotyledons on DEAE-Sephacel and Sephadex G-100 yields almost homogeneous apyrase that shows a band of M(r) 51,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The molecular weight of the native enzyme is also about 51,000. The apyrase preparation is free of nonspecific phosphatases, nucleotidases, and adenosine nucleosidase, as well as dinucleoside polyphosphate-degrading enzymes. The apyrase exhibits a broad pH optimum between 6 and 8. Mg2+ and Ca2+ are required for maximum activity; Zn2+ and Mn2+ are less effective and Co2+, Ni2+, and Cd2+ are without effect. The Km values for ATP and ADP are about 20 microM. All common 5'-nucleoside tri- and diphosphates as well as adenosine 5'-tetraphosphate are substrates.
Guranowski
A
A
Katedra Biochemii, Akademia Rolnicza, Poznań, Poland.
Starzyńska
E
E
Rataj-Guranowska
M
M
Günther Sillero
M A
MA
D000227:Adenine Nucleotides / Q000032:analysis; D001081:Apyrase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010472:Perchlorates; D010944:Plants / Q000737:chemistry / Q000201:enzymology*; D013379:Substrate Specificity
D000227:Adenine Nucleotides; D002413:Cations, Divalent; D010472:Perchlorates; D001081:Apyrase