Dinucleoside 5', 5"'-P1, P3-triphosphate hydrolase from yellow lupin (Lupinus luteus) seeds: purification to homogeneity and hydrolysis of mRNA 5'-cap analogs.
Three separable forms of diadenosine 5',5"'-P1, P3-triphosphate (Ap3A)-degrading activity were revealed when proteins obtained from the meal of yellow lupin seeds by ammonium sulfate precipitation were chromatographed on a DEAE-Sephacel column. The major form, which eluted first at the lowest salt concentration (0.15 M KCl), was free of any activity converting the reaction products, ADP and AMP. Its further purification by gel filtration on Sephadex G-200 and by affinity elution from an AMP-agarose column yielded homogeneous protein as demonstrated on SDS-polyacrylamide gel electrophorograms. The enzyme is a single polypeptide chain of M(r) 41 kDa. Eleven guanosine-containing dinucleoside triphosphates, including analogs of the mRNA 5'-cap structure, have been tested as potential substrates of the lupin "Ap3A hydrolase." All have been hydrolyzed yielding mixtures of corresponding nucleoside mono- and diphosphates. Asymmetrical compounds gave four products; m7Gp3G, et7Gp3G, and bz7Gp3G were hydrolyzed randomly, whereas m7Gp3A, m7Gp3C, and m7Gp3U yielded at least 80% m7GMP plus corresponding NDP and 20% or less NMP plus m7GDP. Hydrolysis of the guanosine-containing hybrids, Ap3G, Cp3G, and Up3G, yielded at least 85% GMP plus corresponding NDP. All dinucleotides containing the m7G-moiety were hydrolyzed 2- to 4.5-fold faster than Ap3A. Thus a general name, "dinucleoside triphosphate hydrolase," is more appropriate for the enzymatic activity described.
Guranowski
A
A
Department of Biochemistry and Biotechnology, Agricultural University, Poznań, Poland.
Starzyńska
E
E
Bojarska
E
E
Stepiński
J
J
Darzynkiewicz
E
E
D017766:Acid Anhydride Hydrolases; D004591:Electrophoresis, Polyacrylamide Gel; D009363:Neoplasm Proteins; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D012314:RNA Cap Analogs / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012639:Seeds; D000099170:Bis(5'-adenosyl)triphosphatase
D017766:Acid Anhydride Hydrolases; D009363:Neoplasm Proteins; D010727:Phosphoric Diester Hydrolases; D012314:RNA Cap Analogs; D012333:RNA, Messenger; D000099170:Bis(5'-adenosyl)triphosphatase