Dinucleoside 5',5'''-P-1,P-3-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 triple prime-P-1,P-3-triphosphate (Ap(3)A)-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 concerting 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 ''Ap(3)A hydrolase.'' All have been hydrolyzed yielding mixtures of corresponding nucleoside mono- and diphosphates. Asymmetrical compounds gave four products; m(7)Gp(3)G, et(7)Gp(3)G, and bz(7)Gp(3)G were hydrolyzed randomly whereas m(7)Gp(3)A, m(7)Gp(3)C, and m(7)Gp(3)U yielded at least 80% m(7)GMP plus corresponding NDP and 20% or less NMP plus m(7)GDP. Hydrolysis of the guanosine-containing hybrids, Ap(3)G, Cp(3)G, and Up(3)G, yielded at least 85% GMP plus corresponding NDP. All dinucleotides containing the m(7)G- moiety were hydrolyzed 2- to 4.5-fold faster than Ap(3)A. Thus a general name, ''dinucleoside triphosphate hydrolase,'' is more appropriate for the enzymatic activity described. (C) 1996 Academic Press, Inc.