DINUCLEOSIDE 5',5'''-P-1,P-3-TRIPHOSPHATE HYDROLASE FROM YELLOW LUPIN(LUPINUS-LUTEUS) SEEDS - PURIFICATION TO HOMOGENEITY AND HYDROLYSIS OF MESSENGER-RNA 5'-CAP ANALOGS
A. Guranowski et al., DINUCLEOSIDE 5',5'''-P-1,P-3-TRIPHOSPHATE HYDROLASE FROM YELLOW LUPIN(LUPINUS-LUTEUS) SEEDS - PURIFICATION TO HOMOGENEITY AND HYDROLYSIS OF MESSENGER-RNA 5'-CAP ANALOGS, Protein expression and purification, 8(4), 1996, pp. 416-422
Three separable forms of diadenosine 5',5 triple prime-P-1,P-3-triphos
phate (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 fur
ther purification by gel filtration on Sephadex G-200 and by affinity
elution from an AMP-agarose column yielded homogeneous protein as demo
nstrated on SDS-polyacrylamide gel electrophorograms. The enzyme is a
single polypeptide chain of M(r) 41 kDa. Eleven guanosine-containing d
inucleoside triphosphates, including analogs of the mRNA 5'-cap struct
ure, have been tested as potential substrates of the lupin ''Ap(3)A hy
drolase.'' All have been hydrolyzed yielding mixtures of corresponding
nucleoside mono- and diphosphates. Asymmetrical compounds gave four p
roducts; m(7)Gp(3)G, et(7)Gp(3)G, and bz(7)Gp(3)G were hydrolyzed rand
omly whereas m(7)Gp(3)A, m(7)Gp(3)C, and m(7)Gp(3)U yielded at least 8
0% m(7)GMP plus corresponding NDP and 20% or less NMP plus m(7)GDP. Hy
drolysis 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 th
an Ap(3)A. Thus a general name, ''dinucleoside triphosphate hydrolase,
'' is more appropriate for the enzymatic activity described. (C) 1996
Academic Press, Inc.