Expression of rat phosphoribosylpyrophosphate synthetase subunits I and II in Escherichia coli. Isolation and characterization of the recombinant isoforms.
The 34-kDa subunit of rat liver phosphoribosylpyrophosphate synthetase is a mixture of the two highly homologous isoforms, PRS I and PRS II. Heretofore, it was not possible to separate the two. We now describe isolation and characterization of the recombinant isoforms, named rPRS I and rPRS II. The respective rat cDNAs were inserted into vectors constructed from pKK233-2 by replacing its replication origin with that of pGEM-1 and expressed in Escherichia coli. The rPRS I and rPRS II were purified to apparent homogeneity with specific activities of 33,400 and 46,200 milliunits/mg, respectively; these values were at least 2.5-fold higher than the highest value for the mammalian enzyme so far reported. Both isoforms showed a similar dependency on Pi as an absolute activator. Sulfate partially substituted for Pi. The maximal activities of rPRS I and rPRS II with sulfate were 43 and 7%, respectively, of those seen with Pi. The two isoforms differed in sensitivity to inhibition by ADP and GDP. Inhibition of rPRS I and rPRS II by 0.3 mM ADP was 87 and 54%, respectively, and inhibition by 1 mM GDP was 93 and 24%, respectively. rPRS II was 180-fold more sensitive than rPRS I to heat inactivation at 49 degrees C.
Ishijima
S
S
Department of Biochemistry, Chiba University School of Medicine, Japan.
Kita
K
K
Ahmad
I
I
Ishizuka
T
T
Taira
M
M
Tatibana
M
M
D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012268:Ribose-Phosphate Pyrophosphokinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013464:Sulfuric Acids / Q000737:chemistry
D007527:Isoenzymes; D011994:Recombinant Proteins; D013464:Sulfuric Acids; D004247:DNA; D012268:Ribose-Phosphate Pyrophosphokinase