Rat liver phosphoribosyl pyrophosphate synthetase: existence of the purified enzyme as heterogeneous aggregates and identification of the catalytic subunit.
Mammalian phosphoribosyl pyrophosphate (PRPP) synthetase has been extensively investigated. However, considerable ambiguity remains concerning its physical and regulatory properties. We purified PRPP synthetase from rat liver and studied some of the physical properties, in parallel with cloning experiments (Taira, M. et. al. [1987] J. Biol. Chem. 262, 14867-14870). 1) The enzyme was purified to a specific activity of 7,280 milliunits/mg, the highest value in the literature for a mammalian PRPP synthetase. The apparent molecular mass was over 1,000 kDa. 2) The final preparation contained 34-kDa, 38-kDa, and 40-kDa protein species, as analyzed by SDS gel electrophoresis. 3) Further attempts at separation using conventional procedures only led to a co-purification of the components. Thus, the purified enzyme appears to exist as complex aggregates composed of heterogeneous components. 4) Gel filtration of the enzyme in the presence of 1 M MgCl2 isolated part of the 34-kDa component, free of other species. The preparation was catalytically active, indicating that this component is the catalytic subunit. 5) The amino acid composition of the 34-kDa subunit and the amino acid sequences of its N-terminal region and of two tryptic peptides were determined. The results are in accord with the results of cDNA analyses.
Kita
K
K
Department of Biochemistry, Chiba University School of Medicine.
Otsuki
T
T
Ishizuka
T
T
Tatibana
M
M
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002384:Catalysis; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006882:Hydroxyapatites; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D008297:Male; D010770:Phosphotransferases / Q000032:analysis*; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D012268:Ribose-Phosphate Pyrophosphokinase / Q000032:analysis* / Q000302:isolation & purification
D000596:Amino Acids; D006882:Hydroxyapatites; D011092:Polyethylene Glycols; D010770:Phosphotransferases; D012268:Ribose-Phosphate Pyrophosphokinase