Partial reconstitution of mammalian phosphoribosylpyrophosphate synthetase in Escherichia coli cells. Coexpression of catalytic subunits with the 39-kDa associated protein leads to formation of soluble multimeric complexes of various compositions.
Rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of 34-kDa catalytic subunits (PRS I and PRS II) and homologous 39- and 41-kDa proteins termed PRPP synthetase-associated proteins (PAPs). While a negative regulatory role was indicated for PAPs, the physiological function of PAPs is less well understood. We attempted to prepare recombinant 39-kDa PAP (PAP39) and to reconstitute the enzyme complex. Free PAP39 was poorly expressed in Escherichia coli, while expression of protein fused with glutathione S-transferase was successful. The purified fusion protein had no PRPP synthetase activity, and bound to dissociated PRS I and PRS II, with a similar affinity. A free form of PAP39 prepared from the fusion protein formed insoluble aggregates. The enzyme complex was then partially reconstituted in situ by coexpression of PAP39 with PRS I or PRS II in E. coli cells. This coexpression led to formation of soluble complexes of various compositions, depending on the conditions. When the relative amount of PAP39 was higher, specific catalytic activities, in terms of the amount of the catalytic subunit, were lowered. PAP39 complexed with PRS I was more readily degraded by proteolysis than seen with PRS II, in vivo and in vitro. These results provide additional, strong evidence for that PAP39 has no catalytic activity in the enzyme complex, but does exert inhibitory effects in an amount-dependent manner, and that composition of the enzyme complex varies, depending on the relative abundance of components present at the site of aggregate formation.
Ishijima
S
S
Department of Biochemistry, Chiba University School of Medicine, Japan. isijima@med.m.chiba-u.ac.jp
Asai
T
T
Kita
K
K
Sonoda
T
T
Tatibana
M
M
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000096:biosynthesis; D006153:Guanosine Diphosphate / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008322:Mammals; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012268:Ribose-Phosphate Pyrophosphokinase / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility
D046911:Macromolecular Substances; C086305:PAP39; D011506:Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D005982:Glutathione Transferase; D012268:Ribose-Phosphate Pyrophosphokinase