Sumio Ishijima

Mammalian phosphoribosyl-pyrophosphate synthetase

PRPP synthetase from rat liver exists as large molecular weight aggregates composed of at least three different components. Cloning of cDNA for the catalytic subunit revealed the presence of two highly homologous isoforms of 34 kDa, designated as PRS I and PRS II. Northern blot analysis showed tissue-differential expression of the two isoform genes. cDNA was expressed in E. coli and studies on the recombinant isoforms showed differences in sensitivity to inhibition by ADP and GDP and to heat inactivation. The rat gene for PRS I has 22 kb and is split into 7 exons. cDNAs for human enzymes were also cloned. Human genes for PRS I and PRS II are localized at different regions on the X-chromosome and their promoter regions were examined. Another component, PRPP synthetase-associated protein of 39 kDa (PAP39), was cloned from cDNA library of the rat liver. The deduced amino acid sequence of PAP39 is remarkably similar to those of PRS I and PRS II. Evidence indicated molecular interaction between PAP39 and the catalytic subunits and an inhibitory effect of PAP39 on the catalytic activity. Expression of the PAP39 gene is tissue-differential like the PRS genes, indicating that the composition of PRPP synthetase may differ with the tissue, hence properties of the enzyme would differ. Further studies on these components and their interaction are expected to reveal various mechanisms governing mammalian PRPP synthetase.

Molecular cloning and sequencing of human cDNA for phosphoribosyl pyrophosphate synthetase subunit II

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit II (PRS II) were isolated. The five overlapping clones contained 2457 base pairs (bp) covering a 954‐bp complete coding region for 318 amino acid residues. Homologies between human and rats PRS II were 99% of the amino acid and 88% of the nucleotides in the coding region. This amino acid homology seems to be the highest so far reported for enzymes involved in nucleotide metabolism and glycolysis. The highly conserved structure may be required for unique catalysis and rigid regulation of this enzyme.

Protease activation following UV irradiation is linked to hypomutability in human cells selected for resistance to combination of UV and antipain

In order to examine the relationship between activation of an antipain-sensitive protease and suppression of mutability in UV (UVC)-irradiated human cells, a human cell variant with the high protease activity induced by UV was established and characterized for its susceptibility to UV-induced mutagenicity. Cells of a hypermutable cell strain, RSa, were mutagenized with ethyl methanesulfonate and irradiated with 10 J/m2 UV, followed by exposure to 20 mM antipain for 34 h. Whereas the combined treatment was totally lethal to RSa cells not treated with ethyl methanesulfonate, one surviving clone was isolated from the mutagenized cells and designated UVAP-1. When fibrinolytic protease activity was measured from extracts of the cell, it was found that the protease activity was elevated promptly after UV irradiation, reaching the maximum at 10 min post-irradiation. This protease activity was inhibited by antipain. After UV irradiation the phenotypic mutation frequencies of UVAP-1 cells were much lower than those of the parent RSa cells, as evaluated by the generation of clones resistant to ouabain-killing. Furthermore, mutation at the K-ras codon 12 in genomic DNA was detected in RSa cells but not in UVAP-1 cells. Thus, the protease activation was correlated with the decreased levels of UV-mutagenicity in UVAP-1 cells, supporting the possible involvement of the antipain-sensitive protease activity in the regulation of cellular mutability following UV irradiation.

Cloning and sequencing of rat cDNA for the 41-kDa phosphoribosylpyrophosphate synthetase-associated protein has a high homology to the catalytic subunits and the 39-kDa associated protein

Rat liver phosphoribosylpyrophosphate synthetase is a complex aggregate of 34-kDa catalytic subunits (PRS I and II) and 39- and 41-kDa associated proteins (PAP39 and 41). When the rat cDNA encoding PAP41 was isolated, the deduced protein sequence was seen to contain 369 amino acids with a calculated molecular mass of 41130. PAP41 has a 79 and 49% identity with PAP39 and PRSs, respectively. When conservative substitutions are included, PAP41 and the three other components have a 66% homology. PAP41 shares some common features with PAP39 and the two proteins form the PAP subfamily. The mRNA of PAP41 is present in all rat tissues we examined.

Rat liver phosphoribosylpyrophosphate synthetase is activated by free Mg2+ in a manner that overcomes its inhibition by nucleotides

Phosphoribosylpyrophosphate synthetase is activated by Pi and free Mg2+ as an essential activator and inhibited by nucleotides, especially ADP and GDP. The rat liver enzyme is a complex aggregate of two highly homologous catalytic subunits (PRS I and PRS II) and two associated proteins (PAP39 and PAP41). PRS I is more sensitive to inhibition by ADP and GDP than is PRS II. The native liver enzyme showed a weaker sensitivity to inhibition by nucleotides than expected from its composition. To further understand the regulation of the liver enzyme, kinetic studies of each subunit component and the liver enzyme regarding Mg2+ activation and inhibition by ADP and GDP were carried out. Assay conditions were designed to keep free Mg2+ at constant concentrations. (1) GDP, as MgGDP, did not affect the apparent Km values of PRS I for MgATP and ribose-5-phosphate but did dramatically increase the apparent Ka value for free Mg2+. (2) In contrast, ADP, as MgADP, increased the Km value for MgATP of PRS I as well as the Ka value for free Mg2+. (3) High concentrations of free Mg2+ almost completely nullified the inhibitory effect of MgGDP and partly that of MgADP on PRS I. (4) At low free Mg2+ concentrations within the physiological range, inhibition by the nucleotides is of physiological significance and conversely, variation in free Mg2+ concentrations critically affects the enzyme activity in the presence of inhibitory nucleotides. (5) The response of PRS II and the native liver enzyme is similar to that of PRS I, while the effects of MgGDP and MgADP were smaller than that on PRS I. (6) We propose that MgGDP binds to a regulatory site of PRS I and PRS II and MgADP to the substrate MgATP site and also the regulatory site. The allosteric interaction of the regulatory site and the Mg2+ binding site is also considered.

Cloning and sequencing of human complementary DNA for the phosphoribosylpyrophosphate synthetase-associated protein 39☆

A human cDNA encoding a human homologue of the rat phosphoribosylpyrophosphate synthetase-associated protein of 39 kDa was isolated. The deduced protein contains 356 amino acids and has calculated molecular mass of 38561. The amino acid sequence is 98% identical to that of the rat. The corresponding mRNA is present in all human tissues examined.

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.

Identification of amino-acid residues linked to different properties of phosphoribosylpyrophosphate synthetase isoforms I and II

The catalytic subunit of rat liver phosphoribosylpyrophosphate synthetase is composed of two isoforms, PRS I and PRS II. The amino-acid sequences differ only by 13 residues, out of which two Lys residues of PRS I at positions 4 and 152 give net additional positive charges to PRS I. Previous work has shown that PRS I is more sensitive to inhibition by ADP and GDP and more stable to heat treatment than is PRS II. To identify amino-acid residues responsible for the different properties, five chimeric enzymes between rat PRS I and PRS II and two mutated enzymes with a single point mutation at position 152 were constructed; these enzymes were produced in Escherichia coli. Changing Lys-4 of PRS I to Val, together with Ile-5 to Leu, completely abolished sensitivity to GDP inhibition of PRS I, indicating that Lys-4 in PRS I is critical for GDP inhibition. The substitutions at position 152 had little effect on GDP inhibition. Characterization of the chimeric enzymes revealed that residues between residues 54-110 and 229-317, namely, Val-55 and/or Ala-81, and Arg-242 and/or Cys-264 of PRS I also contribute to the strong GDP inhibition. Lys-4 was also important for the strong ADP inhibition of PRS I. Regarding the physical properties, chimeric enzymes bearing residues 12-53 of PRS I were stable at 49 degrees C and with digestion with papain and proteinase K. Our observations suggest that Lys-17, Ile-18, and/or Cys-40 of PRS I contribute to stability of the enzyme.

Purification and Characterization of Recombinant Rat Phosphoribosylpyrophosphate Synthetase Subunit I and Subunit II

Phosphoribosylpyrophosphate (PRPP) synthetase catalyzes the formation of PRPP from ATP and ribose 5-phosphate. The enzyme has been purified from bacteria [1, 2] and mammalian tissues [3–5]. The rat liver enzyme exists as complex aggregates of 34-, 38-, and 40-kDa components, the 34-kDa species being the catalytic subunit [5]. The 34-kDa component is actually a mixture of two isoforms, designated as PRS I and PRS II [5, 6]. The two isoforms are composed of 317 amino acid residues, and the sequences are highly conserved, differing only by 13 residues [6]. Furthermore, the amino acid sequences of human [7, 8] and rat [6] PRS II differ only by 3 residues and those of human [8, 9] and rat [6] PRS I are completely conserved. The PRS I and PRS II are encoded by two distinct genes located on X-chromosome [10, 11]. The two genes are expressed in almost all tissues of rats but the mRNA levels differ with the tissues [12]. These observations suggest functional differences between catalytic and/or regulatory properties of PRS I and PRS II. However, separation of the two proteins from the native enzyme was impossible. Therefore, the respective rat cDNAs were expressed in Escherichia coli. The expression vector was designed to produce the unfused proteins. The recombinant isoforms (named rPRS I and rPRS II) were isolated and characterized.

Early mitogenic stimulation of metabolic flux through phosphoribosyl pyrophosphate into nucleotides in Swiss 3T3 cells and requirement of external Magnesium for the response

5-Phosphoribosyl 1-pyrophosphate (PRPP) is a common precursor for the synthesis of all nucleotides and also serves as a critical regulator for the synthesis. In spite of a number of studies in vitro on mammalian PRPP synthetase, our understanding of the regulation of PRPP synthesis in situ is very limited. Various mitogens are known to activate purine and pyrimidine de novo biosynthesis and purine base phosphoribosylation as an early response in quiescent mouse fibroblasts. We aimed at elucidation of the underlying mechanism for the possible increase in PRPP synthesis in mitogen-stimulated mouse fibroblasts in culture. In order to quantitatively follow metabolic flux through PRPP into nucleotides, [ribosyl-14C]inosine was enzymatically prepared and used as a tracer to preferentially label intracellular ribose phosphate. The radioactivity incorporation into cellular nucleotides was measured. Evidence supported the validity of the method. Prior exposure of quiescent Swiss 3T3 cells in culture to epidermal growth factor (EGF) plus insulin for 45-60 min enhanced approximately 2-fold the radioactivity incorporation from [ribosyl-14C]inosine into nucleotides, without increasing the specific radioactivity of intracellular free ribose 5-phosphate. [14C]Uracil incorporation into nucleotides, a measure for PRPP-independent ribose phosphate utilization for nucleotide synthesis, was not increased. These and other results indicate that EGF plus insulin stimulates the metabolic flux through PRPP. A similar stimulation was induced by bombesin and melittin in combination with insulin and by fibroblast growth factor alone. Quiescent Swiss 3T6 cells and human fetal fibroblasts showed a similar stimulation of nucleotide synthesis in response to exposure to serum. For characterization of intracellular signaling pathways, we examined effects of several inhibitors and agents on the stimulation. The divalent cation ionophore A23187 mimicked the response to EGF and insulin in Swiss 3T3 cells, thereby suggesting involvement of divalent cation mobilization in this increase. The effect of the ionophore was not additive to that of the growth factors. Omission of Ca2+ from the incubation medium did not affect the response to EGF and insulin, whereas the omission of Mg2+ did abolish the response. Furosemide, an inhibitor of Mg2+ influx, partially inhibited the stimulated synthesis of nucleotides. Thus, the entry of external Mg2+ into the cells may play a critical role in this signal transduction. These results provided an important access to elucidation of the intracellular mechanisms for the mitogen-induced increase in PRPP and nucleotide syntheses.