Tomoko Sonoda

Metabolic fate of pyrimidines and purines in dietary nucleic acids ingested by mice.

1. In order to study the metabolism and tissue utilization of pyrimidines or purines ingested as dietary nucleic acid components, [14C]uracil, [14C]cytosine-labeled RNA, [14C]thymine-labeled DNA, or [14C]adenine-labeled RNA was fed to mice. 2. Absorption and catabolism of each ingested radioactive material were rapid; 80% or more of the ingested radioactivity was excreted as catabolic products over an 8-h period. 3. Utilization of the ingested radioactive materials for tissue synthesis of nucleic acids was limited under the usual conditions, the extent being 2--5%, 4 h after feeding. Such acid-insoluble radioactivity was localized principally in gastrointestinal tissue, and much lesser amounts, albeit significant, were found in the liver. 4. With increase in the dose of dietary nucleic acids, the amounts of utilized (nucleic acids and nucleotides) and utilizable (nucleosides and free bases) forms of uracil and cytosine and of adenine were increased in all tissues examined. Relationship between the dose and utilization together with additional findings support the view that gastrointestinal tissue and the liver utilize and degrade a greater part of the exogenous nucleic acid bases before their entry into the systemic circulation. 5. The metabolism of DNA thymine was unique in that it was significantly utilized for DNA synthesis in tissues other than the gastrointestinal tissue and liver to a comparative extent. The spleen was particularly active in this respect, and the hyperplastic, hematopoietic spleen was three times more active than the normal spleen. 6. Principal components of partially digested products in the intestinal lumen 1 h after the ingestion were uridine (33%) and cytidine (22%) in the case of [14C]uracil, [14C]cytosine-labeled RNA and inosine (53%) in the case of [14C]adenine-labeled RNA, in accordance with the view that purines and pyrimidines in nucleic acids are absorbed mainly in the form of nucleosides.

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.

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.

Enzyme Regulation of N-Acetylglutamate Synthesis in Mouse and Rat Liver

Ṉ-Acetylglutamate (AGA) synthetase of mammalian liver is known to be stimulated by low concentrations of arginine. The arginine sensitivity of the synthetase was found to show postprandial changes in the liver of DD-Y mice. AGA synthetase activity assayed in the absence of arginine changed only slightly during and after the feeding. With 1 mM arginine, the activity increased and reached a peak value 9 h after the start of feeding. The activation ratios were about 2 and 6 at 0 and 9 h, respectively. Similar changes occurred with 0%, 20%, and 60% casein diets. When the enzymes were partially purified, the respective activation ratios remained the same, suggesting no involvement of a readily dissociable low molecular weight compound. Treatment of mice with cycloheximide did not abolish the increase in the activation ratio. A homogeneous preparation of the synthetase was obtained by a 30,000-fold purification from sonicated mitoplasts of rat liver mitochondria. The molecular weight was 160,000, as estimated on sucrose density gradient centrifugation, with subunits of 57,000 on SDS-gel electrophoresis. The enzyme had a hydrophobic nature, was stabilized by Triton X-100, and contained little phospholipid. Although the molecular basis of the increase in the activation ratio remains to be established, the modification of the nature of AGA synthetase introduces another aspect into the elaborate regulation of urea synthesis.

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.

An improved method for determination of N-acetyl-l-glutamate by its function as an activator of carbamoyl phosphate synthetase I

N-Acetyl-L-glutamate has been examined with regard to its ability to activate carbamoyl phosphate synthetase I (EC 6.3.4.16). Substance(s) inhibitory to carbamoyl phosphate synthetase, present even in the partially purified preparation of rat liver extracts, interfered with the measurement of acetylglutamate. In the experiments using chelating agents, metals were apparently involved in this inhibition. When the partially purified preparation of liver extract was placed on a Chelex 100 column, the inhibitor was eliminated and accurate measurements of acetylglutamate content could be made. Evidence supporting the validity of this improved method is given. A significant difference was observed between acetylglutamate levels determined by the present method and by the one using aminoacylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) to hydrolyze acetylglutamate followed by assay of the glutamate generated. We searched for the presence of glutamate derivatives other than acetylglutamate. When impure tissue preparations containing acetylglutamate were treated with a commercial preparation of aminoacylase, there was an excess amount of glutamate apparently derived from compounds other than acetylglutamate. This can lead to an overestimation of the tissue levels of acetylglutamate.

The synaptic scaffolding protein Delphilin interacts with monocarboxylate transporter 2.

Delphilin, which interacts with a glutamate receptor (GluR) δ2-subunit, is a postsynaptic density scaffolding protein at the cerebellar parallel fiber-Purkinje cell synapses. Delphilin specifically interacts with the GluRδ2 C-terminus via its postsynaptic density-95/discs-large/ZO-1 (PDZ) domain. As a number of PDZ-containing scaffolding proteins bind to several membrane proteins, we expected that Delphilin might also have other binding partners besides GluRδ2. To search for the link between Delphilin and other binding proteins, we carried out screening among candidate membrane proteins localized in Purkinje cells by surface plasmon resonance analyses. As a result, we found that the C-terminus of the monocarboxylate transporter 2 binds specifically and significantly with Delphilin PDZ and there is a probable existence of GluRδ2-Delphilin-monocarboxylate transporter 2 complex in synaptic membranes.