Role of adenine phosphoribosyltransferase in adenine uptake in wild-type and APRT− mutants of CHO

Adenine uptake in cultured Chinese hamster fibroblasts showed biphasic saturation kinetics. The transport system was highly specific for adenine and was competitively inhibited by adenosine. Utilizing mutant clones of Chinese hamster fibroblasts that have either reduced or negligible adenine phosphoribosyltransferase (APRT) activity, we found that (1) adenine was not accumulated against a concentration gradient in the absence of APRT activity and (2) after rapid initial uptake equal to that of the parent the rates of adenine accumulation found for the mutants correlated strongly with their residual APRT activities. Furthermore, using either artificially depressed phosphoribosylpyrophosphate pool size and APRT activities or the mutants with decreased APRT activity, we found that adenine transport was independent of phosphorylation by APRT. These studies suggest that adenine is transported as the free base by facilitated diffusion and is subsequently phosphorylated by APRT.     

Characterization of the biochemical basis of a complete deficiency of the adenine phosphoribosyl transferase (APRT)

Summary In order to study the biochemical basis of a complete deficiency of adenine phosphoribosyl transferase (APRT) the enzyme was purified to homogeneity, its properties were characterized, and antibodies raised. The enzyme is indirectly involved in adenine uptake. Apparently, by forming AMP the internal concentration of adenine is kept low allowing its diffusion.The same APRT is present in various tissues as was revealed by antibody inactivations employing anti-erythrocyte APRT as well as by direct enzyme assays in cells from the APRT deficient patient. In vitro cultured fibroblasts derived from this patient had less than 0.02% enzyme activity. No cross-reacting material was found in erythrocytes obtained from an APRT deficient child.     

Cloning and expression of a mouse adenine phosphoribosyltransferase gene

A functional mouse adenine phosphoribosyltransferase (APRT) gene was identified and cloned by screening a mouse sperm genomic DNA library in lambda Charon 4A. The probe utilized for screening was a restriction fragment encoding much of the hamster APRT gene. Six recombinants that hybridized with the probe were identified, and after digestion with restriction enzymes EcoRI and PvuII revealed three different patterns of digestion for each enzyme. Of the six recombinants, five representing two of the restriction patterns possessed transforming activity. A sixth recombinant, which has a unique restriction pattern, lacks transforming activity but hybridizes well with hamster APRT coding sequences and is a possible candidate for a pseudogene. We used three criteria for conclusively identifying the mouse APRT genes. (1) DNA from the recombinant lambda phage hybridizes with DNA encoding hamster APRT. (2) The recombinant lambda phages and their DNAs transform mouse, hamster and human APRT- cells to the APRT+ phenotype. (3) The hamster and human transformants display APRT activity that migrates with a mobility characteristic of mouse APRT and not of hamster or human. A 3.1-kb EcoRI-SphI restriction fragment which retains transforming activity has been subcloned into the plasmid pBR328. Comparison of restriction enzyme sites with those contained in a mouse APRT cDNA, coupled with loss of transforming activity after enzyme digestion, indicates that the mouse APRT gene is larger than 1.8 kb and contains at least three introns.     

Adenine salvage activity during callus induction and plant growth

Adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) activity was monitored in crude extracts of Arabidopsis thaliana tissues and callus. Changes in APRT activity during germination were determined within different organs of the mature plant and during callus induction. APRT activity was constitutively expressed in all organs examined. There was an increase in APRT activity detected in seeds beginning 3 days following imbibition, after which the level decreased to that found in leaf tissue of mature plants. There was also an increase in APRT activity early during callus induction. A mutant that lacks APRT activity had a diminished capacity for callus induction in both the presence and absence of exogenous cytokinin. The results are consistent with the hypothesis that an increase in APRT activity is associated with actively dividing cells. The significance of these observations is discussed relative to the role of APRT in adenylate and cytokinin metabolism during plant development.     

Partial and complete adenine phosphoribosyltransferase deficiency associated with 2,8-dihydroxyadenine urolithiasis: kinetic and immunochemical properties of APRT

We have studied adenine phosphoribosyltransferase (APRT) in the hemolysates from the families of 2,8-dihydroxyadenine urolithiasis associated with partial deficiency of APRT (the Japanese type) and complete deficiency of APRT (the null type). The APRT in the control subjects was found to be heat-stable at the physiological concentration of phosphoribosylpyrophosphate (PRPP), which was close to the value of its Km for PRPP. The APRT in the Japanese type showed 10 times higher Km values for PRPP and needed a comparably increased level of PRPP for stability in vitro. No change in red cell PRPP was found in the Japanese type of APRT deficiency. The content of APRT enzyme protein was decreased in the hemolysates of the Japanese type, probably due to its lability at the level of PRPP present in the cells. The heterozygote of the null type also had labile enzyme molecules at the physiological PRPP concentration.     

Detection of an amino acid substitution in the mutant enzyme for a special type of adenine phosphoribosyltransferase (APRT) deficiency by sequence-specific protein cleavage.

Generally, if mutant and normal proteins have similar molecular weights and electric charges, they cannot easily be distinguished from one another. We have developed a unique method by which a mutant enzyme of adenine phosphoribosyltransferase (APRT) can easily be distinguished from normal enzyme with nearly identical molecular weight and electric charge. DNA sequencing data have suggested that in this special type of disease (Japanese-type APRT deficiency) there is an amino acid substitution from Met to Thr at position 136 of APRT. Since normal APRT has only one Met residue, the Japanese-type mutant APRT should be a methionine-free protein. Using both an amino acid sequence-specific antiserum against APRT, and specific cleavage of peptide at the methionine residue with BrCN, we could distinguish between normal and mutant proteins. Thus, normal but not mutant APRT was cleaved with BrCN, indicating that the mutant APRT is a methionine-free protein. All tested patients with the Japanese-type APRT deficiency were found to synthesize exclusively methionine-free APRT. Usefulness of this method is not restricted to a single family, as 79% of all the patients with this disease among Japanese, and more than half of all the patients with this disease reported in the world, are likely to have this unique mutation. Thus, not only sequence-specific cleavage of DNA with restriction endonucleases but also that of protein with a chemical agent has been shown to be sometimes useful for the diagnosis and analysis of a genetic disease by careful examination of normal and mutant amino acid sequences.     

Crossovers within a short DNA sequence indicate a long evolutionary history of the APRT*J mutation

Summary Adenine phosphoribosyltransferase (APRT) deficiency causing 2,8-dihydroxyadenine urolithiasis and renal failure is present at a high frequency among the Japanese but not other ethnic groups. A special type of mutant allele, designated APRT*J, with a nucleotide substitution at codon 136 from ATG (Met) to ACG (Thr) is carried by approximately 79% of all Japanese 2,8-dihydroxyadenine urolithiasis patients. We analyzed mutant alleles of 39 APRT deficient patients using a specific oligonucleotide hybridization method after in vitro amplification of a part of the genomic APRT sequence. We found that 24 had only APRT*J alleles. Determination of the haplotypes of 194 APRT alleles from control Japanese subjects and of the 48 different APRT*J alleles indicated that normal alleles occur in four major haplotypes, whereas all APRT*J alleles occur in only two. These results suggest that all APRT*J alleles have a single origin and that this mutant sequence has been maintained for a long period, as calculated from the frequency of the recombinant alleles.     

Rapid method for the diagnosis of partial adenine phosphoribosyltransferase deficiencies causing 2,8-dihydroxyadenine urolithiasis

Summary More than half of the Japanese patients with 2,8-dihydroxyadenine urolithiasis only partially lack adenine phosphoribosyltransferase (APRT), while all the Caucasian patients with the same disease completely lack the enzyme. APRT activities in healthy heterozygotes for the complete APRT deficiencies were at the same levels as the Japanese patients, and simple enzyme assay does not distinguish between these two conditions. We have previously shown, using viable T-cells, that the enzyme was non-functional in the cells from the Japanese patients although they contain considerable APRT activities in the cell extracts. In the present investigations, we devised a rapid method using erythrocytes for the diagnosis of partial APRT deficiencies accompanied by severe impairment in adenine metabolism causing 2,8-dihydroxyadenine lithiasis. Thus, erythrocytes from three different families with 2,8-dihydroxyadenine urolithiasis associated with partial APRT deficiencies incorporated only minimal amounts of radioactive adenine, while normal erythrocytes incorporated significant amounts. These data indicate that severe impairment in adenine metabolism is shown not only in viable T-cells but also in viable erythrocytes. The present procedures provide a rapid method suitable for routine clinical use for the diagnosis of partial APRT deficiencies causing 2,8-dihydroxyadenine lithiasis.     

水稻基因APRT的克隆及其与温敏核雄性不育的关系

在拟南芥中腺嘌呤磷酸核糖转移酶基因(APRT)突变导致植株雄性不育.本文首次报道从水稻(Oryza sativa subsp.indica)中克隆了基因APRT(GenBank登录号AY238894),并将其定位于水稻第4染色体的一个BAC克隆(AL606604)的58 000 bp至63 000 bp区域.该基因长4 220 bp(起始密码子至终止密码子),含7个外显子、6个内含子,编码的APRT蛋白长212个氨基酸残基,与其他物种来源的APRT序列存在很高的同源性.与大麦、小麦、拟南芥1型及其2型的该蛋白同源性分别为54.9%、54.9%、49.6%和59.5%.经保守结构域搜索发现该蛋白中存在APRT催化结构域.从DNA、mRNA两个水平分析了该基因与水稻温敏核雄性不育(TGMS)的关系,结果表明:受温度诱导,水稻"安农S-1"APRT基因的表达变化可能与温敏核雄性不育表现型具相关性.     

Loss of heterozygosity and base substitution at the APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines.

We determined the nature of mutations occurring at the autosomal APRT locus in mismatch-repair-proficient and -deficient colorectal carcinoma cell lines. The analysis of mutations that result in APRT deficiency in a mismatch-repair-deficient strain of DLD-1 heterozygous for this locus enabled us to measure the rate of loss of the wild-type gene through deletion, recombination, or gene conversion as well as the rate of point mutation. The overall rate of mutation at the APRT locus in DLD-1 was elevated 100-fold compared with the mismatch-repair-proficient colorectal carcinoma cell line SW620. Loss of heterozygosity (LOH) at APRT accounted for only 4 to 9% of mutant strains derived from DLD-1, indicating a rate for these types of events of 4 x 10(-7) to 9 x 10(-7). In SW620 the rate of LOH at APRT was about 10-fold higher. LOH was not found at polymorphic markers within the same chromosome subband as APRT, indicating that only a limited portion of the chromosome was affected by these alterations. Chromosome painting of SWS620 mutants revealed that the loss of APRT occurred together with a substantial portion of the long arm of chromosome 16. Differences in the nature of base substitutions at APRT (e.g., the proportion of mutations resulting from transitions or transversions) in these tumor cell lines were also detected. There was also an important similarity---the presence of a mutant APRT gene with multiple base substitutions that may be the result of some sort of error-prone DNA synthesis.     

The Locus for Human Adenine Phosphoribosyltransferase on Chromosome No. 16

Evidence for assigning the locus determining the structure of adenine phosphoribosyltransferase (APRT) to human chromosome No. 16 is presented. Hybrids of APRT-deficient mouse cells and of human fibroblasts having normal APRT were isolated by fusing the parental cells with Sendai virus, blocking de novo purine nucleotide synthesis with azaserine and selecting for hybrids that could use exogenous adenine. The hybrid clones that were studied had only APRT activity that was indistinguishable from human APRT with regard to electrophoretic migration and reaction with antibodies against the partially purified human enzyme. No. 16 was the only human chromosome consistently present in all of the clones, and in one clone, it was the only human chromosome detected. Selection against hybrid cells with 2,6-diaminopurine (DAP) yielded DAP-resistant survivors that lacked chromosome No. 16. One hybrid that originally had an intact No. 16 yielded adenine-utilizing subclones that lacked No. 16 but had a new submetacentric chromosome. The distribution of centromere-associated heterochromatin and the fluorescence pattern indicated that this chromosome consisted of a mouse telocentric chromosome and the long arm of No. 16. Cells having the submetacentric chromosome had human APRT. Both the enzyme and the chromosome were absent in DAP-resistant derivatives. These results suggest that the structure of APRT is defined by a locus on the long arm of human chromosome No. 16.     

The origin of the most common mutation of adenine phosphoribosyltransferase among Japanese goes back to a prehistoric era

The incidence of adenine phosphoribosyltransferase (APRT) deficiency is higher among Japanese nationals than among other ethnic groups, and the most common mutation (APRT*J, ATG to ACG mutation at codon 136) accounts for 68% of the disease-causing genes among Japanese. To investigate the origin of these mutations, we studied the geographical distribution of the mutant genes in Japan. The APRT*J mutation is distributed nearly uniformly in the four main islands of Japan and Okinawa, suggesting a very early origin. The products of PCR amplification between positions 2344 and 2750 of the genomic APRT sequence were examined by SSCP analysis in random blood samples from Japanese, Korean, and Taiwanese nationals. Among 955 random Japanese blood samples, 7 (0.73%) were heterozygous for the APRT*J mutation, giving a calculated heterozygote frequency of 1.1% among Japanese for the entire APRT deficiency. None of 231 Taiwanese samples contained heterozygotes for the APRT*J mutation, while 2 (0.53%) of 356 Korean samples were heterozygous. In addition to the APRT*J sequence, a total of five variant sequences was found. Sequencing one variant revealed a base substitution in intron 4, suggesting therefore that they are harmless mutations. Since the APRT*J mutation is present in Koreans and Okinawans who share ancestors only before the Yayoi era (third century bc to third century ad), the origin of the APRT*J mutation predates 300 bc. Received: 14 May 1996     

Mutants of cultured chinese hamster cells deficient in adenine phosphoribosyl transferase

Spontaneous mutants of cultured Chinese hamster cells (line CHO) deficient in APRT have been isolated by selection in 8-azaadenine (AA). Loss of APRT activity occurs in two discrete steps. In the first step, about 65% of total activity is lost; in the second step, most or all of the remaining activity is lost. Cells totally deficient in APRT are highly resistant to AA and cannot utilize exogenous adenine as a source of purines for cell growth. Cells partially deficient in the enzyme exhibit resistance to AA intermediate to that of wild type and fully deficient cells. Growth of cells partially deficient in APRT is inhibited in medium containing drug by the presence of large numbers of wild type cells, but cells totally deficient in the enzyme are not inhibited by the presence of either partially deficient or wild type cells.Stepwise loss of APRT activity probably has a genetic origin. The mutants exhibit stable phenotypes, and the frequency of fully deficient cells in a partially deficient population is enhanced by treatment with a mutagen. The rate of spontaneous mutation from partial to total deficiency is 3 ± 0.8 × 10^?7 per cell generation, and reversion from full to partial deficiency can occur. Total lack of APRT activity is recessive to its presence, but the specific activity of the enzyme in hybrid cells depends quantitatively upon the specific activities in the two parents.     

APT1, but Not APT2, Codes for a Functional Adenine Phosphoribosyltransferase in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has two separate genes (APT1 and APT2) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT). However, genetic analysis indicated that only APT1 could code for a complementing activity. Cloning and expression of both the APT1 and APT2 genes in Escherichia coli showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity. Northern and Western blot analyses demonstrated that only APT1 was transcribed and translated under normal physiological conditions in yeast. Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event. We conclude that APT1 is the functional gene in S. cerevisiae and that APT2 is a pseudogene.     

2,8-Dihydroxyadenine Urolithiasis: A Not So Rare Inborn Error of Purine Metabolism

Adenine phosphoribosyltransferase (APRT) deficiency is a rare inherited metabolic disorder that leads to the formation and hyperexcretion of 2,8-dihydroxyadenine (DHA) into urine. The low solubility of DHA results in precipitation and formation of urinary crystals and kidney stones. The disease can be present as recurrent urolithiasis or nephropathy secondary to crystal precipitation into renal parenchyma (DHA nephropathy). The diagnostic tools available, including stone analysis, crystalluria, and APRT activity in red blood cells, make the diagnosis easy to confirm when APRT deficiency is suspected. However, the lack of recognition of this metabolic disorder frequently resulted in a delay in diagnosis and treatment with grave consequences. The early recognition and treatment of APRT deficiency are of crucial importance to prevent irreversible loss of renal function. This review summarizes the genetic and metabolic mechanisms underlying DHA stones formation and chronic kidney disease, along with the issues of diagnosis and management of APRT deficiency. Moreover, we report the mutations in the APRT gene responsible for APRT deficiency in 51 French patients (43 families) including 22 pediatric cases (18 families) among the 64 patients identified in the biochemistry laboratories of Necker Hospital, Paris (1978–2013).     

A mutant allele common to the type I adenine phosphoribosyltransferase deficiency in Japanese subjects

Adenine phosphoribosyltransferase (APRT) deficiency is a genetic disorder which causes 2,8-dihydroxy-adenine urolithiasis. The estimated incidence of heterozygosity in Caucasian and Japanese populations is 1%. Mutant alleles responsible for the disease have been classified as APRT*Q0 (type I) and APRT* (type II). In our previous study, we demonstrated in APRT*J a single common base change which accounts for 70% of the Japanese mutants. The present report describes the analysis of an APRT*Q0 mutation in Japanese subjects. Two nucleotide substitutions common to all seven affected alleles from four unrelated subjects (three homozygotes and a heterozygote) were identified: G----A at nucleotide position 1453 and C----T at 1456. The G----A altered the amino acid Trp98 to a stop codon. The C----T did not alter Ala99. These point mutations were demonstrated by sequence analysis of polymerase chain reaction (PCR)-amplified genomic DNA and cDNA. The G----A change at 1453 results in the elimination of a PflMI site in the APRT gene. PflMI digests, which were used to confirm the G----A transition, can be useful in screening for this specific mutation.     

Identification of Two Novel Mutations in Adenine Phosphoribosyltransferase Gene in Patients with 2,8‐Dihydroxyadenine Urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.     

Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.     

2,8-Dihydroxyadenine lithiasis in a Japanese patient heterozygous at the adenine phosphoribosyltransferase locus.

All reported cases of 2,8-dihydroxyadenine (DHA) lithiasis have been due to functional homozygous deficiency of adenine phosphoribosyltransferase (APRT). Here we describe the first case of DHA lithiasis in a patient who has functional APRT activity in cultured lymphoblasts. The patient is heterozygous for Japanese-type (type II) APRT deficiency as demonstrated by starch-gel electrophoresis and DNA sequence analysis. We also demonstrate the use of starch-gel electrophoresis for differentiation between the type II mutant enzyme and the wild-type enzyme.