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.

Molecular cloning of a human cDNA for the 41-kDa phosphoribosylpyrophosphate synthetase-associated protein.

A human cDNA encoding 41-kDa phosphoribosylpyrophosphate (PRPP) synthetase (PRS)-associated protein (PAP41) was cloned from two expressed sequence tag (EST) clones having the nucleotide similarity of 61.5 and 70.0% to human PAP39 cDNA. The predicted open reading frame of 1107 base pairs (bp) has the nucleotide identity of 91.8% to rat PAP41 and encodes a protein of 369 amino acids with a calculated molecular weight (MW) of 40,925. The deduced amino acid sequence exhibits the 98.9% identity to rat PAP41 and 72.2, 50.6, and 50.0% identity with human PAP39, PRS I, and PRS II, respectively, but lacks the PRPP binding site. Southern blot analysis suggested that the PAP41 gene exists as a single copy in the human genome. The single PAP41 mRNA of about 2.1 kb was shown to be present in five human cell lines by Northern blot analysis.

Kinetic and regulatory properties of rat liver phosphoribosylpyrophosphate synthetase complex are partly distinct from those of isolated recombinant component catalytic subunits.

Rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of two catalytic subunits (PRS I and II, in a ratio of approximately 4:1) and two catalytically inactive PRPP synthetase-associated proteins. To better understand the significance of the complex structure, the properties of the native liver enzyme were compared with those of homologous aggregates of recombinant PRS I and PRS II (rPRS I and rPRS II). (1) The specific activity per catalytic subunits of the liver enzyme was about 2.5 times lower than that of rPRS I over a wide pH range. Km values for substrates and Ka values for Pi and Mg2+ of the three enzymes were similar. (2) Specific activity of the liver enzyme for the reverse reaction was about 2 times lower than those of rPRSs. Km values for substrates of the three enzymes were comparable. (3) The liver enzyme was more stable than were rPRSs when incubated at a high temperature or in the absence of stabilizing agents. (4) The liver enzyme was markedly less sensitive to inhibition by nucleotides compared to rPRS I. GDP at 1 mM inhibited the liver enzyme and rPRS I by 32 and 93%, respectively. This effect is not ascribable to molecular interaction between rPRS I and II, as reconstitution of the two did not alter the sensitivity to nucleotide inhibition. (5) Our observations suggest that complex aggregation states of the native enzyme not only suppress the activities but also stabilize the catalytic subunits and the associated proteins and remarkably reduce the sensitivity to inhibition by nucleotides.

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.

Effects of insulin-like growth factor-1 on endotoxin translocation in burned rats receiving total parenteral nutrition.

The purpose of this study was to investigate the effect of insulin-like growth factor-1 (IGF-1) on the translocation of endotoxin from the gut of burned rats. Twenty-eight male Sprague-Dawley rats (7-wk-old) were subjected to 20% full-thickness scald burns and were divided into two groups. One group received 4 mg.kg-1.d-1 of IGF-1 (IGF-1 group, n = 14), and the other received saline (control group, n = 13). All rats were fed exclusively by total parenteral nutrition (TPN). On the second postburn day, rats were killed. The amount of endotoxin in the liver and spleen were measured. RNA from the terminal ileum was extracted, and Northern blot analyses of alpha-tubulin, beta-actin, cell division cycle-2 (cdc2), and immunoglobulin-A (IgA) were performed. Nitrogen balance was improved (p < 0.001), and the wet weight of intestine and its mucosa were increased significantly in the burned rats that received IGF-1. Gene expression of alpha-tubulin and beta-actin were not changed. Cdc2 was elevated (P < 0.05), but IgA was decreased (P < 0.05) in the IGF-1 group. Levels of endotoxin in the liver and spleen were significantly reduced (P<0.05) by the administration of IGF-1. A negative correlation between the levels of endotoxin in the liver and the weight of the intestinal mucosa was observed. In conclusion, IGF-1 improved nitrogen balance, promoted the proliferation of intestinal mucosa and reduced the translocation of endotoxin.

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.

[Mammalian phosphoribosylpyrophosphate synthetase].

PRPP synthetase from rat liver exists as large molecular weight aggregates composed of at least four different components, i.e., two isoforms of 34 kDa catalytic subunits, PRSI and PRSII, and two associated proteins of 39 kDa and 41 kDa (PAP39 and PAP41). The four proteins have remarkably similar amino acid sequences and form a relatively large group of PRS family. PAPs, however, have extra regions of 40 to 50 amino acid residues, totally dissimilar to sequences of the catalytic subunits and thus form a subfamily. PAPs suppress the catalytic activity of PRS. They are very likely to be enzymatically inactive. The relative amounts of the mRNAs of the four components vary with the tissues, hence composition and properties of PRPP synthetase would also differ. Future studies on the physiology and pathology of PAPs are critical in elucidation of pathogenesis of some types of hyperuricemia.

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.

Promoter region of the rat phosphoribosylpyrophosphate synthetase-associated protein 39.

The 5' region of the 39-kDa rat phosphoribosylpyrophosphate synthetase-associated protein (PAP39) gene was isolated and sequenced. The promoter region of the rat PAP39 is GC-rich and contains potential binding sites for regulatory factors. Its promoter activity was demonstrated by transfection of the promoter region in fusion with the chloramphenicol acetyltransferase gene into rat pheochromocytoma PC12 cell.

Promoter region of the mouse cyclin-dependent kinase 5-encoding gene.

While cyclin-dependent kinases, such as CDC2 and CDK2, are key regulators of cell-cycle progression, cyclin-dependent kinase 5 (CDK5) is highly expressed in mature neurons with no evident cell-cycle regulation. The 5'-region of the mouse CDK5 gene was isolated and sequenced. The isolated clone included exons 1 through 7. The 5'-flanking region has a high G+C content. There is no TATA box around the transcriptional start points (tsp), as determined by primer extension analysis. One CCAAT box, one AP-1-binding site, two AP-2-binding sites, and one cAMP-responsive element are located upstream from the tsp. Promoter/cat fusion assays showed that the 5.8-kb fragment of this 5'-flanking sequence possessed the promoter activities expressing cat in rat PC12 pheochromocytoma cells. The effect of deletions of the promoter suggested the presence of two negative control elements located from -2.9 kb to -546 bp, and from -212 to -155 upstream from the 5' end of the tsp. Two positive elements from bp -300 to -212 and from -155 to -41 were also detected. In the element from bp -300 to -212, there was a putative NF-IL6-binding sequence. Thus, the CDK5 promoter region contains multiple positive and negative cis-acting regulatory elements, an arrangement which suggests that the regulation of transcription of CDK5 is under complex control.

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.

Expression of CDK5 (PSSALRE kinase), a neural cdc2-related protein kinase, in the mature and developing mouse central and peripheral nervous systems.

CDK5 is a cdc2-related protein kinase that is known to be highly expressed in mature brain. In this study, we obtained a mouse CDK5 cDNA by screening an adult mouse cDNA library. Northern blot analysis demonstrated that the mouse CDK5 mRNA was expressed especially highly in brain, and moderately in kidney, testis and ovary. In brain the expression of CDK5 is already seen at embryonal 12.5 days (E12.5), and it gradually increases through the embryonal stage. After birth, the expression is maintained at a high level to adulthood. In situ hybridization demonstrated that the expression of CDK5 mRNA was distributed in neurons throughout the brain, spinal cord and peripheral ganglia, especially in the hippocampal pyramidal cells, cerebellar Purkinje cells, cortical neurons, olfactory mitral cells, mesencephalic and motor trigeminal nuclei and trigeminal ganglion. In any portion, no apparent expression was observed in glia. During development, the expression of CDK5 was already seen at E12.5 intensely in trigeminal and dorsal root ganglia, and moderately and diffusely in the central nervous system. The expression pattern of CDK5 is quite in contrast with that of CDC2. The fact that CDK5 is expressed in terminally differentiated non-dividing neurons predicts an alternative function(s) in addition to controlling the cell cycle.

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.

Rapid mobilization of intracellular Mg2+ by bombesin in Swiss 3T3 cells: mobilization through external Ca(2+)- and tyrosine kinase-dependent mechanisms.

We examined the effects of growth factors on intracellular free Mg2+ concentrations ([Mg2+]i) in single Swiss 3T3 fibroblasts, using microfluorometry of a Mg(2+)-sensitive dye mag-fura-2. We had already noted an increase in [Mg2+]i after exposure to bombesin for 30-60 min [Ishijima, S., Sonoda, T., & Tatibana, M. (1991) Am. J. Physiol. 261 (Cell Physiol. 30), C1074-C1080]. In the present work, we found that bombesin also induced early changes in [Mg2+]i. The [Mg2+]i reached peak values within 15 s in most cells, and the significant rise lasted only for 1-2 min. The extent of the increase varied from cell to cell (0-600 microM above basal). On the average, the [Mg2+]i was increased from basal 0.33 to 0.54 mM. Since the time course was similar to that of [Ca2+]i changes, and the dye mag-fura-2 also binds Ca2+, we evaluated Ca2+ interference with measurement of [Mg2+]i. The contribution of Ca2+ binding would be below 20% of the mag-fura-2 signal. The bombesin-induced [Mg2+]i increase was not dependent on external Mg2+, but the omission of external Ca2+ decreased by 60% the [Mg2+]i increase, and the Ca2+ channel blocker, nicardipine inhibited by 90% the [Mg2+]i response. This inhibition was partially reversed by raising the concentration of external Ca2+. Two structurally distinct tyrosine kinase inhibitors, genistein and lavendustin A, almost completely inhibited the [Mg2+]i response. These results suggest that bombesin rapidly induces Mg2+ mobilization from the intracellular pool, through external Ca(2+)- and tyrosine kinase-dependent mechanisms.

A novel 39-kDa phosphoribosylpyrophosphate synthetase-associated protein of rat liver. Cloning, high sequence similarity to the catalytic subunits, and a negative regulatory role.

The rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of the 34-kDa catalytic subunits (PRS I and II) and other 39- and 41-kDa proteins (Kita, K., Otsuki, T., Ishizuka, T., and Tatibana, M. (1989) J. Biochem. (Tokyo) 105, 736-741), which are termed here PRPP synthetase-associated proteins (PAPs). We have cloned the cDNA for the major one of 39 kDa (PAP39) from a rat liver cDNA library. Nucleotide sequencing showed that the clone encoded 356 amino acids containing sequences of all five peptides derived from PAP39. Surprisingly, the deduced amino acid sequence is markedly similar to those of the 34-kDa catalytic subunits. Excluding two regions (about 45 residues in total), PAP39 has a 48% identity with PRS I. Northern analysis detected a major 1.9-kilobase transcript in all 16 rat tissues examined, and the relative amounts of PAP39 mRNA to PRS I mRNA varied with tissues. Covalent cross-linking experiments gave definitive evidence for molecular interaction of PAP39 with the catalytic subunits. Immunoprecipitation experiments revealed that all the catalytic subunits existed as complexes containing PAP39. When PAPs were eliminated from the rat liver enzyme complex by gel filtration in the presence of 1 m MgCl2, a lyotrope, or by mild tryptic treatment, the enzyme activity of the remaining catalytic subunits increased. Based on these results, we propose that PAP39, the major component of PAPs, plays a negative regulatory role in PRPP synthesis and hence is an important factor controlling nucleotide syntheses in general.

Promoter regions of the human X-linked housekeeping genes PRPS1 and PRPS2 encoding phosphoribosylpyrophosphate synthetase subunit I and II isoforms.

The 5' regions of the human phosphoribosylpyrophosphate synthetase subunit I and II genes (PRPS1 and PRPS2, respectively) were isolated and sequenced. A comparison of the nucleotide sequences between human and rat PRPS1 genes revealed that the sequences around the transcription initiation sites were conserved over 56 nucleotides, and that a TATA-like sequence, a CCAAT box and three putative Sp1 binding sites were present at almost the same positions in the GC-rich sequences. Two major transcription initiation sites were localized in the human PRPS1, one of the two was located 27 nucleotides downstream from the TATA-like sequence, while the upstream initiation site was in the TATA-like sequence. The promoter region of the human PRPS2 gene was also GC-rich and contained a TATA-like sequence, four Sp1 binding sites and a homopyrimidine stretch. The initiation sites were localized at 90 nucleotides upstream from the ATG initiation codon. Chloramphenicol acetyltransferase (CAT)/promoter fusion assays showed that a 2.0 kb region (human PRPS1) and a 1.1 kb region (human PRPS2) possessed the promoter activities in four cell lines. The CAT activities in the three human cell lines tended to correlate with the steady-state mRNA levels of the PRPS1 and PRPS2 genes. These results suggest that the 5' flanking regions cloned contribute to the cell-differential expression of these two genes.

Mitogen-induced early increase in cytosolic free Mg2+ concentration in single Swiss 3T3 fibroblasts.

Events related to the early mobilization of Mg2+ in mammalian cells in response to external stimuli are not well characterized. We examined changes in cytoplasmic free Mg2+ concentrations ([Mg2+]i) after mitogenic stimulation in single mouse Swiss 3T3 fibroblasts, using digital ratio imaging microscopy of the fluorescent probe mag-fura-2. Stimulation with bombesin or epidermal growth factor (EGF) in combination with insulin led to a significant increase in mean [Mg2+]i levels from basal 0.22 mM to 0.29-0.35 mM after 30-60 min. The response showed some heterogeneity among individual cells with respect to the extent of the increase; approximately 10% of the cells showed no [Mg2+]i response. Bombesin or EGF alone induced a significant increase in [Mg2+]i but was less effective than when combined with insulin. In medium without added Mg2+, the increase in [Mg2+]i was considerably decreased, either with bombesin plus insulin or EGF plus insulin. These results provide direct evidence for the mobilization of Mg2+ as an early cellular response to growth factors.

Expression of rat phosphoribosylpyrophosphate synthetase subunits I and II in Escherichia coli. Isolation and characterization of the recombinant isoforms.

The 34-kDa subunit of rat liver phosphoribosylpyrophosphate synthetase is a mixture of the two highly homologous isoforms, PRS I and PRS II. Heretofore, it was not possible to separate the two. We now describe isolation and characterization of the recombinant isoforms, named rPRS I and rPRS II. The respective rat cDNAs were inserted into vectors constructed from pKK233-2 by replacing its replication origin with that of pGEM-1 and expressed in Escherichia coli. The rPRS I and rPRS II were purified to apparent homogeneity with specific activities of 33,400 and 46,200 milliunits/mg, respectively; these values were at least 2.5-fold higher than the highest value for the mammalian enzyme so far reported. Both isoforms showed a similar dependency on Pi as an absolute activator. Sulfate partially substituted for Pi. The maximal activities of rPRS I and rPRS II with sulfate were 43 and 7%, respectively, of those seen with Pi. The two isoforms differed in sensitivity to inhibition by ADP and GDP. Inhibition of rPRS I and rPRS II by 0.3 mM ADP was 87 and 54%, respectively, and inhibition by 1 mM GDP was 93 and 24%, respectively. rPRS II was 180-fold more sensitive than rPRS I to heat inactivation at 49 degrees C.