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PubMed1994

Polynucleotide phosphorylase of Escherichia coli induces the degradation of its RNase III processed messenger by preventing its translation.

Polynucleotide phosphorylase, a 3' to 5' processive exoribonuclease is post-transcriptionally autocontrolled and it was previously shown that this control is dependent on a 5' processing by RNase III. In this paper, the mechanism of regulation is analyzed by studying the properties of a pnp-lacZ translational gene fusion. It is shown that this message is stable, even when processed by RNase III, and that the degradation rate is directly linked to the intracellular concentration of polynucleotide phosphorylase or to the pnp-lacZ messenger translation rate. Mutations able to decrease the level of repression are all located in the ribosome loading site. Taken together, these results suggest that polynucleotide phosphorylase is able to recognize specifically the processed messenger and to prevent its translation, thus allowing degradation of the message.

Base Sequence↗
PubMed1977

Effects of polyamines on the degradation of ribonucleic acids by polynucleotide phosphorylase of Micrococcus luteus.

The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8] from Micrococcus luteus have been studied. Although the breakdown of all the synthetic polynucleotides tested was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C) greater than poly(A) greater than poly(U) at pH 7.5. However, the difference in degree of stimulation among polynucleotides decreased as the pH or monovalent cation concentration was increased. In the presence of heparin, an inhibitor of polynucleotide phosphorylase hydrolysis of polynucleotides, spermidine clearly stimulated the breakdown of poly(C) and poly(A), while the breakdown of poly(U) was stimulated only slightly by the addition of spermidine. Although binding of [14C]spermine to polynucleotide phosphorylase was observed by gel filtration, the amount of spermine bound to the enzyme was much less than that to RNA.

Binding Sites↗
PubMed1978

Metabolism of cytokinin: ribosylation of cytokinin bases by adenosine phosphorylase from wheat germ.

As part of the study of cytokinin metabolic pathways, an enzyme, adenosine phosphorylase (EC 2.4.2.-), which catalyzed the ribosylation of N(6)-(Delta(2)-isopentenyl)adenine, N(6)-furfuryladenine, and adenine to form the corresponding nucleosides, was partially purified from wheat (Triticum aestivum) germ. The pH optimum for the ribosylation of the cytokinins and adenine was from 6.5 to 7.8; for guanine and hypoxanthine it was from 7.0 to 8.5 At pH 7.2 (63 millimolar N-2-hydroxyethyl piperazine-N'-ethanesulfonic acid) and 37 C the K(m) for N(6)-(Delta(2)-isopentenyl)adenine was 57.1 micromolar; N(6)-furfuryladenine, 46.5 micromolar; adenine, 32.2 micromolar; and the V(max) for N(6)-(Delta(2)-isopentenyl)adenine, N(6)-furfuryladenine, and adenine were 134.7, 137.1, and 193.1 nanomoles per milligram protein per minute, respectively. The equilibrium constants of the phosphorolysis of N(6)-(Delta(2)-isopentenyl)adenosine and adenosine by this enzyme indicated that the reaction strongly favored nucleoside formation. This enzyme was shown to be distinct from inosine-guanosine phosphorylase based on the differences in the Sephadex G-100 gel filtration behaviors, pH optima, and the product and p-hydroxymercuribenzoate inhibitor studies. These results suggest that adenosine phosphorylase may play a significant role in the regulation of cytokinin metabolism.

Journal Article↗
PubMed1973

Polynucleotide phosphorylase has a role in growth of Escherichia coli.

Mutants having low levels of polynucleotide phosphorylase activity grow poorly at 45 C. All revertants isolated for their ability to grow better at that temperature also regained higher levels of polynucleotide phosphorylase and the ability to be induced for tryptophanase. Thus, a physiological role is implied for the enzyme polynculeotide phosphorylase.

Adenosine Diphosphate↗
PubMed1991

A paradoxical increase of a metabolite upon increased expression of its catabolic enzyme: the case of diadenosine tetraphosphate (Ap4A) and Ap4A phosphorylase I in Saccharomyces cerevisiae.

The APA1 gene in Saccharomyces cerevisiae encodes Ap4A phosphorylase I, the catabolic enzyme for diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). APA1 has been inserted into a multicopy plasmid and into a centromeric plasmid with a GAL1 promoter. Enhanced expression of APA1 via the plasmids resulted in 10- and 90-fold increases in Ap4A phosphorylase activity, respectively, as assayed in vitro. However, the intracellular concentration of Ap4A exhibited increases of 2- and 15-fold, respectively, from the two different plasmids. Intracellular Ap4A increased 3- to 20-fold during growth on galactose of a transformant with APA1 under the control of the GAL1 promoter. Intracellular adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G) also increased in the transformant under these conditions. The chromosomal locus of APA1 has been disrupted in a haploid strain. The Ap4A phosphorylase activity decreased by 80% and the intracellular Ap4A concentration increased by a factor of five in the null mutant. These results with the null mutant agree with previous results reported by Plateau et al. (P. Plateau, M. Fromant, J.-M. Schmitter, J.-M. Buhler, and S. Blancquet, J. Bacteriol. 171:6437-6445, 1989). The paradoxical increase in Ap4A upon enhanced expression of APA1 indicates that the metabolic consequences of altered gene expression may be more complex than indicated solely by assay of enzymatic activity of the gene product.

Cloning, Molecular↗
PubMed1994

The gene coding for polynucleotide phosphorylase in Photorhabdus sp. strain K122 is induced at low temperatures.

Photorhabdus sp. strain K122 was found to produce higher levels of the protein CAP87K when cultured at 9 degrees C than when cultured at 28 degrees C. NH2-terminal sequencing of this protein revealed homology with the NH2 terminus of Escherichia coli polynucleotide phosphorylase. A 4.5-kb DNA fragment from strain K122 was cloned and sequenced and found to have 75% identity to the E. coli rpsO-pnp operon coding for ribosomal protein S15 and polynucleotide phosphorylase, respectively. Predicted proteins encoded by this sequence were found to have 86% identity with ribosomal protein S15 and polynucleotide phosphorylase from E. coli, and the genes were called rpsO and pnp, respectively. Quantitation of rpsO and pnp mRNA transcripts from K122 revealed that there was a 2.4-fold increase in the level of pnp mRNA and a 1.9-fold decrease in the level of rpsO mRNA at 9 degrees C relative to 28 degrees C. Primer extension analysis revealed the positions of possible promoters controlling the expression of rpsO and pnp in K122, suggesting that the genes are expressed independently. The increase in the level of pnp mRNA at 9 degrees C was not due to any relative increase in its stability compared with that of the rpsO transcript. However, there was evidence to suggest that it may be a result of a cold-inducible promoter, P2, in the intergenic region between rpsO and pnp. Several features of P2 support the suggestion that it may be cold inducible.

Amino Acid Sequence↗
PubMed2000

Cloning and characterization of the glucooligosaccharide catabolic pathway beta-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana.

Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-beta-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95 degrees C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k(cat)/K(m) values are 155.2, 76.0, and 9.9 mM(-1) s(-1) for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-beta-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85 degrees C. It has specific activity of 11.8 U/mg and a K(m) of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K(m) for cellobiose (compared to GghA's K(m) of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.

Amino Acid Sequence↗
PubMed1993

LPS inhibits PI-phospholipase C but not PC-phospholipase D or phosphorylase activation by vasopressin and norepinephrine.

Rats were infused with endotoxin (50 micrograms/100 g body wt) for 3 h, and the parenchymal cells of the liver were maintained in primary culture for 1-3 h. The effects of vasopressin, norepinephrine, and glucagon on the activation of phosphatidylinositol (PI)-phospholipase C, phosphatidylcholine (PC)-phospholipase D, and glycogen phosphorylase a were investigated. Activation of PI-phospholipase C was markedly reduced, particularly with norepinephrine. This confirms that one of the early metabolic impairments seen in acute endotoxin treatment is inhibition of PI-phospholipase C activity. However, the ability of vasopressin, norepinephrine, and glucagon to stimulate glycogen phosphorylase a and PC-phospholipase D was not affected by this endotoxin treatment. We conclude that activation of phosphorylase a by vasopressin and norepinephrine is not entirely dependent on the activation of PI-phospholipase C and inositol trisphosphate formation.

Animals↗
PubMed2002

Characterization of trehalose phosphorylase from Bacillus stearothermophilus SK-1 and nucleotide sequence of the corresponding gene.

A bacterial trehalose phosphorylase (TPase; EC 2.4.1.64) was purified from the culture supernatant of Bacillus stearothermophilus SK-1 to apparent homogeneity, and some properties were investigated. Furthermore, a gene from SK-1 responsible for the TPase was cloned by Southern hybridization with a degenerate oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The Mr of the enzyme was estimated to be 150,000 by gel filtration and 83,000 by SDS-PAGE, so the enzyme is likely to be a homodimer. The enzyme had optimum activity at pH 7.0-8.0 or nearby and the optimum temperature was about 75 degrees C. The deduced amino acid sequence of the SK-1 TPase encodes a theoretical protein with a Mr of 87,950. Alignment of amino acid sequences with a maltose phosphorylase from Lactobacillus brevis the crystal structure and active site of which had been analyzed suggested that these two phosphorylases evolved from a common ancestor. The Escherichia coli cells harboring the plasmid containing the cloned TPase gene had about 100 times the activity of SK-1.

Amino Acid Sequence↗
PubMed2002

Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium, Thermotoga maritima MSB8.

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80 degrees C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70 degrees C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) kcat followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the kcat of 8.0 s(-1) had the highest (1.1 x 10(4) M(-1) s(-1)) kcat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.

Enzyme Stability↗
PubMed1987

Nucleotide sequence of the pnp gene of Escherichia coli encoding polynucleotide phosphorylase. Homology of the primary structure of the protein with the RNA-binding domain of ribosomal protein S1.

The pnp gene is located at 69 min on the Escherichia coli chromosome adjacent to the rpsO gene which encodes the ribosomal protein S15. In this paper, we present the sequence of a 3030-nucleotide DNA fragment containing the open reading frames coding for ribosomal protein S15 and polynucleotide phosphorylase. Translation of pnp is initiated by 5'-UUG-3' codon separated by 7 nucleotides from a good ribosome binding site. Codon usage in this gene is typical of highly expressed proteins of E. coli. Some of the transcripts of the pnp gene terminate just after the stem of the terminator t2 visible in the nucleotide sequence. However, a very strong read-through occurs at this site, thus permitting many of the pnp transcripts to extend beyond this transcription terminator. We also describe the primary structure homologies between a 69-amino-acid stretch of polynucleotide phosphorylase and the four homologous stretches of ribosomal protein S1 which form its RNA binding site. The possibility that this 69-amino-acid stretch constitutes the polynucleotide binding domain of polynucleotide phosphorylase is discussed.

Amino Acid Sequence↗
PubMed2002

Expression of uridine and thymidine phosphorylase genes in human breast carcinoma.

Uridine phosphorylase (UPase) and an angiogenic enzyme, thymidine phosphorylase (dThdPase) are involved in degradation of the pyrimidine nucleosides through phosphorolysis. The expression levels of UPase and dThdPase are higher in human solid tumors including breast carcinomas than in normal tissues. To clarify the correlation between the expression levels of UPase and dThdPase genes and the clinicopathological factors, mRNA levels of these enzymes were examined by RT-PCR in 43 breast carcinomas. UPase gene expression was not correlated with dThdPase gene expression (regression coefficient R = 0.032). Although the expression level of the dThdPase gene was correlated with angiogenesis, detected by immunostaining endothelial cells (R = 0.66), that of UPase gene was not (R = 0.044). These results suggest that UPase does not have a strong angiogenic activity. The UPase gene expression levels in tumors of patients who relapsed were significantly higher than in those from patients who did not (p = 0.039). Although the expression levels of neither UPase or dThdPase were associated with age, pT, pN, pM, estrogen or progesterone receptor positivity, the patients with the higher levels of UPase gene expression had worse survival (p = 0.0038) than those with lower levels. In contrast, the expression of dThdPase gene was not related to relapse or survival of these patients with breast carcinoma. Our findings suggest that the expression level of UPase gene may be an independent prognostic marker in human breast carcinoma.

Adult↗
PubMed1990

Myopathic evolution of an exertional muscle pain syndrome with phosphorylase b kinase deficiency.

The symptoms of a myopathy permanently affecting limb girdle muscles are reported in a 31-year-old woman who has been presenting an exertional muscle pain syndrome with myoglobinuria for 20 years. Investigations revealed a slightly decreased utilization of glycogen in muscle, while its storage affected only rare type 2 fibers. Active phosphorylase was undetectable and phosphorylase b kinase activity was clearly decreased in muscle cells, but normal in erythocytes, lymphocytes and cultured fibroblasts.

Adult↗
PubMed1993

Expression, purification, characterization, and deletion mutations of phosphorylase kinase gamma subunit: identification of an inhibitory domain in the gamma subunit.

A catalytic fragment, gamma 1-298, derived from limited chymotryptic digestion of phosphorylase b kinase (Harris, W.R. et al., J. Biol. Chem., 265: 11740-11745, 1990), is reported to have about six-fold greater specific activity than does the gamma subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the gamma subunit, full-length wild-type and seven truncated forms of gamma were expressed in E. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar (gamma 1-353 and gamma 1-341) or less (gamma 1-331 and gamma 1-276) specific activity than does the full-length wild-type gamma, gamma 1-386. Three truncated forms, gamma 1-316, gamma 1-300, and gamma 1-290 have molar specific activities approximately twice as great as those of the full-length wild-type gamma and the nonactivated holoenzyme. All recombinant gamma s exhibit similar Km values for both substrates, i.e., about 18 microM for phosphorylase b and about 75 microM for MgATP. Three truncated gamma s, gamma 1-316, gamma 1-300, and gamma 1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (Vmax/Km) than that of the full-length wild-type gamma and a 3.5- to 4.5-fold greater efficiency than that of the truncated gamma 1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of gamma, which is located at gamma 301-331. gamma 1-290, but not gamma 1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the gamma subunit to exhibit phosphotransferase activity. Both gamma 1-290 and gamma 1-300 have several properties similar to full-length wild-type gamma, including metal ion responses (activation by free Mg2+ and inhibition by free Mn2+), pH dependency, and substrate specificities.

Amino Acid Sequence↗
PubMed1989

The binding of beta-glycerophosphate to glycogen phosphorylase b in the crystal.

The binding of beta-glycerophosphate (glycerol-2-P) to glycogen phosphorylase b in the crystal has been studied by X-ray diffraction at 3 A resolution. Glycerol-2-P binds to the allosteric effector site in a position close to that of AMP, glucose-6-P, UDP-Glc, and phosphate. In this position, glycerol-2-P is stabilized through interactions of its phosphate moiety with the guanidinium groups of Arg 309 and Arg 310 which undergo conformational changes, and the hydroxyl group of Tyr 75, while the same residues and solvent are involved in van der Waals interactions with the remaining part of the molecule. Kinetic experiments indicate that glycerol-2-P partially competes with both the activator (AMP) and the inhibitor (glucose 6-phosphate) of phosphorylase b. A comparison of the positions of glycerol-2-P, AMP, glucose 6-phosphate, UDP-Glc, and Pi at the allosteric site is presented.

Adenosine Monophosphate↗
PubMed1983

Heterotropic interactions of AMP and glucose binding sites in phosphorylase a are destroyed by limited proteolysis.

Subtilisin BPN' hydrolyses a single peptide bond in phosphorylase a. The two proteolytic fragments are attached to each other by noncovalent bonds in solution as shown by gel filtration and ultracentrifugation studies. The subtilisin nicked phosphorylase a is inactive, however, still binds AMP and glucose as judged by equilibrium dialysis and fluorescence experiments. The modified enzyme can be dephosphorylated by protein phosphatase and AMP is an effective inhibitor of the dephosphorylation reaction. Glucose cannot cancel the AMP inhibition as well as cannot expel AMP from the nucleotide binding site. Thus a single nick in the polypeptide chain breaks the "communication" between the two ligand binding domains.

Adenosine Monophosphate↗
PubMed1987

A phospho-oligosaccharide mimics insulin action on glycogen phosphorylase and pyruvate kinase activities in isolated rat hepatocytes.

A phospho-oligosaccharide which is the polar head group of a novel insulin-sensitive glycophospholipid has recently been involved in insulin action. We have investigated the insulin-like effects of this phospho-oligosaccharide on both glycogen phosphorylase a and pyruvate kinase activities of hepatocytes incubated in the presence of glucagon (0.1 nM). Similarly to insulin, the phospho-oligosaccharide antagonized glucagon-dependent activation of glycogen phosphorylase, as well as the inactivation of pyruvate kinase caused by this hormone. The antagonistic action of the phospho-oligosaccharide on glucagon effects was dose-dependent. Furthermore, it partially antagonized glucagon-stimulated cyclic AMP levels. These results support the hypothesis that this phospho-oligosaccharide mediates at least some insulin actions in hepatocytes.

Animals↗
PubMed1983

Structure-activity relationship of ligands of the pyrimidine nucleoside phosphorylases.

Eighty-seven pyrimidine base and nucleoside analogs were evaluated as inhibitors of uridine phosphorylase (UrdPase) and thymidine phosphorylase (dThdPase). These findings, together with an extensive literature review, have allowed construction of structure-activity relationships for the binding of ligands to UrdPase and dThdPase and provide a basis for the rational design of new inhibitors of these enzymes. Additionally, 2,6-pyridinediol and 6-benzyl-2-thiouracil have been identified as being potent inhibitors of UrdPase and dThdPase respectively.

Animals↗