Partial purification and properties of rat liver mitochondrial polynucleotide phosphorylase.
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SUBMITTER: See YP
PROVIDER: S-EPMC1174413 | biostudies-other | 1972 Nov
REPOSITORIES: biostudies-other
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Project description:1. Polynucleotide phosphorylase has been isolated and partially purified from crude preparations of guinea-pig liver nuclei. 2. The enzyme is particulate and associated with RNA and lipids characteristic of membranes. 3. It has phosphorolysis and exchange activities, but the latter may be due to a contaminating enzyme. 4. The phosphorolysis activity is dependent on bivalent cations, preferably Mg(2+), has a pH optimum between 8.6 and 9.2 and is inhibited by potassium chloride and sodium chloride. 5. The enzyme catalyses phosphorolysis of poly A, poly C, poly U, rRNA and tRNA. Poly G is only phosphorolysed to a very small extent and DNA is not a substrate. 6. The enzyme appears to lack nucleoside diphosphate polymerization activity.
| S-EPMC1179382 | biostudies-other
Project description:The mitochondrial enzyme phosphate-dependent glutaminase was partially purified from rat liver. The enzyme had Mr 290 000 as judged by chromatography on Sephacryl S-300. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the preparation, glutaminase was tentatively identified with a peptide of Mr 73 500. The concentration-dependence on glutamine was highly sigmoidal, with half-maximum velocity at 22 mM-glutamine. Half-maximum activity was obtained with 5 mM-phosphate. The enzyme required ammonia as an obligatory activator, in agreement with previous reports on intact and sonicated mitochondria. These findings further differentiate liver glutaminase from the phosphate-dependent glutaminase present in kidney and several other tissues.
| S-EPMC1153663 | biostudies-other
Project description:In bacteria, polynucleotide phosphorylase (PNPase) is one of the main exonucleolytic activities involved in RNA turnover and is widely conserved. In spite of this, PNPase does not seem to be essential for growth if the organisms are not subjected to special conditions, such as low temperature. We identified the PNPase-encoding gene (pnp) of Pseudomonas putida and constructed deletion mutants that did not exhibit cold sensitivity. In addition, we found that the transcription pattern of pnp upon cold shock in P. putida was markedly different from that in Escherichia coli. It thus appears that pnp expression control and the physiological roles in the cold may be different in different bacterial species.
| S-EPMC180990 | biostudies-literature
Project description:1. The partial purification of purine nucleoside phosphorylase from rabbit erythrocytes is described. 2. Analytical and preparative isoelectric focusing gave a pI value for the enzyme of 4.65. 3. Gel-chromatography and sucrose-density-gradient-centrifugation techniques gave estimates of the molecular weight in the range 75000-83000. 4. Lineweaver-Burk plots of kinetic data were non-linear at high inosine concentrations. Extrapolation of the linear part of such plots yielded a Km value for inosine of about 70 micrometer for the rabbit erythrocyte and liver enzymes. 5. A Hill interaction coefficient of 0.75 was obtained, suggesting negative co-operativity with respect to the binding of inosine. 6. Treatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) caused partial inactivation, and subsequent Lineweaver-Burk plots with inosine as substrate displayed complete linearity, with an increase in Km value for inosine to 200 micrometer. 7. Starch-gel electrophoresis did not reveal the presence of secondary isoenzymes; all tissue extracts examined gave electrophoretic patterns similar to those obtained with the partially purified enzyme from erythrocytes. 8. Results of hybridization studies with nucleoside phosphorylase from human foetal liver suggest that the rabbit enzyme is also a trimer.
| S-EPMC1183717 | biostudies-other
Project description:Polynucleotide phosphorylase (PNPase) plays synthetic and degradative roles in bacterial RNA metabolism; it is also thought to participate in bacterial DNA transactions. Here we used chimeric polynucleotides, composed of alternating RNA and DNA tracts, to analyze whether and how Mycobacterium smegmatis PNPase discriminates RNA from DNA during the 3'-phosphorolysis reaction. We find that a kinetic block to 3'-phosphorolysis of a DNA tract within an RNA polynucleotide is exerted when resection has progressed to the point that a 3'-monoribonucleotide flanks the impeding DNA segment. The position of the pause one nucleotide before the first deoxynucleotide encountered is independent of DNA tract length. However, the duration of the pause is affected by DNA tract length, being transient for a single deoxynucleotide and durable when two or more consecutive deoxynucleotides are encountered. Substituting manganese for magnesium as the metal cofactor allows PNPase to "nibble" into the DNA tract. A 3'-phosphate group prevents RNA phosphorolysis when the metal cofactor is magnesium. With manganese, PNPase can resect an RNA 3'-phosphate end, albeit 80-fold slower than a 3'-OH. We discuss the findings in light of the available structures of PNPase and the archaeal exosome·RNA·phosphate complex and propose a model for catalysis whereby the metal cofactor interacts with the scissile phosphodiester and the penultimate ribose.
| S-EPMC3791318 | biostudies-literature
Project description:The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5'-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III-processed pnp mRNA, thus exposing the transcript to degradative pathways. More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus destroying the double-stranded structure at the 5' end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5' double-stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double-stranded structure at its 5' end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus, the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5' fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript.
| S-EPMC2648360 | biostudies-literature
Project description:Neisseria meningitidis autoaggregation is an important step during attachment to human cells. Aggregation is mediated by type IV pili and can be modulated by accessory pilus proteins, such as PilX, and posttranslational modifications of the major pilus subunit PilE. The mechanisms underlying the regulation of aggregation remain poorly characterized. Polynucleotide phosphorylase (PNPase) is a 3'-5' exonuclease that is involved in RNA turnover and the regulation of small RNAs. In this study, we biochemically confirm that NMC0710 is the N. meningitidis PNPase, and we characterize its role in N. meningitidis pathogenesis. We show that deletion of the gene encoding PNPase leads to hyperaggregation and increased adhesion to epithelial cells. The aggregation induced was found to be dependent on pili and to be mediated by excessive pilus bundling. PNPase expression was induced following bacterial attachment to human cells. Deletion of PNPase led to global transcriptional changes and the differential regulation of 469 genes. We also demonstrate that PNPase is required for full virulence in an in vivo model of N. meningitidis infection. The present study shows that PNPase negatively affects aggregation, adhesion, and virulence in N. meningitidis.
| S-EPMC4862713 | biostudies-literature
Project description:Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3'-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction PNPase acts as a polymerase, using NDPs as substrates to add NMPs to the 3'-OH terminus of RNA chains while expelling inorganic phosphate. The apparent essentiality of PNPase for growth of M. tuberculosis militates for mycobacterial PNPase as a potential drug target. A cryo-EM structure of Mycobacterium smegmatis PNPase (MsmPNPase) reveals a characteristic ring-shaped homotrimer in which each protomer consists of two RNase PH-like domains and an intervening α-helical module on the inferior surface of the ring. The C-terminal KH and S1 domains, which impart RNA specificity to MsmPNPase, are on the opposite face of the core ring and are conformationally mobile. Single particle reconstructions of MsmPNPase in the act of poly(A) synthesis highlight a 3'-terminal (rA)4 oligonucleotide and two magnesium ions in the active site and an adenine nucleobase in the central tunnel. We identify amino acids that engage the 3' segment of the RNA chain (Phe68, Arg105, Arg112, Arg430, Arg431) and the two metal ions (Asp526, Asp532, Gln546, Asp548) and we infer those that bind inorganic phosphate (Thr470, Ser471, His435, Lys534). Alanine mutagenesis pinpointed RNA and phosphate contacts as essential (Arg105, Arg431, Lys534, Thr470+Ser471), important (Arg112, Arg430), or unimportant (Phe68) for PNPase activity. Severe phosphorolysis and polymerase defects accompanying alanine mutations of the enzymic metal ligands suggest a two-metal mechanism of catalysis by MsmPNPase.
| S-EPMC8284320 | biostudies-literature
Project description:A prominent enzyme in organellar RNA metabolism is the exoribonuclease polynucleotide phosphorylase (PNPase), whose reversible activity is governed by the nucleotides diphosphate-inorganic phosphate ratio. In Chlamydomonas reinhardtii, PNPase regulates chloroplast transcript accumulation in response to phosphorus (P) starvation, and PNPase expression is repressed by the response regulator PSR1 under these conditions. Here, we investigated the role of PNPase in the Arabidopsis (Arabidopsis thaliana) P deprivation response by comparing wild-type and pnp mutant plants with respect to their morphology, metabolite profiles, and transcriptomes. We found that P-deprived pnp mutants develop aborted clusters of lateral roots, which are characterized by decreased auxin responsiveness and cell division, and exhibit cell death at the root tips. Electron microscopy revealed that the collapse of root organelles is enhanced in the pnp mutant under P deprivation and occurred with low frequency under P-replete conditions. Global analyses of metabolites and transcripts were carried out to understand the molecular bases of these altered P deprivation responses. We found that the pnp mutant expresses some elements of the deprivation response even when grown on a full nutrient medium, including altered transcript accumulation, although its total and inorganic P contents are not reduced. The pnp mutation also confers P status-independent responses, including but not limited to stress responses. Taken together, our data support the hypothesis that the activity of the chloroplast PNPase is involved in plant acclimation to P availability and that it may help maintain an appropriate balance of P metabolites even under normal growth conditions.
2009-09-12 | GSE18071 | GEO
Project description:Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5'-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics.
| S-EPMC1164719 | biostudies-other