Poly(A) synthesis in T2L phage-infected Escherichia coli. A combination of polynucleotide phosphorylase and ATPase.

In crude extracts of T2L phage-infected Escherichia coli cells an enzyme activity was found that produced poly(A) from ATP as substrate. Purification of the extract led to the isolation of two enzymes, a polynucleotide phosphorylase and an ATPase. The polynucleotide phosphorylase possessed the same properties as the well-known enzyme from uninfected cells and its molecular weight was about 265 000. The ATPase was purified to over 90% purity; its molecular weight was estimated to be about 165 000 with three subunits of 55 000. The characterization of this enzyme showed that it was different from any ATPase known so far. Mg2+ cannot be replaced by Ca2+, as it can from the membrane-bound ATPases. The only product yielded by the enzyme was ADP; it was very specific for ATP, other ribonucleotide triphosphates being practically unaffected. The rate of ATP splitting was found to be very high, the turnover number being 2.51 X 10(4) min-1 at 37 degrees C. Even at 0 degree C the enzyme was still active. The optimal assay conditions for ATPase turned out to be very similar to those of polynucleotide phosphorylase. Thus the combination of the two enzymes very efficiently produced poly(A) from ATP. In this combination the polynucleotide phosphorylase was the rate-limiting enzyme, since its turnover number was about 40 times lower than that of the ATPase. The evaluation of a variety of properties of the poly(A)-synthesizing constituent found in the crude extracts led us to conclude that this activity arises from the combined action of ATPase and polynucleotide phosphorylase, and is not due to a poly(A) polymerase.     

Thermostable polynucleotide phosphorylases from Bacillus stearothermophilus and Thermus aquaticus.

Polynucleotide phosphorylase from Bacillus stearothermophilus has been purified to homogeneity. Polyacrylamide gel electrophoresis run under denaturing conditions indicates that the enzyme is a tetramer with subunits of apparent molecular weight 51,000 daltons. A partial purification of polynucleotide phosphorylase from Thermus aquaticus has also been effected. The two enzymes show similar catalytic properties, which differ little from those of mesophilic polynucleotide phosphorylases. The use of thermostable polynucleotide phosphorylases for in vitro nucleic acid synthesis is discussed.     

Distribution of enzymes of nucleic acid biosynthesis and their precursors during fractionation of Escherichia coli MRE-600 extract

Distribution of the enzymes of template-dependent and template-independent polynucleotide syntheses (DNA-polymerase I, RNA-polymerase, polynucleotide phosphorylase) as well as those of the biosynthesis of nucleic acids precursors (nucleotide kinases, acetokinase and nucleoside deoxyribosyltransferases) during fractionation of Escherichia coli MRE-600 cell extract was studied. On the basis of the results obtained a technological scheme was developed that enabled to combine routine procedures of purification of the above mentioned enzymes.     

Oligoribonuclease is distinct from the other known exoribonucleases of Escherichia coli.

Oligoribonuclease, an exoribonuclease specific for small oligoribonucleotides, was initially characterized 20 years ago (S. K. Niyogi and A. K. Datta, J. Biol. Chem. 250:7307-7312, 1975) and shown to be different from RNase II and polynucleotide phosphorylase. Here we demonstrate, using mutant strains and purified enzymes, that oligoribonuclease is not a manifestation of RNases D, BN, T, PH, and R, exoribonucleases discovered subsequently. Thus, oligoribonuclease is the eighth distinct exoribonuclease discovered in Escherichia coli. We also show that oligoribonuclease copurifies with polynucleotide phosphorylase.     

Synthesis of Polynucleotides by Microorganisms

Polynucleotides could be synthesized from nucleoside diphosphates by microorganisms belonging to genera Pseudotnonas, Serratia, Xatuhonwnas, Proteus, Aerobacter, Bacillus, and Brevibacterium. These strains were rich in polynucleotide phosphorylase easily extractable from cells and poor in both nuclease and nucleoside-diphosphate-degrading enzymes. Polynucleotide phosphorylase was effectively extracted from the bacterial cells, that had been once soaked in saturated saline solution, with hypotonic solution. Synthesis of polynucleotides was observed not only when the substrates were incubated with polynucleotide phosphorylase preparation isolated from the bacterial cells, but also when the substrates were added directly to the bacterial cultures.     

Isolation and characterization of specialized transducing lambda phages carrying ribosomal protein genes of Escherichia coli

Summary Insertion in an episome of a kanamycine-resistant element (Tn5) at the polynucleotide phosphorylase gene level, results, after transduction into a wild strain, by the loss of activities specific to polynucleotide phosphorylase. A low phosphorolytic activity is nevertheless detectable in crude extracts, but no longer in extracts slightly purified after heat treatment at 54°C. The part played by other enzymes in these activities is discussed. Bacterial growth is not affected by introduction of the mutation.     

Isolation and characterization of a polynucleotide phosphorylase from Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens BaM-2 produces large amounts of extracellular enzymes, and the synthesis of these proteins appears to be dependent upon abnormal ribonucleic acid metabolism. A polynucleotide phosphorylase (nucleoside diphosphate:polynucleotide nucleotidyl transferase) was identified, purified, and characterized from this strain. The purification scheme involved cell disruption, phase partitioning, differential (NH4)2SO4 solubilities, agarose gel filtration, and diethylaminoethyl-Sephadex chromatography. The purified enzyme demonstrated the reactions characteristic of polynucleotide phosphorylase: polymerization, phosphorolysis, and inorganic phosphate exchange with the beta-phosphate of a nucleotide diphosphate. The enzyme was apparently primer independent and required a divalent cation. The reactions for the synthesis of the homopolyribonucleotides, (A)n and (G)n, were optimized with respect to pH and divalent cation concentration. The enzyme is sensitive to inhibition by phosphate ion and heparin and is partially inhibited by rifamycin SV and synthetic polynucleotides.     

Study on the structure-function relationship of polynucleotide phosphorylase: model of a proteolytic degraded polynucleotide phosphorylase.

It is already known that modification of E. coli polynucleotide phosphorylase by endogenous proteolysis induces drastic changes in both phosphorolysis and polymerisation reactions. The structural parameters of the proteolysed polynucleotide phosphorylase are described. The phosphorolysis of polynucleotide, which is quite progressive for the native enzyme, is shown to be only partially progressive for the degraded enzyme, owing to the loss of polymer attachment sites.     

A study of the localization of polynucleotide phosphorylase within rat liver cells and of its distribution among rat tissues and diverse animal species

1. Rat liver polynucleotide phosphorylase was localized in the mitochondrion, but may also occur in the nucleus. 2. The mitochondrial enzyme was found in rat heart, kidney, liver, muscle and spleen. 3. Mitochondrial polynucleotide phosphorylase is also present in calf, chicken, guinea-pig and rabbit liver and in goldfish muscle. 4. A possible physiological role for the enzyme in the control of the intramitochondrial ADP concentration is suggested.     

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.     

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.     

Quaternary structure and proteolysis of the polynucleotide phosphorylase from C. perfringens]

This report describes structural studies on purified polynucleotide phosphorylase from C. perfringens. A method is described for the purification of the enzyme which yields a product equivalent in activity to the native polynucleotide phosphorylase from E. coli. These studies revealed a molecular heterogeneity arising from successive stages of proteolysis, to which this enzyme is especially sensitive; unusally, the enzyme is obtained as a mixture of variable proportions of the native and proteolysed forms. We found in all cases a trimeric basic structure composed of the native (alpha) or proteolysed (lapha) or proteolysed (alpha', alpha") catalytic sub-units, However, the enzyme is rather easily dissociated into its sub-units, a phenomenon which seems to accompany proteolysis (Table). Under the action of either endogenous proteases or trypsin, two enzymatic forms are obtained: their quaternary structures seem analogous, but they differ in their catalytic properties from each other and from the initial enzyme. With some care at each step of purification, the polynucleotide phosphorylase of E. coli can be obtained exclusively in its native form. The greater susceptibility to proteolysis of the enzyme from C. perfrigens and the relationship between such degradation and quaternary structure seem to be at the origin of the peculiar behavior of this polynucleotide phosphorylase.     

The preferential loss of the polylysine- or polyornithine-stimulated activity of Clostridium perfringens polynucleotide phosphorylase during proteolysis

1. An improved method for the purification of Clostridium perfringens polynucleotide phosphorylase (nucleoside diphosphate-polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is described. The product was stable and was highly stimulated by polylysine or polyornithine. 2. It migrated as a single enzyme during sucrose-density-gradient centrifugation, and no separation of polymerization and phosphorolytic activities was observed. 3. Trypsin digestion caused a rapid, preferential loss of the polylysine- or polyornithine-stimulated activity, which was prevented by low concentrations of polyornithine. 4. The protection by polyornithine was not specific. 5. It is concluded that charge effects on the clostridial polynucleotide phosphorylase itself are primarily responsible for the stimulation of this enzyme by polylysine or polyornithine.     

细菌RNA稳定性调控相关元件的研究进展

RNA降解体（细菌RNA降解的主要执行者）是一种多亚基的蛋白质复合物,主要由RNA解螺旋酶、聚核苷酸磷酸化酶（polynucleotide phosphorylase,PNPase）、内切核酸酶（ribonuclease E,RNase E）以及糖酵解途径中的烯醇化酶、磷酸果糖激酶等组成,参与核糖体RNA（ribosome RNA,rRNA）的加工以及信使RNA（messenger RNA,mRNA）的降解。此外,RNA分子伴侣Hfq和调控小RNA（small RNA,sRNA）在RNA稳定性调控中也发挥着重要作用。综述了细菌RNA稳定性调控相关功能元件,特别是降解体蛋白及RNA分子伴侣Hfq的最新进展,以期为研究细菌RNA稳定性及其参与的代谢调控提供理论参考。     

Immunoglobulin M biosynthesis. Production of intermediates and excess of light chain in mouse myeloma MOPC 104E

1. Conditions have been established for the estimation of molecular weights of proteins by analytical gel filtration and sucrose-density-gradient centrifugation in 2.5m-potassium chloride-1m-sodium chloride; Halobacterium cutirubrum polynucleotide phosphorylase, DNA-dependent RNA polymerase and RNA-dependent RNA polymerase have been studied by these methods. 2. The RNA-dependent polymerase has also been studied by density-gradient centrifugation in the absence of salt. 3. All three proteins are of unusually low molecular weight compared with similar enzymes from non-halophilic bacteria.     

Blue-dextran--Sepharose affinity chromatography: recognition of a polynucleotide binding site of a protein.

Native Escherichia coli polynucleotide phosphorylase can be retained on blue-dextran--Sepharose. The bound enzyme cannot be displaced by its mononucleotide substrates such as ADP, UDP, CDP, GDP and IDP, but it is easily eluted by its polymeric substrates. Under identical conditions, lactate dehydrogenase, bound on blue-dextran--Sepharose, is not eluted by poly(I) but can be specifically displaced by NADH. On the other hand, the trypsinized polynucleotide phosphorylase, known to be an active enzyme which has lost its polynucleotide site, does not bind to the affinity column. The native polynucleotide phosphorylase can also be tightly bound to poly(U)--agarose and displaced from it only by high salt concentration. The trypsinized enzyme is not bound at all on poly(I)--AGAROSe. Moreover, the native enzyme linked on blue-dextran--Sepharose, remains active indicating a free access of nucleoside diphosphates to the active center. These results taken together show that the dye ligand is not inserted onto the mononucleotide binding site and suggest rather that it binds to the polynucleotide binding region. The implications of this study and the application of blue-dextran--Sepharose affinity chromatography to other proteins having affinity for nucleic acids are discussed.     

A deoxyadenylate kinase activity associated with polynucleotide phosphorylase from Micrococcus luteus

We report here the presence of two enzymatic activities associated with highly purified preparations of polynucleotide phosphorylase from Micrococcus luteus. The first, a nuclease activity, which is not separated from the phosphorylase on hydroxylapatite, may be due to substitution of H2O for phosphate in the phosphorolysis reaction. The second activity, a deoxyadenylate kinase, the bulk of which is not resolved from the phosphorylase using gel filtration, sucrose density gradient centrifugation, DEAE-Sephadex, or hydroxylapatite chromatography, may represent a new activity of polynucleotide phosphorylase or be due to an enzyme which is tightly bound to the phosphorylase. Several properties of the kinase are described and its possible significance with respect to the overall enzyme mechanism is discussed.