Nucleic acid enzymology of extremely halophilic bacteria. <i>Halobacterium cutirubrum</i> polynucleotide phosphorylase

Peterkin, Pearl I.; Fitt, Peter S.

doi:10.1042/bj1210613

Cited by 25 publications

(12 citation statements)

References 32 publications

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“…The peaks ofMn2+-independent and Mn2+-stimulated activity (see below) corresponded exactly. The enzyme is therefore much smaller than any known bacterial or eukaryote alkaline phosphatase and is the fourth unusually small enzyme to be isolated from H. cutirubrum, the other three being polynucleotide phosphorylase, DNA-dependent RNA polymerase and RNA-dependent RNA polymerase (Fitt et al, 1975;Louis & Fitt, 1971a,b, 1972Louis et al, 1971;Peterkin & Fitt, 1971). Further work will be necessary toestablishifa significant proportion ofH.…”

Section: Resultsmentioning

confidence: 98%

“…Cell culture H. cutirubrum N.R.C. 34001 cells were grown in Gochnauer & Kushner's (1969) complex medium as F previously described (Fitt et al, 1975;Peterkin & Fitt, 1971). They were harvested in mid-exponential phase after 48h of growth, when the culture has an E660 of approx.…”

Section: Methodsmentioning

confidence: 99%

“…phosphatase is already fully active at an ionic strength about one-quarter to one-half that usually necessary with enzymes from extreme halophiles (Larsen, 1967;Kushner, 1968;Louis & Fitt, 1972;Peterkin & Fitt, 1971). Table 7 shows the substrate specificity of H. cutirubrum alkaline phosphatase.…”

Section: Protein Cytochrome C A-chymotrypsinogenmentioning

confidence: 99%

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Isolation and properties of a small manganese-ion-stimulated bacterial alkaline phosphatase

Fitt¹,

Peterkin²

1976

Biochemical Journal

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1. An alkaline phosphatase was partially purified from extracts of Halobacterium cutirubrum. 2. The enzyme has a mol.wt. of 15 500 and is therefore less than one-quarter of the size of other known bacterial alkaline phosphatases. 3. It is stimulated up to ten-fold by Mn2+, but not by Ca2+ or Mg2+. 4. The activities with and without Mn2+ cannot be separated by gel filtration and have similar restricted substrate specificities. 5. The only substrates for the enzyme that have so far been found are p-nitrophenyl phosphate, 5'-dATP, 5'-dTMP and 5'-dTTP.

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Section: Resultsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

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Isolation and properties of a small manganese-ion-stimulated bacterial alkaline phosphatase

Fitt¹,

Peterkin²

1976

Biochemical Journal

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“…Other halophilic enzymes, however, show narrow pH optima even at high salt concentrations. Among these are the aspartate transcarbamylase (85), polynucleotide phosphorylase (99) and citrate synthase (25) of H. cutirubrum, ornithine carbamoyltransferase of H. salinarium (37), and glycerol dehydrogenase of Ps. salinaria (6).…”

Section: Long-range Electrostaticmentioning

confidence: 99%

Salt-dependent properties of proteins from extremely halophilic bacteria.

Lanyi¹

1974

Bacteriological Reviews

433

222

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113 69 a Hydrophobicity index (in kcal/residue) calculated as described by Bigelow (14), who used the free energies of transfer for amino acids from water to ethanol (118). Ratio of volume for polar and nonpolar residues, according to Fisher (41). cDialysis of ribosomes against buffer containing Mg'+, and a low concentration of monovalent cations, followed by sedimenting the RNA-rich particles. dDialysis of ribosomes against 0.3 mM Mg2+, centrifugation; supernatant is S-1. Incubation in 3.5 M LiCl-ethylenediaminetetraacetic acid (EDTA), centrifugation. Supernatant is S-2, followed by incubation in 3.5 M LiCl-urea, which yielded supernatant S-3. ' Cell envelopes were dialyzed against 0.01 M EDTA. Centrifugation at 105,000 x g yielded red pellet (J. K. Lanyi and E. L. Bugna, unpublished data). ' Exhaustive dialysis of cell membranes against distilled water, sedimentation at 105,000 x g for 20 to 30 h.

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“…The enzyme initially was discovered by Grunberg-Manago et al in 1955 (3) in Azotobacter vinelandii. Subsequently, its enzymatic activity was detected among eubacteria (4,5), Archea (6), eukaryotic microbes (7), plants (8), and animal cells (9). ''Degradosome'' complexes containing PNPase, RNase E, the RhlB helicase, enolase, and other proteins have been isolated from E. coli and other bacteria (10)(11)(12)(13) and recently have been shown to exist also in vivo (14) and to function as ribonucleolytic machines (15,16).…”

mentioning

confidence: 99%

RhlB helicase rather than enolase is the β-subunit of the Escherichia coli polynucleotide phosphorylase (PNPase)–exoribonucleolytic complex

Lin

Lin‐Chao²

2005

Proc. Natl. Acad. Sci. U.S.A.

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Escherichia coli polynucleotide phosphorylase (PNPase), a protein that has both ribonucleolytic and synthetic capabilities, binds, along with the 48-kDa glycolytic enzyme enolase, the 50-kDa DEAD-box protein RhlB helicase and other cellular proteins to the C-terminal ''scaffold'' region of RNase E to form a complex termed the RNA degradosome. PNPase itself has been reported to exist as a complex (␣ 3␤2) containing trimers of a catalytic subunit (␣) and dimers of another subunit (␤). The ␤-subunit has been believed to be enolase; we report here that it is instead the RhlB helicase. Whereas interaction between PNPase-␣ and enolase was observed in bacteria that synthesize RNase E having a scaffold region, immunoprecipitates from cells expressing PNPase-␣, RhlB, and enolase from single-copy chromosomal loci, plus a mutant RNase E protein lacking its C-terminal half, showed direct association of PNPase-␣ only with RhlB. Using affinity chromatography, we found that PNPase-␣ and RhlB form a ribonucleolytically active complex corresponding to the mass calculated previously for ␣3␤2 (i.e., 377-380 kDa), whereas no association between PNPase-␣ and enolase was detected. Chromosomal deletion of the eno gene had no effect on the ability of PNPase to degrade either single-or double-stranded RNAs. Collectively, our findings show that direct interaction between PNPase-␣ and RhlB occurs physiologically in the absence of the RNase E C-terminal region, that enolase association with PNPase-␣ is a consequence of the interaction of both proteins with RNase E, and that, contrary to current notions, enolase is not the ␤-subunit of E. coli PNPase complex.degradosome ͉ RNase E P olynucleotide phosphorylase (PNPase) is a major 3Ј to 5Ј exoribonuclease of Escherichia coli and functions both in the degradation of mRNA and stable RNA species and as a poly(A) polymerase (1, 2). The enzyme initially was discovered by Grunberg-Manago et al. in 1955 (3) in Azotobacter vinelandii. Subsequently, its enzymatic activity was detected among eubacteria (4, 5), Archea (6), eukaryotic microbes (7), plants (8), and animal cells (9). ''Degradosome'' complexes containing PNPase, RNase E, the RhlB helicase, enolase, and other proteins have been isolated from E. coli and other bacteria (10-13) and recently have been shown to exist also in vivo (14) and to function as ribonucleolytic machines (15,16). In yeast and animal cells, several PNPase-related 3Ј to 5Ј exonucleases have been identified and shown, along with an RNA helicase, to form an ''exosome '' complex (17-19). Because of its occurrence in a broad spectrum of organisms and its involvement in a variety of ribonucleolytic complexes (for recent reviews, see refs. 20 and 21), the identification and characterization of protein complexes containing PNPase continue to be of general interest.In crude cell extracts of E. coli, PNPase displays heterogeneity (22). Early in its history, PNPase was shown by Portier (23) to exist in two active forms, A or B, having molecular masses of Ϸ252 and 365 kDa, respective...

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Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum polynucleotide phosphorylase

Cited by 25 publications

References 32 publications

Isolation and properties of a small manganese-ion-stimulated bacterial alkaline phosphatase

Isolation and properties of a small manganese-ion-stimulated bacterial alkaline phosphatase

Salt-dependent properties of proteins from extremely halophilic bacteria.

RhlB helicase rather than enolase is the β-subunit of the Escherichia coli polynucleotide phosphorylase (PNPase)–exoribonucleolytic complex

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