DNA-dependent RNA polymerase from Pseudomonas aeruginosa

1987 ◽  
Vol 65 (9) ◽  
pp. 776-782 ◽  
Author(s):  
Brenda Allan ◽  
Andrew M. Kropinski
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzyme Activity ◽  
Rna Polymerase ◽  
Actinomycin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Subunit Structure ◽  
Partial Activity ◽  
Inhibited Enzyme

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having β′ (157 000), β (148 000), σ (87 000), and α2 (45 000) subunits as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+ produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5–9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 °C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.

C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7

1994 ◽  
Vol 14 (9) ◽  
pp. 6164-6170
Author(s):  
P P Sadhale ◽  
N A Woychik
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rna Polymerase Iii ◽  
Sodium Dodecyl ◽  
Polymerase Iii ◽  
Conditional Mutant ◽  
5.8S Rrna ◽  
Cell Growth And Viability

We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.

Effects of Some Vitamins on Lactoperoxidase Enzyme Activity

2009 ◽  
Vol 79 (3) ◽  
pp. 188-194 ◽  
Author(s):  
Melda Sisecioglu ◽  
Murat Cankaya ◽  
Hasan Ozdemir
Keyword(s):  
Ascorbic Acid ◽  
Enzyme Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Exchange Chromatography ◽  
Inhibition Mechanism ◽  
Vitamin K3 ◽  
Vitro Effect

Objective: The present paper investigates the in vitro effect of L-ascorbic acid (vitamin C), menadione sodium bisulfate (vitamin K3), and folic acid on purified lactoperoxidase (LPO). Methods: This enzyme was purified from bovine milk by Amberlite CG 50 resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography. Results: Rz (A412/A280) value for the purified LPO was found to be 0.8. Lactoperoxidase was purified 20.45-fold with a yield of 28.8 %. Purity of enzyme was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method and a single band was observed. All tested vitamins caused inhibition of the enzyme activity and displayed a competitive type of inhibition mechanism. IC50 values of these three vitamins were 2.03 µM, 0.025 mM, and 0.0925 mM, and the Ki constants were 0.508±0.257 µM, 0.0107±0.0044 mM, and 0.0218±0.0019 mM respectively. Conclusion: The vitamins discussed here displayed inhibition-type competition with LPO enzyme at varying concentrations. Our study showed that L-ascorbic acid exhibited a much higher inhibitory effect at lower concentrations, so it was evidently a more potent inhibitor than other vitamins tested.

Inactivation of 6-phosphofructo-2-kinase during anaerobiosis in the marine whelk Busycon canaliculatum

1991 ◽  
Vol 260 (6) ◽  
pp. R1168-R1175
Author(s):  
L. Bosca ◽  
K. B. Storey
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase ◽  
Molecular Mass ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Busycon Canaliculatum ◽  
Native Molecular Mass ◽  
Dependent Protein

6-Phosphofructo-2-kinase (PFK-2) was analyzed in four organs of the anoxia-tolerant marine gastropod mollusk Busycon canaliculatum. Whelk PFK-2 resembled the nonhepatic enzyme from mammals with highest activity occurring in gill (22 pmol.min-1.g-1). Hepatopancreas PFK-2 was purified over 8,000-fold to a final specific activity of 11 mU/mg protein (at 20 degrees C) and gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a dimer with a native molecular mass of 142 kDa and a subunit molecular mass of 67 kDa. The purified enzyme showed negligible fructose-2,6-bisphosphatase (FBPase-2) activity, although the activity ratio of PFK-2 to FBPase-2 was 0.625 in crude extracts. In response to environmental anoxia, the activity of PFK-2 dropped in all organs to 34-56% of the corresponding aerobic value (half-time was 2 h in gill), and the Michaelis constant for fructose 6-phosphate increased by 50% (to 92 microM in gill). These changes paralleled decreases in organ fructose 2,6-bisphosphate concentration and pyruvate kinase activity and contribute to the overall glycolytic rate depression induced by anoxia in this facultative anaerobe. In vitro treatment of the anoxic form of hepatopancreas PFK-2 with alkaline phosphatase increased enzyme activity, suggesting that the aerobic and anoxic enzyme forms are interconverted by reversible protein phosphorylation. However, the protein kinase involved in this process is not yet known; incubation of aerobic PFK-2 with Mg-ATP plus adenosine 3',5'-cyclic monophosphate-dependent protein kinase or protein kinase C did not alter enzyme activity.

scholarly journals In vitro characterization of aminoglycoside adaptive resistance in Pseudomonas aeruginosa.

1996 ◽  
Vol 40 (6) ◽  
pp. 1387-1393 ◽  
Author(s):  
J A Karlowsky ◽  
M H Saunders ◽  
G A Harding ◽  
D J Hoban ◽  
G G Zhanel
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Protein ◽  
Cytoplasmic Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proton Motive Force ◽  
Motive Force ◽  
Adaptive Resistance ◽  
Resistant Cells

Aminoglycoside adaptive resistance was characterized in one reference strain and four clinical isolates of Pseudomonas aeruginosa. Adaptive resistance was initiated with a 2-h gentamicin or tobramycin exposure at the MIC. Each P. aeruginosa strain demonstrated an adaptive-resistance period of between 8 and 12 h when tested with both aminoglycosides. Aminoglycoside adaptive resistance was shown to correlate with a decrease in [3H] gentamicin accumulation and a small (5%) but significant (P < 0.05) reduction in proton motive force. The mean generation time of P. aeruginosa during peak levels of adaptive resistance (i.e., maximum reductions in aminoglycoside killing) was not significantly different from that of control organisms (P < 0.05). No changes in outer membrane protein or lipopolysaccharide sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles were noted when control, adaptively resistant, and postadaptively resistant cells were compared. Cytoplasmic membrane profiles of adaptively resistant cells, however, demonstrated several band changes when compared with control and postadaptively resistant cells. We conclude that the decrease in aminoglycoside accumulation associated with adaptive resistance in P. aeruginosa may be, in part, a function of reductions in proton motive force and/or cytoplasmic membrane protein changes. However, the importance of these changes requires further investigation.

scholarly journals Microbial metabolism of amino alcohols. Purification and properties of coenzyme B12-dependent ethanolamine ammonia-lysae of Escherichia coli

1978 ◽  
Vol 175 (2) ◽  
pp. 555-563 ◽  
Author(s):  
Carol M. Blackwell ◽  
John M. Turner
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Coenzyme B12 ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Protein Components ◽  
Inhibited Enzyme

1. The 120-fold purification of ethanolamine ammonia-lyase from Escherichia coli extracts, to apparent homogeneity, is described. Ethanolamine, dithiothreitol, glycerol and KCl protected the apoenzyme from inactivation. 2. At the optimum pH7.5, Km values for ethanolamine and coenzyme B12 were 44μm and 0.42μm respectively. The Km for ethanolamine was markedly affected by pH, transitions occurring at pH7.0 and 8.35. 3. The enzyme was specific for ethanolamine as substrate, none of the 18 analogues tested being active. l-2-Aminopropan-l-ol (Ki 0.86μm), dl-1-aminopropan-2-ol (Ki 2.2μm) and dl-1,3-diaminopropan-2-ol (Ki 88.0μm) inhibited competitively. 4. Enzyme activity was inhibited, irreversibly and non-competitively, by the coenzyme analogues methylcobalamin (Ki 1.4nm), hydroxocobalamin (Ki 2.1nm) and cyanocobalamin (Ki 4.8nm). 5. Iodoacetamide inhibited in the absence of ethanolamine, but only slightly in its presence. p-Hydroxymercuribenzoate inhibited markedly even in the presence of ethanolamine. Dithiothreitol and 2-mercaptoethanol (less effectively) restored activity to the enzyme dialysed against buffer containing ethanolamine. 6. Although K+ ions stabilized the enzyme during dialysis or storage, they were not necessary for activity. 7. Gel filtration showed the enzyme to be of high molecular weight, ultracentrifugal studies giving s20,w of 16.4 and an estimated mol.wt. 560400. The isoelectric point for the apoenzyme was approx. pH5.0. inhibited enzyme activity at concentrations above 1m (95% inhibition at 3m) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated protein subunits of mol.wt. 61400. 8. Immunological studies showed that the E.coli enzyme was closely related to those of other enterobacteria, but only distantly to that of Clostridium sp. A double precipitin band suggested that the apoenzyme may be made up of two protein components.

scholarly journals C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7.

1994 ◽  
Vol 14 (9) ◽  
pp. 6164-6170 ◽  
Author(s):  
P P Sadhale ◽  
N A Woychik
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rna Polymerase Iii ◽  
Sodium Dodecyl ◽  
Polymerase Iii ◽  
Conditional Mutant ◽  
5.8S Rrna ◽  
Cell Growth And Viability

We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.

scholarly journals Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

1982 ◽  
Vol 2 (4) ◽  
pp. 412-425 ◽  
Author(s):  
S I Reed ◽  
J Ferguson ◽  
J C Groppe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Mutagenesis ◽  
Coding Region ◽  
Loop Analysis ◽  
Partial Digestion ◽  
Polyadenylic Acid

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

scholarly journals Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi

1977 ◽  
Vol 167 (1) ◽  
pp. 71-75 ◽  
Author(s):  
R F Matagne ◽  
J P Schlösser
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Enzyme Preparation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Activity Analysis ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Argininosuccinate Lyase

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.

Identification of pilin pools in the membranes of Pseudomonas aeruginosa

1982 ◽  
Vol 152 (2) ◽  
pp. 687-691
Author(s):  
T H Watts ◽  
E A Worobec ◽  
W Paranchych
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Gel Electrophoresis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Outer Membranes

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.

scholarly journals Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration

1977 ◽  
Vol 163 (2) ◽  
pp. 369-378 ◽  
Author(s):  
P R Dunkley ◽  
H Holmes ◽  
R Rodnight
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synaptic Membrane ◽  
Hydroxyl Groups ◽  
Phosphorylated Proteins

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.

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