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161.
162.
An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide M
r
=150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrane protein M
r
=150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are coregulated and that the active enzyme has a role in regulating its own synthesis.Non-standard Abbreviations SDS
sodium dodecyl sulphate
- PAGE
polyacrylamide gel electrophoresis
- DOC
sodlum deoxycholate 相似文献
163.
Molybdenum is required for induction of nitrate reductase and of NAD-linked formate dehydrogenase activities in suspensions of wild type Paracoccus denitrificans; tungsten prevents the development of these enzyme activities. The wild type forms a membrane protein M
r150,000 when incubated with tungsten and inducers of nitrate reductase and this is presumed to represent an inactive form of the enzyme. Suspensions of mutant M-1 did not develop nitrate reductase or formate dehydrogenase activities but the membrane protein M
r150,000 was formed under all conditions tested, including without inducers and without molybdenum. Analysis of membranes, solubilized with deoxycholate, by polyacrylamide gel electrophoresis under nondenaturing conditions showed that the mutant protein had similar electrophoretic mobility to the active nitrate reductase formed by the wilde type. Autoradiography of preparations from cells incubated with 55Fe showed that the mutant and wild type proteins contained iron. However, in similar experiments with 99Mo, incorporation of molybdenum into the mutant protein was not detectable.We conclude that mutant M-1 is defective in one or more steps required to process molybdenum for incorporation into molybdoenzymes. This failure affects the normal regulation of nitrate reductase protein with respect to the role of inducers.Non-Standard Abbreviations DOC
deoxycholate
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulfate 相似文献
164.
Michael G. Clark Sally D. Neville Dallas G. Clark 《Biochemical and biophysical research communications》1981,103(2):690-697
Hepatocytes were prepared from a strain of rats deficient in hepatic phosphorylase kinase and were used to assess the role of this enzyme in the adrenergic regulation of pyruvate kinase and gluconeogenesis. Epinephrine (10 μM) stimulated glucose output and gluconeogenesis from 1.8 mM lactate but did not significantly affect the concentration of hepatocyte glycogen. In addition epinephrine treatment led to an inhibition of pyruvate kinase. The stimulation of gluconeogenesis and the inhibition of pyruvate kinase by epinephrine were blocked by both α- and β-antagonists: similar effects with epinephrine were observed in cells from control animals. It is concluded that mechanisms for the adrenergic regulation of pyruvate kinase and gluconeogenesis are similar in hepatocytes from both phosphorylase kinase-deficient and normal rats. 相似文献
165.
Proteolytic removal of the COOH terminus of the T4 gene 32 helix-destabilizing protein alters the T4 in vitro replication complex 总被引:7,自引:0,他引:7
The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. 1. Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32I protein for this synthesis. 2. Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. 3. Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. 4. The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3'-OH end of a polynucleotide chain and are thereby inhibited. Eukaryotic helix-destabilizing proteins may also have similar functional domains essential for the control of their activities. 相似文献
166.
A single overlay plaque assay was designed and evaluated for infectious hematopoietic necrosis virus. Epithelioma papillosum carpio cells were grown in normal atmosphere with tris(hydroxymethyl)aminomethane- or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered media. Plaques were larger and formed more quickly on 1- to 3-day-old cell monolayers than on older monolayers. Cell culture medium with a 10% addition of fetal calf serum (MEM 10) or without serum (MEM 0) were the most efficient virus diluents. Dilution with phosphate-buffered saline, saline, normal broth, or deionized water reduced plaque numbers. Variations in the pH (7.0 to 8.0) of a MEM 0 diluent did not affect plaque numbers. Increasing the volume of viral inoculum above 0.15 ml (15- by 60-mm plate) decreased plaquing efficiency. Significantly more plaques occurred under gum tragacanth and methylcellulose than under agar or agarose overlays. Varying the pH (6.8 to 7.4) of methylcellulose overlays did not significantly change plaque numbers. More plaques formed under the thicker overlays of both methylcellulose and gum tragacanth. Tris(hydroxymethyl)aminomethane and HEPES performed equally well, buffering either medium or overlay. Plaque numbers were reduced when cells were rinsed after virus adsorption or less than 1 h was allowed for adsorption. Variation in adsorption time between 60 and 180 min did not change plaque numbers. The mean plaque formation time was 7 days at 16 degrees C. The viral dose response was linear when the standardized assay was used. 相似文献
167.
Defective F pili and other characteristics of Flac and Hfr Escherichia coli mutants resistant to bacteriophage R17.
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Mutants resistant to the donor-specific bacteriophage R17 were isolated from Hfr and Flac-containing strains of Escherichia coli K-12. Thirty-five mutants were examined for the presence of F pili by electron microscopy. The pilus morphology was studied, as were the abilities of the cells to retract their pili and to synthesize new pili. Measurements were made of the efficiency of the conjugal deoxyribonucleic acid transfer and of M13 and R17 phage infection. All mutants had noticeable defects in pilus production, structure, or function. Mutants were found which produced unusually long pili, displayed wide variations in the number of pili per cell, and were deficient in pilus retraction and synthesis. Evidence is presented that there may be two pathways of pilus retraction. 相似文献
168.
The physiological and morphological characteristics of eighty-two strains of Hanseniaspora and Kloeckera, representing twenty-nine described species, were examined. These results along with DNA base composition and DNA/DNA reassociation experiments revealed that the genus Hanseniaspora comprises six distinct species, viz. H. valbyensis, H. uvarum, H. guilliermondii, H. occidentalis, H. osmophila and H. vineae, with K. japonica, K. apiculata, K. apis, K. javanica, K. corticis and K. africana, respectively, as their imperfect states. 相似文献
169.
170.