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Reconstitution of RecBC DNase activity from purified Escherichia coli RecB and RecC proteins 总被引:8,自引:0,他引:8
I D Hickson C N Robson K E Atkinson L Hutton P T Emmerson 《The Journal of biological chemistry》1985,260(2):1224-1229
The Escherichia coli RecB protein, normally synthesized in low amounts, has been amplified by linkage of the recB gene to the phage lambda leftward promoter in an expression plasmid. From strains harboring this plasmid, RecB protein has been purified to homogeneity by a simple procedure which includes affinity chromatography on a column of RecC protein bound to agarose. The purified RecB protein has DNA-dependent ATPase activity but no exonuclease activity. RecC protein alone has neither ATPase nor exonuclease activity. However, when combined together, the RecB and RecC proteins show the ATP-dependent double-stranded exonuclease properties characteristic of the RecBC DNase. 相似文献
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Mike A. Steer Jayson M. Semmens 《Journal of experimental marine biology and ecology》2003,290(2):165-178
The influence of both predator and prey size on the shift from a pulling to a drilling predatory response was examined in the intertidal octopus Octopus dierythraeus, using an experimental program. Additionally, selective drilling, where particular regions of the prey are targeted, was examined for a variety of bivalve and gastropod prey. O. dierythraeus always initially attempted to pull bivalves apart. Shells that were eventually drilled were always subjected to significantly more pulling attempts than those that could be pulled apart, indicating that octopus are willing to expend more energy to access the flesh quickly. There was no defined threshold where bivalve size caused an octopus to switch from a pulling to a drilling response. Instead, there was a broad size range where the octopus could adopt either handling method and it varied for each individual. Octopus may only able to pull open bivalves before the molecular ratchet or ‘catch’ mechanism that many bivalves possess is engaged. This might explain the lack of a relationship between either octopus or bivalve size and the success of pulling, as it is likely that when the bivalves were presented to individual octopus they were either setting the ‘catch’ mechanism, or had already engaged it. O. dierythraeus demonstrated selective drilling on a variety of molluscan prey, with penetration sites differing between prey species. O. dierythraeus targeted the valve periphery, which was the thinnest part of the shell, therefore minimizing handling time. O. dierythraeus always drilled gastropods, but did not target the thinnest regions of the shells, with drill site varying according to the morphology of the prey. Elongate species with pronounced aperture lips were drilled in the apical region, close to the columella on the side of the opercula whereas nonelongate species were drilled immediately above the aperture. The location of drilling sites may represent a trade-off between targeting the most effective places to inject paralyzing secretions and the mechanically simplest places to drill. 相似文献
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Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes 总被引:2,自引:0,他引:2
Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity. 相似文献
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Extracellular production of a heat-stable somatic antigen (HSSA) by Bacillus thuringiensis subsp. dendrolimus strain T84A1-A [flagellar (H) serotype 4a: 4b] was serologically detected. In Ouchterlony tests, the HSSA antiserum gave single precipitin lines against both untreated and heat-treated culture supernatants. These two precipitin lines fused completely. When colonies of strain T84A1-A were grown on nutrient agar plates containing the homologous HSSA antiserum, precipitin halos were formed around the colonies. Of 27 type strains of B. thuringiensis subspecies tested, only the type strains of B. thuringiensis subsp. sotto (H serotype 4a: 4b) and B. thuringiensis subsp. israelensis (H serotype 14) formed [precipitin halos on nutrient agar plates containing antiserum against the HSSA of strain T84A1-A. 相似文献