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11.
The purification and specificity of a neutral endopeptidase from rabbit kidney brush border 总被引:41,自引:18,他引:23
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1. A neutral peptidase, previously shown to be located in the brush border of the proximal tubule, and assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain was purified from rabbit kidney. 2. The starting material for the purification was a microsomal pellet prepared from a homogenate of cortical tissue. The membrane-bound enzymes were solubilized by treatment with toluene and trypsin. About half the neutral peptidase activity was released by this treatment in a form that no longer sedimented with the microsomal pellet and which penetrated polyacrylamide gels when subjected to disc electrophoresis. Other treatments with detergents or proteolytic enzymes either inactivated the peptidase or failed to convert it into a genuinely soluble form. 3. Chromatography with successive columns of Sephadex G-200, DEAE-cellulose and hydroxyl-apatite yielded an enzyme that was free of other brush-border peptidase activities and which was homogeneous on disc electrophoresis and ultracentrifugation. 4. The purified enzyme attacked [(125)I]iodoglucagon at a rate comparable with that for [(125)I]iodoinsulin B chain. It did not appear to attack proteins (insulin, albumin and casein) that had been similarly iodinated. 5. Unlabelled insulin B chain and unlabelled glucagon were substantially hydrolysed by the endopeptidase, whereas insulin and albumin released only trivial amounts of ninhydrin-reacting material. The resistance of insulin to attack by endopeptidase, even after prolonged incubation, was confirmed by biological and immunoassay. 6. The specificity of the peptidase was determined by analysis of the products after incubating unlabelled insulin B chain, and some oligopeptide substrates, including pentagastrin, with the enzyme. All of the bonds readily cleaved were those involving the alpha-amino group of hydrophobic residues, i.e. x-Leu-, x-Val-, x-Tyr-, x-Phe- and x-Met-, provided that the residues were not C-terminal. 7. The enzyme showed only endopeptidase activity. Substrates suitable for aminopeptidases, carboxypeptidases or esterases were not attacked. 相似文献
12.
Isolation of cytomegalovirus from a cohort of 100 infants throughout the first year of life 总被引:3,自引:0,他引:3
13.
It is known that plaque formation by some rhinoviruses is greatly enhanced by increasing the concentration of MgCl(2). The mechanism of this action was studied by investigating the effects of MgCl(2) on rhinovirus HGP adsorption, growth, clumping, thermal stability, and cell susceptibility to viral cytopathic effect. The latent period was at most 7 hr, whether virus was propagated in cells maintained in 0.8 or 30 mm MgCl(2), but virus release was 8- to 310-fold greater in the presence of 30mm MgCl(2), depending on initial multiplicity. Intracellular virus content appeared unaffected by Mg(++) and reached maximal yield (plateau phase) at about 10 hr. Viral adsorption was increased when cells were maintained in 30mm Mg(++). It is likely that the two effects of magnesium, enhanced adsorption and increased virus release, both contribute to enhancement of plaque formation. 相似文献
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15.
We previously identified three well-dispersed mutations, E978-K, F989-L and D1009-R within the haemolysin A signal region, located at positions –46, –35 and –15, with respect to the C-terminus, respectively. Each mutation reduces the efficiency of secretion two- to threefold leaving 30–45% of the wild-type activity. We have constructed by in vitro manipulations double mutants of HlyA carrying all combinations of these mutations and a triple mutant carrying all three mutations. The effects on secretion were determined and the results, including residual levels of secretion with the triple mutant of only 0.6%, compared with the wild type, indicated that these residues may interact to form a single function in the wild-type signal. To test this further, we developed a secretion competition assay in order to classify signal mutations. We demonstrated that a CIZ-HlyA fusion protein, containing the C-terminal 81 kDa of HlyA fused to virtually the whole LacZ protein, strongly inhibits the secretion of the wild-type HlyA co-expressed In the same cell. The properties of the fusion indicate that it blocks the translocator. The three mutations singly and in combinations were recombined in vitro into the 3′-end of the hybrid gene. In every case, the presence of a mutation in the secretion signal of the hybrid protein alleviated the inhibition of secretion of the co-expressed HiyA. All the mutations are therefore essentially recessive and we propose that they all affect an early function, probably recognition of the translocator, rather than a subsequent step involved in translocation or final release of the toxin to the medium. This would indicate that residues involved in recognition for steps 相似文献
16.
Glen P Kenny Julien Periard W Shane Journeay Ronald J Sigal Francis D Reardon 《Journal of applied physiology》2003,95(6):2355-2360
The hypothesis that the magnitude of the postexercise onset threshold for sweating is increased by the intensity of exercise was tested in eight subjects. Esophageal temperature was monitored as an index of core temperature while sweat rate was measured by using a ventilated capsule placed on the upper back. Subjects remained seated resting for 15 min (no exercise) or performed 15 min of treadmill running at either 55, 70, or 85% of peak oxygen consumption (V(o2 peak)) followed by a 20-min seated recovery. Subjects then donned a liquid-conditioned suit used to regulate mean skin temperature. The suit was first perfused with 20 degrees C water to control and stabilize skin and core temperature before whole body heating. Subsequently, the skin was heated ( approximately 4.0 degrees C/h) until sweating occurred. Exercise resulted in an increase in the onset threshold for sweating of 0.11 +/- 0.02, 0.23 +/- 0.01, and 0.33 +/- 0.02 degrees C above that measured for the no-exercise resting values (P < 0.05) for the 55, 70, and 85% of V(o2 peak) exercise conditions, respectively. We did note that there was a greater postexercise hypotension as a function of exercise intensity as measured at the end of the 20-min exercise recovery. Thus it is plausible that the increase in postexercise threshold may be related to postexercise hypotension. It is concluded that the sweating response during upright recovery is significantly modified by exercise intensity and may likely be influenced by the nonthermal baroreceptor reflex adjustments postexercise. 相似文献
17.
Kenneth T. Izutsu Sahba Fatherazi Carol M. Belton Dolphine Oda Frank D. Cartwright George E. Kenny 《In vitro cellular & developmental biology. Animal》1996,32(6):361-365
Summary The relations between K+ channel and Cl− channel currents and mycoplasma infection status were studied longitudinally in HSG cells, a human submandibular gland cell
line. The K+ channel currents were disrupted by the occurrence of mycoplasma infection: muscarinic activation of K+ channels and K+ channel expression as estimated by ionomycin- or hypotonically induced K+ current responses were all decreased. Similar decreases in ionomycin- and hypotonically induced responses were observed for
Cl− channels, but only the latter decrease was statistically significant. Also, Cl− currents could be elicited more frequently than K+ currents (63% of cases versus 0%) in infected cells when tested by exposure to hypotonic media, indicating that mycoplasma
infection affects K+ channels relatively more than Cl− channels. These changes occurred in the originally infected cells, were ameliorated when the infection was cleared with sparfloxacin,
and recurred when the cells were reinfected. Such changes would be expected to result in hyposecretion of salivary fluid if
they occurredin vivo. 相似文献
18.
Christian Chervaux Nathalie Sauvonnet Annick Le Clainche Brendan Kenny A. Lesley Hunt Jenny K. Broome-Smith I. Barry Holland 《Molecular & general genetics : MGG》1995,249(2):237-245
An in frame gene fusion containing the coding region for mature -lactamase and the 3-end of hylA encoding the haemolysin secretion signal, was constructed under the control of a lac promoter. The resulting 53 kDa hybrid protein was specifically secreted to the external medium in the presence of the haemolysin translocator proteins, HlyB and HlyD. The specific activity of the -lactamase portion of the secreted protein (measured by the hydrolysis of penicillin G), approximately 1 U/g protein, was close to that of authentic, purified TEM--lactamase. This is an important example of a hybrid protein that is enzymatically active, and secreted via the haemolysin pathway. Previous studies have indicated that haemolysin is secreted directly into the medium, bypassing the periplasm, to which -lactamase is normally targeted. This study indicated, therefore, that normal folding of an active -lactamase, can occur, at least when fused to the HlyA C-terminus, without the necessity of entering the periplasm. Despite the secretion of approximately 5 g/ml levels of the active -lactamase fusion into the medium, there was maximally only a 50% detectable increase in the LD50 for resistance to ampicillin at the individual cell level. This result suggests that, normally, resistance to ampicillin requires a high concentration of the enzyme close to killing targets, i.e. in the periplasm, in order to achieve significant levels of protection.These authors made an equal contribution to this work 相似文献
19.
20.
Proliferating cell nuclear antigen is required for DNA excision repair. 总被引:95,自引:0,他引:95
Fractionation of extracts from human cell lines allows nucleotide excision repair of damaged DNA to be resolved into discrete incision and polymerization stages. Generation of incised intermediates depends on the XP-A protein, a polypeptide that recognizes sites of damaged DNA, and on the human single-stranded DNA-binding protein HSSB. The proliferating cell nuclear antigen (PCNA) is required for the DNA synthesis that converts the nicked intermediates to completed repair events. This need for PCNA implies that repair synthesis is carried out by DNA polymerase delta or epsilon. The ability to visualize repair intermediates in the absence of PCNA facilitates dissection of the multiprotein reaction that leads to incision of damaged DNA in a major pathway of cellular defense against mutagens. 相似文献