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1.
"Pertussis toxin induces tachycardia and impairs the increase in blood pressure produced by alpha 2-adrenergic agonists" 总被引:4,自引:0,他引:4
Administration of purified pertussis toxin to rats induced persistent tachycardia, (observed in conscious rats but not after pithing); as little as 0.05 microgram/100 g produced a significant effect. Pertussis toxin-treatment did not affected the pressor response produced in the pithed rats by the alpha 2-adrenergic agonist methoxamine but markedly diminished the pressor effect of the alpha 2-adrenergic agonists clonidine and azepexole. A role of adenylate cyclase inhibition in the action of postsynaptic vascular alpha 2-adrenergic receptors is suggested. 相似文献
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3.
Procedures are described for measurement of total protein-bound —SH groups and apparent thiolester linkages in mitochondrial preparations. The procedure for total —SH is based on reaction with radioactive p-mercuribenzoate in 6 m guanidine hydrochloride at pH 9 containing 1 m ammonia buffer. The procedure for apparent thiolesters is based on alkylation of available — SH groups in 6 m guanidine hydrochloride, protein precipitation with perchloric acid, and ammonolysis at pH 9 to liberate — SH from any thiolester present.No variations in total —SH groups measured in Submitochondrial particles or in mitochondria were noted with presence or absence of ATP, ADP, substrates, or uncouplers of oxidative phosphorylation. Previous reports of changes in measured — SH groups thus appear to reflect solely changes in the reactivity of — SH groups and not in the total — SH groups present.Submitochondrial particles contained less than 0.5 nmole of apparent thiolester per mg of protein; mitochondria contained 1 to 2 nmoles. No variation of apparent thiolester content was noted with change in conditions affecting oxidative phosphorylation. The data do not, of course, eliminate possible changes in thiolester content below the sensitivity of the detection procedures. 相似文献
4.
Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI 总被引:2,自引:0,他引:2
P J Greene B T Ballard F Stephenson W J Kohr H Rodriguez J M Rosenberg H W Boyer 《Gene》1988,68(1):43-51
Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti-EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus. 相似文献
5.
Receptor and G-protein-dependent regulation of turkey erythrocyte phosphoinositidase C 总被引:1,自引:0,他引:1
C P Downes C P Berrie P T Hawkins L Stephens J L Boyer T K Harden 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》1988,320(1199):267-280
Several lines of experimental evidence indicate the involvement of a guanine nucleotide-dependent protein (G-protein) in the hormone-stimulated hydrolysis of phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2). However, the shortcomings of available procedures for cell-free assay of hormone-stimulated phosphoinositidase C (PIC) have limited our current understanding of the molecular and mechanistic details of PIC regulation. We recently have proposed that turkey erythrocyte membranes may provide a valuable model system for studies of G-protein-dependent PtdIns(4,5)P2 hydrolysis. The membranes can be simply prepared from [3H]inositol-labelled erythrocytes and they contain a PIC activity that hydrolyses endogenous phosphoinositides and is exquisitively sensitive to guanine nucleotides. PtdIns(4,5)P2 is the principal substrate for this enzyme, there being relatively little direct hydrolysis of phosphatidylinositol 4-phosphate and no detectable hydrolysis of PtdIns. The membranes also contain a purinoceptor of the P2y subclass that is efficiently coupled to PtdIns(4,5)P2 hydrolysis both in intact cells and in the isolated membranes. 2-Methylthioadenosine trisphosphate (2-methyl-S-ATP), a specific P2y receptor agonist, has no effect upon PtdIns(4,5)P2 hydrolysis in the absence of guanine nucleotides, but greatly enhances both the potency and efficacy of PIC activation by guanine nucleotides such as GTP gamma S. GTP gamma S alone stimulates PIC activity only after a prolonged time-lag; the effect of increasing doses of 2-methyl-S-ATP is progressively to shorten this lag phase. These results suggest that the mechanism of G-protein activation involves acceleration of a nucleotide exchange reaction as has been demonstrated for the activation of adenylate cyclase in the same membrane preparation. As well as contributing valuable information on the substrate specificity of PIC and its mode of regulation by hormones, turkey erythrocytes provide a plentiful source of plasma membranes and may be useful for purification of the appropriate G-protein and PIC activities. 相似文献
6.
Expression kinetics of the lactose (lac) operon in Escherichia coli are reviewed for both wild-type and recombinant cell cultures under chemostatic conditions. A unified model which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression is summarized and supporting data is shown to verify its accuracy. The synthesis of alpha-amylase with a recombinant form of Bacillus subtilis is also reviewed to point out generic features in transport regulation, the lac operon model providing a point of departure. While there are many similarities in the influence of transport on both regulating models, there are also important differences. In a chemostat system, the synthesis of alpha-amylase is nongrowth associated, while beta-galactosidase is a growth-associated enzyme. Nevertheless, transport regulation is an important feature in both instances. 相似文献
7.
Photophosphorylation in Attached Leaves of Helianthus annuus at Low Water Potentials 总被引:5,自引:2,他引:3
The in situ response of photophosphorylation and coupling factor activity to low leaf water potential (ψL) was investigated using kinetic spectroscopy to measure the flash-induced electrochromic absorption change in attached sunflower (Helianthus annuus L. cv IS894) leaves. The electrochromic change is caused by the formation of an electric potential across the thylakoid membrane associated with proton uptake. Since depolarization of the thylakoid membrane following flash excitation is normally dominated by proton efflux through the coupling factor during ATP formation, this measurement can provide direct information about the catalytic activity of the coupling factor. Under low ψL conditions in which a clear nonstomatal limitation of net photosynthesis could be demonstrated, we found a strong inhibition of coupling factor activity in dark-adapted leaves which was probably caused by an increase in the energetic threshold for the activation of the enzyme at low ψL. While this result supported earlier in vitro findings, we further discovered that the light-dependent reduction of coupling factor reversed any observable effect of low ψL on the energetics of activation or on photophosphorylation competence. Furthermore, coupling factor was reduced, even in severely droughted sunflower, almost immediately upon illumination. Based on these measurements, we conclude that the nonstomatal limitation of photosynthesis observed by us and others in droughted plants cannot be explained by impaired coupling factor activity. 相似文献
8.
Reductive release of ferritin iron: a kinetic assay 总被引:1,自引:0,他引:1
Ferritin iron release, a process of considerable interest in biology and medicine, occurs most readily in the presence of reducing agents. Here is described a kinetic assay for measuring the rate of ferritin iron removal promoted by various reductants. The new procedure uses ferrozine as a chromophoric, high-affinity chelator for the product, Fe(II). The initial rate of iron release is quantified by continuous spectrophotometric measurement of the Fe(ferrozine)2/3+ complex which absorbs maximally at 562 nm. The initial rate of iron mobilization is dependent on reductant concentration, but not on the concentration of the chelating agent, ferrozine. Saturation kinetics are observed for all reductants, including dihydroxyfumarate, cysteine, caffeic acid, ascorbate, and glutathione. Superoxide dismutase greatly inhibits ferritin iron release by ascorbate, but has little or no effect on the reducing action of dihydroxyfumarate, cysteine, caffeic acid, or glutathione. Ferritin iron removal by dihydroxyfumarate was inhibited by various metal ions. This new assay may be used for rapid screening of test compounds for treatment of iron overload and for investigation of the mechanistic aspects of ferritin iron reduction. 相似文献
9.
Catalytic properties of chloroplast F1-ATPase modified at catalytic or noncatalytic sites by 2-azido adenine nucleotides 总被引:1,自引:0,他引:1
When heat-activated F1-ATPase from chloroplasts was repeatedly exposed to Mg2+ and 2-azido-ATP, followed by separation from medium nucleotides and photolysis, a total of two sites per enzyme, both catalytic and noncatalytic, were labeled. In a coupled assay with pyruvate kinase about half the activity was lost when one site per enzyme was modified. However, increased modification resulted in no further loss of activity. In contrast, methanol-sulfite activation of the enzyme showed a loss of most of the catalytic capacity when one site per enzyme was modified. Predominant labeling of either one catalytic or one noncatalytic site caused a loss of most of the activity in either assay. An indication that the enzyme modified at one site retained some catalytic activity was verified by measurement of the [18O]Pi species formed when [gamma-18O]ATP was hydrolyzed by partially derivatized enzyme. With either catalytic or noncatalytic site modification, the distributions of [18O]Pi species formed showed that the modified enzyme had different catalytic characteristics. An interpretation is that with modification by azido nucleotides at either catalytic or noncatalytic sites, capacity for rapid catalysis is largely lost but the remaining sites retain weak modified catalytic properties. 相似文献
10.
Control of erythroid differentiation: asynchronous expression of the anion transporter and the peripheral components of the membrane skeleton in AEV- and S13-transformed cells 总被引:7,自引:3,他引:4 下载免费PDF全文
Chicken erythroblasts transformed with avian erythroblastosis virus or S13 virus provide suitable model systems with which to analyze the maturation of immature erythroblasts into erythrocytes. The transformed cells are blocked in differentiation at around the colony-forming unit-erythroid stage of development but can be induced to differentiate in vitro. Analysis of the expression and assembly of components of the membrane skeleton indicates that these cells simultaneously synthesize alpha-spectrin, beta-spectrin, ankyrin, and protein 4.1 at levels that are comparable to those of mature erythroblasts. However, they do not express any detectable amounts of anion transporter. The peripheral membrane skeleton components assemble transiently and are subsequently rapidly catabolized, resulting in 20-40-fold lower steady-state levels than are found in maturing erythrocytes. Upon spontaneous or chemically induced terminal differentiation of these cells expression of the anion transporter is initiated with a concommitant increase in the steady-state levels of the peripheral membrane-skeletal components. These results suggest that during erythropoiesis, expression of the peripheral components of the membrane skeleton is initiated earlier than that of the anion transporter. Furthermore, they point a key role for the anion transporter in conferring long-term stability to the assembled erythroid membrane skeleton during terminal differentiation. 相似文献