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1.
Murine lymphocytes oxidized by galactose oxidase were radiolabeled by reduction with NaB3H4. The labeled cells were incubated with Con A and the Con A-Con A receptor complexes formed in situ on the viable cells were isolated by immuno-precipitation with anti-Con A serum and fixed Staphylococcus aureus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography analysis of the precipitates demonstrated four high-molecular-weight glycoproteins which were oxidized by GO and which bound Con A. These same four glycoproteins were also oxidized and labeled by . [3H]Tyrosine biosynthetic labeling identified these four plus several other Con A receptors. Because Con A sterically inhibits GO mitogenic stimulation, these four glycoproteins are likely to represent the necessary sites of oxidative mitogenic action and are good candidates for the targets of Con A mitogenesis. 相似文献
2.
The oxidation of unsaturated fatty acid micelles by the superoxide free radical (), during γ irradiation in the presence of formate, is kinetically distinct from oxidation by hydroxyl free radicals (HO.). The evidence suggests that a direct reaction between () and lipid hydroperoxide initiates a chain oxidation process in the micelles. While tetranitromethane, which reacts rapidly with (), protects the micelles from oxidation, active superoxide dismutase is no more effective than its apoprotein, due to lack of penetration of the micellar environment. We discuss these findings in the light of recent literature, and with reference to their possible significance for biological systems. 相似文献
3.
Peter Nicholls 《BBA》1976,430(1):13-29
1. Formate inhibits cytochrome oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome . The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome ) and in the half-reduced species (). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of minus , free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome ) and azide (which induces peak shifts of cytochrome towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome is faster than its rate of dissociation from , especially in the presence of cytochrome . The for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]2.5. Formate inhibition of ascorbate plus oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion. 相似文献
4.
R S Himmelwright N C Eickman E I Solomon 《Biochemical and biophysical research communications》1978,84(2):300-305
Half hemocyanin is shown to undergo a unique change at the Cu(II)?Cu(I) active site with temperature, exhibiting class II mixed valent properties at low temperature (The appearance of an intense near IR intervalence-transfer transition and a delocalized EPR spectrum). This requires a Cu(II)NNNCu(I) bridging geometry. The effects of CO coordination to half , combined with the presence of a low energy charge transfer transition, demonstrate that azide is also bridging at room temperature. Finally, half is found to be capable of coordination of a second at the copper(II) site. 相似文献
5.
Interaction of lymphocytes and macrophage cell line cells (M1 cells). I. Functional maturation and appearance of Fc receptors im M1 cells 总被引:2,自引:0,他引:2
M1 cells, which are cell line cells established from myeloid leukemia cells of the SL strain mouse, can differentiate from blast cells () to mature macrophages () within 48 hr, when they are cultured with conditioned medium (CM) obtained from murine embryonic fibroblasts. While cells have no phagocytic activity nor Fc receptor (FcR), cells possess both characteristics. The appearance of FcR is temperature-dependent and inhibited by a metabolic inhibitor, cycloheximide. FcR on cells is resistant to trypsin and pronase. cells improve the viability of macrophage-depleted SL splenic lymphocytes and restore the in vitro secondary plaque forming cell response of macrophage-depleted spleen cells to particulate and soluble antigens. cells lack this macrophage-substituting capacity. Mm1 cells, mutant cells from M1 cells, having FcR and higher phagocytic activity than cells, are also devoid of this capacity. 相似文献
6.
The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent Km values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g1 = 2.007 and g11 = 2.051, which are characteristic of . The generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes. 相似文献
7.
8.
Quercetin inhibited a dog kidney ( preparation without affecting for ATP or for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the ( activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the for ATP and the for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on , for substrate, and for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations. 相似文献
9.
The apparent Arrhenius energy of activation () of the water osmotic permeability () of the basolateral plasma cell membrane of isolated rabbit proximal straight tubules has been measured under control conditions and after addition of 2.5 mM of the sulfhydryl reagent, para-chloromercuribenzenesulfonic acid (pCMBS), of mersalyl and of dithiothreitol. (kcal/mol) was (controls) and (pCMBS), while decreased with pCMBS to of its control value. Mersalyl also decreased both in vitro and in vivo (using therapeutical doses). These actions of pCMBS and mersalyl were quickly reverted with 5 mM dithiothreitol and prevented by 0.1 M thiourea. for free viscous flow is 4.2 and greater than 10 for non-pore-containing lipid membranes. By analogy with these membranes and with red blood cells, where similar effects of pCMBS on are observed, it is concluded that cell membranes of the proximal tubule are pierced by aqueous pores which are reversibly shut by pCMBS. Part of the action of mercurial diuretics can be explained by their action on . 相似文献
10.
Bernard Lacour Tilman Drüeke Evelyne Pierandreï Bernadette Nabarra Jean-Louis Funck-Brentano 《生物化学与生物物理学报:生物膜》1981,648(2):151-161
Parathyroid hormone (PTH) and calcitonin exert well known effects on the renal tubule which are thought to involve specific hormone receptors and adenyl cyclase. In the intestine, it is not clear whether the action of PTH and calcitonin is only indirect or also direct, and their mechanisms of action are much less well established. In the present study, possibly direct effects of PTH and calcitonin on Na+ transport in isolated intestinal epithelial cells of rats were investigated. In the presence of bovine PTH (1.2 I.U./ml) in the incubation medium, the Na+ efflux rate constant () of isolated enterocytes was significantly reduced when compared to that in control experiments with the hormone vehicle only. The mean depression of induced by bovine PTH was 26% as compared to the control (100%) and to that induced by ouabain (4.0mM) which was 44%. No depressant effect of bovine PTH on was observed when the isolated enterocytes were incubated with ouabain (4.0 mM). Thus, bovine PTH appeared to inhibit the ouabain-sensitive Na+ pump. When incubating the isolated epithelial cells in an EGTA-containing Ca2+-free medium, bovine PTH lost its capacity to inhibit (). Thus, the presence of extracellular Ca2+ appeared necessary for the inhibitory effect of bovine PTH. In contrast to its effect on , bovine PTH induced no change in net Na+ uptake by isolated enterocytes. Moreover, no significant effect on enterocyte Na+ transport could be demostrated for salmon or porcine calcitonin at two different concentrations in the incubation medium. Neither bovine PTH nor salmon calcitonin induced significant changes in enterocyte cyclic AMP or cyclic GMP concentrations. It was concluded that bovine PTH, but not calcitonin, exerted a direct inhibitory effect on the ouabain-sensitive of isolated rat enterocytes. The effect of bovine PTH occured without measurable activation of the cyclic nucleotide system but needed the presence of Ca2+ in the incubation medium to be operative. 相似文献
11.
(1) A quantitative study has been made of the binding of ouabain to the ( in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ( will bind ouabain either in the presence of Mg2+ and Pi, (‘Mg2+, Pi’ conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the ) of ouabain to the M. sexta brain ( under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ( has a higher for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ( can bind ouabain. 相似文献
12.
A method for calculating the rate constant () for the oxidation of the primary electron acceptor (A1) by the secondary one (A2) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: and , respectively.The proposed method has also been used for the estimation of the value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P+ at a rate exceeding this for the transfer of electron from A1 to A2. The method of Parson cannot be used in this case. The value of has been found to be .The activation energies for the A1 to A2 electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively. 相似文献
13.
W R Scowcroft A H Gibson J D Pagan 《Biochemical and biophysical research communications》1976,73(2):516-523
Nitrogenase activity in agar cultures of cowpea rhizobia, strain 32H1, was rapidly inhibited by but this was relieved by increased O2 tension. Inhibition was more rapid than that caused by inhibitors of protein synthesis and was not relieved by methionine sulfoximine or methionine sulfone. Under conditions were nitrogenase activity was inhibited by , glutamine synthetase and glutamate synthase were substantially unaffected. Glutamate dehydrogenase was undetected in either nitrogenase active or inhibited cultures. These results indicate that inhibition of nitrogenase activity in strain 32H1 is not effected through glutamine synthetase regulation of nitrogenase synthesis. 相似文献
14.
G W Ruddock J A Raleigh C L Greenstock 《Biochemical and biophysical research communications》1981,102(1):554-560
A steady-state competition system has been developed to investigate the reactions of the superoxide radical anion () with various peroxides, including the so-called Haber-Weiss reaction. Potassium superoxide dissolved in an oxygen-free solution of DMSO containing 18-dicyclohexyl-6-crown, is the source of . High pressure liquid chromatography is used as an assay system for reactivity, to detect and quantitate the yield of anthracene, formed as a major product in the reaction between and 9,10-dihydroanthrancene. Decrease in anthracene yields, in the presence of peroxide, may be used to indicate a possible competing reaction between and added peroxide. Complications involving peroxide-stimulated formation of anthraquinone derivatives are discussed. No evidence for a competing reaction between and peroxide can be detected up to a 10-fold excess of peroxide over 9,10-dihydroanthracene. 相似文献
15.
Inhibitors of glutamine synthetase cause derepression of nitrogenase biosynthesis in the presence of in the photosynthetic bacterium Rhodopseudomonas capsulata. A new derepressor of nitrogenase biosynthesis, β-N-oxalyl-L-α,β-diaminopropionic acid (ODAP), is here compared with the widely used L-methionine-DL-sulfoximine (MSX). With both compounds, a quantitative correlation has been observed between inhibition of glutamine synthetase and derepression of nitrogenase biosynthesis. We also find that both MSX and ODAP inhibit nitrogenase activity in vivo in R. capsulata. The latter effect seems to be indirect and related to the previously reported reversible inhibition of nitrogenase activity in vivo by . As a control it was observed that neither nor MSX nor ODAP inhibit nitrogenase activity in vivo in Clostridium pasteurianum. 相似文献
16.
The changes in polymer-solvent interactions that occur when native calf thymus DNA is dialyzed against Na2SO4 solutions of a given ionic strength and buffer concentration but of varying concentrations in methylmercuric hydroxide have been investigated with the help of solution density measurements at 25 °C and pH 6.8–7.0. From measurements executed under equilibrium dialysis conditions at the three salt levels 5 mm, 0.05 m, and 0.5 m Na2SO4 (m refers to molality) and in the presence of 5 mm cacodylic acid buffer, the density increments for native calf thymus DNA were determined as a function of CH3HgOH concentration. was found not to vary with organomercurial concentration, irrespective of the concentration of supporting electrolyte, until a certain CH3HgOH concentration level has been reached, viz., , beyond which increases strongly with increasing concentration of CH3HgOH. As is shown by optical melting, becomes a function of organomercurial concentration the moment DNA undergoes denaturation brought about by the complexing of CH3HgOH with the various N-binding sites of the base residues in the DNA double helix.Polymer-solvent interactions, expressed in terms of preferential water interactions (“net hydration”) and preferential salt interactions (“salt solvation”), were derived from the data in combination with data obtained on the preferential interaction of CH3HgOH with denatured DNA and data on the partial specific volumes of all major solution components, gathered from density measurements on solutions with fixed concentrations of diffusible components. Evidence is presented which shows that denaturation in general decreases the net hydration while salt becomes preferentially associated with the polyelectrolyte. This process is further amplified by the interaction of CH3HgOH with denatured DNA: Methylmercurated DNA alters the redistribution of diffusible components at dialysis equilibrium to such an extent that in a formal sense large amounts of water are rejected from the immediate vicinity of the polymer. The molecular implications of these findings are explored. The results are further discussed in the light of previous findings where the methylmercury-induced denaturation of DNA had been studied with the help of buoyant density measurements in a Cs2SO4 density gradient and by velocity-sedimentation in a variety of sulfate media. 相似文献
17.
Howard M. Katzen Denis D. Soderman Barbara G. Green 《Biochemical and biophysical research communications》1981,98(2):410-416
Concanavalin A (Con A) precipitates detergent-solubilized insulin receptors (free of or bound to []insulin) prepared from fat cell membranes resulting in a loss of []insulin binding capacity (or bound hormone) in the soluble fraction. The losses can be recovered by redissolving the precipitates with methyl-α-D-mannoside; the sugar also inhibits precipitation. []insulin also binds to insoluble Con A-occupied receptors. At all concentrations of Con A tested, the initial amounts of free or hormone-bound receptors were completely accounted for by their distribution between soluble and insoluble states. We conclude that Con A and insulin can co-bind to independent sites on the insulin receptor without inhibiting each other and that previously reported decreases in insulin binding to solubilized insulin receptors were likely due to precipitation by Con A of the receptors. 相似文献
18.
Proton inventory investigations of the hydrolysis N-acetylbenzotriazole at pH 3.0 (or the equivalent point on the pD rate profile) have been conducted at two different temperatures and at ionic strengths ranging from 0 to 3.0 M. The solvent deuterium isotope effects and proton inventories are remarkably similar over this wide range of conditions. The proton inventories suggest a cyclic transition state involving four protons contributing to the solvent deuterium isotope effect for the water-catalyzed hydrolysis. The hydrolysis data are described by the equation with , where ko is the observed first-order rate constant in protium oxide, n is the atom fraction of deuterium in the solvent, kn is the rate constant in a protium oxide-deuterium oxide mixture, and is the isotopic fractionation factor. 相似文献
19.
The fall in transepithelial electrical resistance which accompanies aldosterone stimulation of short-circuit current () in toad urinary bladder has been studied further to evaluate the possible causal role of this response in hormonal stimulation of Na+ transport. A steady-state change in tissue conductance was found to depend upon both the simultaneous stimulation of transport by the steroid and the metabolic state of the tissue. Changes in metabolic state alone did not alter resistance. A sustained increase in Na+ transport, dependent on pretreatment with aldosterone and elicited by addition of glucose, could be obtained without a sustained decrease in resistance. Amiloride, an inhibitor of Na+ uptake, produced changes in that were linearly correlated with its effects on tissue conductance. On the basis of the relationship with amiloride, the response to aldosterone was about two-fold higher than would be predicted from its effects on conductance alone. Despite the apparent lack of a simple quantitative dependence of the change in on the change in conductance when the response is fully developed, the results suggest that conductance changes may mediate the initial or early stage of the response. 相似文献
20.
The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity , EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity had an apparent half saturation constant of for free calcium, a maximum reaction velocity of ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K+, Na+, ouabain, KCN, dicyclohexylcarbodiimide and NaN3, had no significant effect on the activity of high-affinity . Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol . Since the kinetic properties of the high-affinity showed a close resemblance to those of erythrocyte plasma membrane , the high-affinity of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells. 相似文献