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Substrate- and alkali-metal-ion-induced pK shifts in intestinal brush-border sucrase, according to the three-protons model.
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To define adequately enzyme activation/inhibition mechanisms as a function of pH, it is necessary to characterize the effector-induced pK shifts on both the free enzyme and on the enzyme-substrate complex. On the basis of our recent three-protons model for sucrase [Vasseur, van Melle, Frangne & Alvarado (1988) Biochem. J. 251, 667-675], we show how the 'fundamental' pK values, deduced from the classical double-logarithmic transformations, are insufficient to generate the required information. This insufficiency derives from the fact that, for sucrase, the acid ionization constant, K1, is a molecular constant that involves complex, V-type plus K-type, activatory and inhibitory kinetic effects. As a consequence, substrate-induced pK shifts cannot be interpreted correctly only by using the fundamental pK approach, because an unequal number of key protons is involved, depending on whether the free enzyme or the enzyme-substrate complex is considered. We demonstrate how this problem can be solved by using the 'theoretical' pK values, derived from the reciprocals of the Michaelis pH functions, i.e. Cha's fractional concentration factors. The procedure we propose, which is general, has the advantage of yielding all the macroscopic pK values for any given model, as calculated from the microscopic pK values. Furthermore, it permits predicting pK shifts as a function of [S] and/or [A] (where S is the substrate and A is the allosteric modifier), an objective that cannot be attained by using the double-logarithmic plot approach. Finally, we describe the relation existing between the fundamental and the theoretical pK values. 相似文献
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G Semenza 《European journal of biochemistry》1969,8(4):518-529
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Current concepts of pH-dependent enzyme function are expanded to consider enzymes with up to four key proton families. In an earlier paper the authors extended classical theory to explain the existence, in the acid ionization reaction, of two functionally distinct, V and K, proton families, exemplified by the 1988 sucrase three-proton-families model of Vasseur et al. They now propose that enzymes having two distinguishable proton families at each side of the pH-activity curves exist in nature although there is no previously published evidence of their existence. The resulting, more general, four-proton-families model is treated as a useful framework from which submodels can be derived by simplification, the simplest being the 1911 linear model of Michaelis and Davidsohn, which took into account two proton families out of the theoretical maximum of four proposed here. It is shown that whether a three-proton-families or a four-proton-families model can explain sucrase better is not merely a question of theory but also involves the practical question of having enough data, at each side of the pH spectrum, to permit making an unequivocal choice between the two alternatives. The paper concludes with a discussion of substrate-induced pK shifts according to both models. 相似文献
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Monique Vasseur Charles Tellier Francisco Alvarado 《Archives of biochemistry and biophysics》1982,218(1):263-274
The activation of rabbit intestinal brush-border sucrase in the pH range 4.8 to 9.2 was studied as a function of sucrose concentration and temperature in a metal-free, n-butylamine universal buffer, both in the absence and in the presence of sodium. When sodium was absent, enzyme activation involved the simultaneous loss of two key protons (pK1 of about 5.6), thus yielding a high-affinity, catalytically active enzyme conformation. Inactivation followed when a third key proton (pK2 of about 8.4) was lost. When sodium was present, kinetic analysis in the pH range 4.8 to 7.2 revealed that sodium activation involves distinct effects on the two kinetic parameters, Vm and Km. The Vm parameter seemed to conform to the classical rules of pH-dependent enzyme activation and implicated the release of a single proton whose apparent pK (pK1y, about 5.6) was little affected by sodium. On the contrary, the Km parameter was strongly influenced by sodium. Here, activation of rabbit sucrase seemed to involve release of a different proton whose apparent pK (pK1x also of about 5.6 in the absence of sodium) was strongly shifted to more acid values by saturating sodium concentrations. The functional distinction between the above two protons explains the existence of strong affinity-type activating effects of sodium on rabbit sucrase, previously shown to be pH independent (F. Alvarado and A. Mahmood, 1979, J. Biol. Chem.254, 9534–9541). 相似文献
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1-5-D-Gluconolactone is a competitive inhibitor of both sucrase and isomaltase. Substitution of the 1H and 2H at C1 of the glucosyl moiety in p-CL-phenyl-alpha-D-glucopyranoside leads to a decrease in kcat of both sucrase and isomaltase, the k1H/k2H ranging between 1.14 and 1.20. Treatment of the association constants and of the kcat values for a number of p-substituted phenyl-alpha-D-glucopyranosides on the basis of the Hammet-Hansch equation has allowed the estimation of the importance of hydrophilicity-hydrophobicity as well as of the magnitude of the p values for both substrate-enzyme interaction and catalysis in both sucrase and isomaltase. The magnitude of the secondary deuterium effect as well as the low values of p in both sucrase and isomlatase are strongly indicative of the rate-limiting step going through the formation of an oxocarbonium ion. In conjunction with other observations reported previously, the data presented here led to the suggestion of the main lines of a reaction mechanism for the two glucosidases: prptonation of the glycosidic oxygen is followed by the liberation of the "aglycone" with formation of an oxocarbonium ion, which is temporarily stabilized by a carboxylate group. 相似文献
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A detergent solubilised sucrase from monkey small intestine has been purified 388-fold to gel electrophoretic homogeneity with an overall recovery of 36%. The molecular weight of the enzyme was 263 kDa by gel filtration. Electrophoresis in the presence of SDS indicates that the enzyme is a hetero-dimer. Mixed substrate inhibition studies and inhibition by PCMB and Tris suggest the presence of two catalytically active sites in the form of maltase and sucrase with isomaltase activity being common to both sites. Polyclonal antiserum against the purified enzyme showed a single continuous precipitin line with the purified antigen. 相似文献
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Purification and properties of rabbit intestinal sucrase 总被引:2,自引:0,他引:2
Y Takesue 《Journal of biochemistry》1969,65(4):545-552
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S Matsushita 《Comparative biochemistry and physiology. B, Comparative biochemistry》1983,76(3):465-470
The sucrase was purified from the small intestinal mucosa of the adult chick. Purification procedure involved solubilization with papain, ethanol precipitation, chromatography on Sephadex G-200 and DEAE-Sephadex. Several characters of the chick intestinal sucrase resembled those of the intestinal sucrase-isomaltase complex of some mammals (rabbit, rat and human). 相似文献
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Y Watanabe H Kawamoto T Imaizumi N Sakagoshi K Iwakura H Morita T Iwasaki H Yoshida 《Cellular signalling》1991,3(1):59-64
Addition of lithium ion to the inhibitory GTP-binding (Gi) protein resulted in a decrease of its ADP-ribosylation by islet-activating protein (pertussis toxin, IAP). The possibility that this decrease was due to dissociation of the Gi protein trimer was examined. Results showed that lithium ions had no appreciable effect on either the Gi protein trimer or its dissociation into its three subunits induced by Mg2+ and GTP gamma S. Next, the effect of lithium ions on Gi protein-mediated adenylate cyclase inhibition and alpha 2-adrenoceptor in human platelet membranes was examined. Lithium ion was found to impair adenylate cyclase inhibition of alpha 2-adrenoceptor stimulation of forskolin-stimulated enzyme activities. The monovalent ion also abolished guanine nucleotide modulation (GTP shift) of agonist binding, while it had no remarkable effects on antagonist binding in alpha 2-adrenoceptor of human platelet membranes. These results suggested that lithium ion caused functional change of the Gi protein without remarkable change of its dissociation, causing modulation in a coupling between alpha 2-adrenoceptor and Gi protein. 相似文献
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The effect of harmaline, a plant alkaloid has been studied on rat intestinal brush border sucrase activity. Stimulation of sucrase activity by Na+ was found to be pH-dependent. At neutral pH, 20 mM Na+ stimulated sucrase activity by reducing K(m) by 30%, while at acidic pH (5.2), the activity increased 4-fold compared to Na+-free enzyme. At 1.0 mM, harmaline markedly inhibited (67%) the enzyme activity at pH 5.2 in the absence of Na+. However, inhibition was reduced in presence of 20 mM sodium, whereas 4.0 mM harmaline was required to inhibit the enzyme activity by 65%. In the absence of Na+ ions, harmaline inhibition of sucrase activity was of competitive type, but it changed to non-competitive type in presence of 20 mM Na+ at pH 5.2. Sucrase-harmaline interactions as a function of pH, both in presence and absence of Na+ revealed a shift in pH optima of the enzyme towards a higher pH in presence of 4 mM and 1 mM harmaline respectively. The observed inhibition was reversible in nature and was only partially overcome by sodium, lithium, potassium, cesium, rubidium and ammonium ions. These findings suggest that harmaline also inhibits rat brush border sucrase and that the presence of Na+ site is not a pre-requisite for the inhibition. 相似文献
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Inhibition of porcine small intestinal sucrase by valienamine 总被引:2,自引:0,他引:2
Valienamine, an aminocyclitol, has been isolated from the enzymolysis broth of validamycins. The absolute configuration of valienamine is similar to that of alpha-D-glucose. The inhibitory effect of this amino-sugar analog of alpha-D-glucose, valienamine, on porcine small intestinal sucrase was examined. Valienamine was found to be potent, competitive reversible inhibitor of porcine small intestinal sucrase in vitro with an IC50 value of 1.17 x 10(-3)M. Valienamine also exhibited dose-dependent, instantaneous inhibition of porcine small intestinal sucrase. The inhibition of porcine small intestinal sucrase by valienamine was pH-independent. 相似文献
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Susumu Matsushita 《Development genes and evolution》1984,193(4):211-218
Summary Allantoic endoderm of 3.5-day chick embryos was cultured in recombination with digestive-tract mesenchymes of 6-day chick embryos, and the differentiation of the endoderm was studied, with special attention being given to the appearance of brush-border (BB) antigens and sucrase. Irrespective of the origin of the associated digestive-tract mesenchymes, the allantoic endoderm differentiated into a columnar epithelium, expressing BB antigens and sucrase, and also into a BB antigen-negative pseudostratified or stratified epithelium of cuboidal or columnar cells with PAS or alcian blue staining in the apical portion or a BB antigen-negative stratified squamous epithelium. These results suggest that 3.5-day allantoic endoderm has the potency to differentiate into intestinal and cloacal epithelium. 相似文献