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Solvent isotope effect on bile formation in the rat.

ABSTRACT: 2H2O affects many membrane transport processes by solvent and kinetic isotope effects. Since bile formation is a process of osmotic filtration where such effects could be important, we investigated the effects of 2H2O on bile formation in the in situ perfused rat liver. Dose finding experiments showed that at high concentrations, 2H2O increased vascular resistance and induced cholestasis; at 60% 2H2O however, a clear dissociation between the vascular and biliary effects was observed. Therefore, further experiments were carried out at this concentration. The main finding was a reduction in bile salt-independent bile flow from 0.99 +/- 0.04 to 0.66 +/- 0.04 microliters.min-1.g-1 (P < 0.001). This was associated with a 40% reduction in biliary bicarbonate concentration (P < 0.001). Choleretic response to neither taurocholate nor ursodeoxycholate was altered by 2H2O; in particular, there was a similar stimulation of bicarbonate secretion by ursodeoxycholate in the presence of 60% 2H2O. To further elucidate this phenomenon, the effect of 2H2O on three proteins potentially involved in biliary bicarbonate secretion was studied in vitro. 2H2O slightly inhibited cytosolic carboanhydrase and leukocyte Na+/H(+)-exchange, these effects reached statistical significance at 100% 2H2O only, however. In contrast, Cl-/HCO(3-)-exchange in canalicular membrane vesicles was already inhibited by 50% (P < 0.001) at 60% 2H2O. Finally, there was a slight reduction in biliary glutathione secretion while that of the disulphide was not affected. Our results are compatible with an inhibition of canalicular Cl-/HCO(3-)-exchange by 2H2O. Whether this is due to altered hydration of the exchanger and/or of the transported bicarbonate remains to be determined.

SUBMITTER: Elsing C

PROVIDER: S-EPMC1136760 | biostudies-other | 1995 Apr

REPOSITORIES: biostudies-other

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Project description:1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch [(1981) Biochemistry 20, 3189-3196]. It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS)

Project description:PurposeTransforming growth factor beta-induced protein (TGFBIp) aggregates into the phenotypic amyloid fibrils and/or non-amyloid deposits in corneal dystrophies and other disorders. While significant progress has been made in molecular genetics to successfully establish the link between the missense mutations of TGFBI and TGFBIp-related corneal dystrophies, the underlying mechanism for the abnormal aggregation remains elusive due to the lack of insights into the conformational perturbations induced by mutations. In the present study, we examined the effects of denaturants and a co-solvent on recombinant TGFBIp, with a focus on protein conformational changes and amyloid fibril formation.MethodsRecombinant TGFBIp was subjected to various spectroscopic studies, such as far-ultraviolet circular dichroism (far-UV CD), intrinsic tryptophan fluorescence and quenching, and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence, under various denaturing conditions (urea and guanidine hydrochloride [GndHCl], acidic pH, and trifluoroethanol [TFE, co-solvent]). A thioflavin T (ThT) fluorescence assay was used to determine the fibril formation of TGFBIp. In addition, a rabbit polyclonal antibody against the oligomer precursors that initiate the formation of amyloid fibrils was also used in dot blot experiments to detect the formation of prefibrillar precursors.ResultsThe purified recombinant TGFBIp is in the folded state according to its intrinsic tryptophan fluorescence analyses. A single-step unfolding process was observed in the GndHCl denaturation experiment. Results from far-UV CD, intrinsic tryptophan fluorescence, and ANS fluorescence experiments showed that TFE exerted its solvent effects by initially unfolding and transforming TGFBIp to a beta-sheet-enriched conformer at 20%. When increased to 40%, TFE changed TGFBIp into a non-native alpha-helix conformer. Although GndHCl and TFE led to protein unfolding, enhanced fibril formation could only be observed in the presence of TFE and at acidic pH, according to the ThT fluorescence assays. The paradigmatic protofibrillar TGFBIp oligomers were also detected during the fibril formation by the dot blot experiment.ConclusionsOur results suggest that protein unfolding may serve as the prerequisite but is not sufficient for the fibrillogenesis. Other factors, such as the solvent used, fragmentation, or pH, may also be crucial for the formation of TGFBIp fibrils.

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Solvent isotope effect on bile formation in the rat.

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