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Experiments allowing Na+-dependent short-term uptake measurements by ileal brush border vesicles were described. Glucose uptake was compared with taurocholate uptake in the presence of NaCl, NaSCN and Na2SO4. In contrast to the observation made with glucose, taurocholate transport was the same for the three electrolytes, indicating electroneutral taurocholate transport.
The effect of anhydro-4-epitetracycline on sodium gradient-dependent D-glucose transport of rabbit renal brush-border membrane vesicles was studied. The purity of isolated brush-border membrane vesicles as judged by enzyme activities was not different between normal control and anhydro-4-epitetracycline-administered rabbits. There was no difference in estimate of intravesicular volume, either. When NaCl was used for sodium gradient, the overshoot of D-glucose uptake into brush-border membrane vesicles isolated from anhydro-4-epitetracycline-treated rabbits was significantly smaller than that of normal control rabbits. In the cases of NaSCN or Na2SO4, the former was also smaller than the latter, but not significantly so. To avoid the possible effect of membrane potential on D-glucose uptake, the voltage-clamp method was applied. Even in the voltage-clamped condition, the overshoot of D-glucose uptake into vesicles from anhydro-4-epitetracycline-treated rabbits was decreased compared to that of normal rabbits. In vitro incubation of brush-border membrane vesicles with 20 mM anhydro-4-epitetracycline caused no alteration in sodium gradient-dependent D-glucose uptake. Our results demonstrate that there exists a disorder in sodium gradient-dependent D-glucose uptake of renal brush-border membrane in anhydro-4-epitetracycline-treated rabbits, and suggest that this disorder is one of the underlying mechanisms of experimental Fanconi syndrome.
Although a number of molecules are known to function as current-carrying proton carriers across lipid bilayer membranes, no such hydroxide ion carriers have been found to date. We report that (C6F5)2Hg, which can function as a chloride ion carrier, can also carry a hydroxide ion. In 100 mM Na2SO4 solutions, membranes treated with (C6F5)2Hg are almost ideally selective for H+/OH- between pH 6.0 and 9.5. Membrane conductance varies linearly with [OH-] over this pH range and with the square of the (C6F5)2Hg concentration. The presumed current-carrying species is the dimer [(C6F5)2Hg]2OH-, which, along with the neutral molecule (C6F5)2Hg, shuttles back and forth within the bilayer. In 0.2 M NaCl at pH 9.5, the OH- and Cl- conductances are approximately equal. Thus, the carrier displays an approximately 10(4)-fold preference for OH- over Cl-.
The phase behaviour of L-alpha-1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) was examined in aqueous dispersions containing a range of sodium salts. The phase properties of the lipid exhibited a graded response to the presence of simple anions analogous to that of the Hofmeister series encountered in the study of the solution properties of proteins. Salts early in the series (such as Na2SO4 and NaCl) gave rise to substantial decreases in the temperature (Th) of the transition from the lamellar liquid-crystal to inverted hexagonal phase (L alpha----HII) and small increases in the temperature (Tm) of the transition from the lamellar gel to lamellar liquid-crystal phase (L beta----L alpha). Salts towards the end of this series (such as NaI and NaSCN) led to increases in Th and decreases in Tm. Similar effects were seen in lipid dispersions containing a series of non-ionic co-solutes. In both cases, the relative efficiency of perturbation of the lipid phase properties reflected the relative ability of the anion or co-solute to influence the structure of the bulk water. X-ray diffraction measurements indicated that these effects were probably mediated through the ability of the co-solutes to bring about changes in the extent of the lipid/water interface.
A cerebroside sulphatase (cerebroside-3-sulphate 3 sulphohydrolase, EC 3.1.6.8) assay based on radio thin-layer chromatography is described. The substrate was labelled by the catalytic addition of tritium to cerebroside sulphate. Using this assay the cerebroside sulphatase activity of sulphatase A (Aryl-sulphate sulphohydrolase, EC 3.1.6.1) from human liver and kidney in the absence of activators was investigated. The pH optimum of this reaction depends on the buffer concentration, being pH 4.5 at 50 mM and 5.3 at 10 mM sodium formate. With the latter concentration the apparent Km for cerebroside sulphate is 0.06 mM; SO2-4 and nitrocatechol sulphate inhibit noncompetitively with a Ki of 4.51 mM for Na2SO4 and 0.43 mM for nitrocatechol sulphate. The cerebroside sulphatase activity of sulphatase A is highly dependent on the ionic strength. The optimum sodium formate concentration is 10 mM, and the cerebroside suophatase activity decreases rapidly with increasing buffer concentration. The same concentration dependence is observed in the inhibitory effect of cerebroside sulphate on the arylsulphatase reaction. The inhibition decreases at increasing buffer concentrations, becoming an activation at 70 mM sodium formate. The progress curve of the cerebroside sulphatase reaction shows a deviation from linearity similar to that of the arylsulphatase reaction. Investigation of the effect of preincubation with cerebroside sulphate on the arylsulphatase activity of the enzyme shows that cerebroside sluphatase activity and inactivation of the enzyme by cerebroside sulphate occur simultaneously. These observations are interpreted as supporting the assumption that cerebroside suophate and arylsulphates are degraded at an identical active site on the same enzyme. Differences in the properties of the cerebroside sulphatase and the arylsulphatase reaction of the enzyme may be attributed to the differences in the physiocochemical state of the two substrates.
The activity of two purified homogeneous phosphoprotein phosphatases types P I and P II) (phosphoprotein phosphohydrolase, EC 3.1.3.16) from rabbit liver (Khandelwal, R.L., Vandenheede, J.R., and Krebs, E.G. (1976) J. Biol. Chem. 251, 4850-4858) were examined in the presence of divalent cations, Pi, PPi, nucleotides, glycolytic intermediates and a number of other compounds using phosphorylase a, glycogen synthase D and phosphorylated histone as substrates. Enzyme activities were usually inhibited by divalent cations with all substrates; the inhibition being more pronounced with phosphorylase a. Zn2+ was the most potent inhibitor among the divalent cations tested. The enzyme was competitively inhibited by PPi (Ki = 0.1 mM for P I and 0.3 mM for PII), Pi (Ki = 15 mM for P I and 19.8 mM for P II) and p-nitrophenyl phosphate (Ki = 1 mM and 1.4 mM for P I and P II, respectively) employing phosphorylase a as the substrate. The compounds along with a number of others (Na2SO4, citrate, NaF and EDTA) also inhibited the enzyme activity with the other two substrates. Severe inhibition of the enzyme was also observed in the presence of the adenine and uridine nucleotides; monophosphate nucleotides being more inhibitory with phosphorylase a, whereas the di- and triphosphate nucleotides showed more inhibition with glycogen synthase D and phosphorylated histone. Cyclic AMP had no significant effect on enzyme activity with all the substrates tested. Phosphorylated metabolites did not show any marked effect on the enzyme activity with phosphorylase a as the substrate.
Purified chondrocytic alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and inorganic pyrophosphate. Optimal activities for p-nitrophenyl phosphate, ATP and inorganic pyrophosphate are found at pH 10.5, 10.0 and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations. p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasng substrate concentration. Heat inactivation studies indicate that both phosphorolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ (0.1-10.0 mM) and Mn2+ (0.01-0.1 mM) show a small stimulation of p-nitrophenyl phosphate-splitting activity at pH 10.5. Levamisole, Pi, CN-, Zn2+ and L-phenylalanine are all reversible inhibitors of the phosphomonoesterase activity. Pi is a competitive inhibitor with a Ki of 10.0 mM. Levamisole and Zn2+ are potent non-competitive inhibitors with inhibition constants of 0.05 and 0.04 mM, respectively. The chondrocytic alkaline phosphatase is inhibited irreversibly by Be2+, EDTA, EGTA, ethane-1-hydroxydiphosphonate, dichloromethane diphosphonate, L-cysteine, phenyl-methylsulfonyl fluoride, N-ethylmaleimide and iodoacetamide. NaCL, KCL and Na2SO4 at 0.5-1.0 M inhibit the enzyme. At pH 8.5, the cleavage of inorganic pyrophosphate (pyrophosphate phosphohydrolase, EC 3.6.1.1) by the chondrocytic enzyme is slightly enhanced by low levels of Mg2+ and depressed by concentrations higher than 1mM. Ca2+ show only inhibition. Similar effects of Mg2+ and Ca2+ on the associated ATPase (ATP phosphohydrolase, EC 3.1.6.3) activity were observed. Arrhenius studies using p-nitrophenyl phosphate and AMP as substrates have accounted for the ten-fold difference in V in terms of small differences in both the enthalpies and entropies of activation which are 700 cal/mol and 2.3 cal/degree per mol, respectively.
The dependence of alpha-chymotrypsin thermostability and catalytic activity on the degree of its amino groups modification has been studied. Modification was carried out by both alkylation (using acrolein with further reduction of Schiff bases by sodium borohydride) and acylation (with siccinic or acetic anhydrides). It has been determined that modification of the majority of titrated amino groups (approximately 80%) only has a slight effect on the first-order rate-constant characterizing the monomolecular process of enzyme thermoinactivation (50 degrees C, pH 8). Thermostability sharply increases (by 120 times) only for a degree of modification higher than 80%, but, nevertheless, the complete substitution of all the titrated amino groups again leads to enzyme destabilization. The conclusion has been drawn that there is only one or two amino groups out out approximately fifteen titrated ones, the modification of which plays a key role in the lateration by the enzyme of its thermostability. The degree of the stabilization effect has been studied relative to both the nature and concentration of the salt added Na2SO4, NaCl, KCl, CCl3COOK, (CH3)4NBr. Ultraviolet absorption (280 nm) of chymotrypsin has also been elucidated with respect to the degree of alkylation of its NH2-groups. The data obtained allowed the conclusion to be drawn that enzyme modification leads to a decrease in the non-electrostatic (hydrophobic) interactions on the surface layer of the globule. As a result, a protein conformation more stable in respect to denaturation (unfolding), is formed.
Bovine liver D-glycerate dehydrogenase (D-glycerate:NAD (NADP) oxidoreductase, EC 1.1.1.29) adapts its kinetic behaviour to a sequential mechanism. The presence of NaCl causes an appreciable variation in the Km and V values. relative to the both substrates in the hydroxypyruvate/D-glycerate dehydrogenase/NADH system, which does not happen in the D-glycerate/D-glycerate dehydrogenase/NAD system. The former system is inhibited by high concentrations of NaCl and activated by low salt concentrations. The hydroxypyruvate concentration causing substrate inhibition increases as the concentration of NaCl increases; excess NADH inhibition is independent of the salt concentration. The variation of the initial rates of both systems, in the presence of chlorides having monovalent and divalent cations, or sodium halides, Na2SO4 and NaNO3 (at constant ionic strength) suggests that the anions have a specific action on the enzyme. An increase in the NaCl concentration causes a displacement of the optimum D-glycerate dehydrogenase pH (with hydroxypyruvate and NADH as substrates) towards the acid area. The enzyme stability, at varying pH, varies with the salt concentration.
Pseudomonas cytochrome oxidase (EC 1.9.3.2) is composed of two subunits. Each subunit has a molecular weight of approx. 63000 and, according to the iron determination, contains two hemes. Cytochrome oxidase was subjected to various dissociation procedures to determine the stability of the dimeric structure. Progressive succinylation of 14 to 68% of the lysine residues of the enzyme increases the amount of the protein appearing in the subunit form (S20,W approximately 4 S) from 18 to 92%. At a high degree of succinylation a component with a sedimentation coefficient of approx. 2 S appears. The subunits with sedimentation coefficients of approx. 4 S and 2 S are also formed when the pH is below 4 or above 11. The same molecular weight (63000) was found for these two components in sodium dodecylsulphate electrophoresis. No dissociation of cytochrome oxidase was observed in salt solutions like 3 M NaC1 and 1 M Na2SO4, or in 6 M urea. The slight decrease in the sedimentation coefficients in NaC1 solutions is partly explained by preferential hydratation of the protein.
The structure of spectrin from human erythrocytes has been investigated by the EPR technique measuring the mobility of the protein spin label, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. Conformational changes in the protein induced by variation of the concentrations of NaCl, Na2SO4, KCl, CaCl2 and MgCl2 and of pH have been studied. It could be demonstrated that both Ca2+ and Mg2+ give rise to structural changes by binding to specific sites, whereas the monovalent cations (K+, Na+) seem to act via ionic strength. A model is used to correlate the spin label mobility with the radius of the protein. In the Ca2+- and Mg2+-binding experiments, the decrease in the spin label mobility has been interpreted on the basis of the theory of multiple chemical equilibria. These experiments have been compared with EPR spectra measured at different pH values. The results support the model in that binding of H+, Ca2+ or Mg2+ reduces the charges located on the protein surface: the 'discharging' reduces the repulsive forces on the surface of the molecule and consequently, the protein contracts in discrete steps.
Previously reported inhibitors of benzo[a]pyrene (BaP) mutagenicity in Salmonella typhimurium strain TA98 were tested for their effectiveness against the mutagenicity of 6-methyl-benzo[a]pyrene (6-CH3-BaP), 6-hydroxymethyl-benzo[a]pyrene (6-CH2OH-BaP) and 6-acetoxymethyl-benzo[a]pyrene (6-CH3COOCH2-BaP). Dose-response curves obtained for phenothiazine (PTH), 2-chlorophenothiazine (2Cl-PTH), phenylisothiocyanate (PHN), phenethylisothiocyanate (PNE), trans-retinol (TR) and disulfiram (TETD) showed a variety of degrees of inhibition of mutagenicity. Additionally, glutathione (GSH) was found to inhibit the mutagenicity of 6-CH3COOCH2-BaP, and the mutagenicity of 6-CH2OH-BaP was enhanced by the addition of supplemental ATP, Na2SO4 and EDTA. Only 2Cl-PTH was equally as good an inhibitor of 6-CH3-BaP and BaP, reducing revertant colonies to less than 50% of control at 10 X BaP concentration. To probe the mechanism of inhibition, the effect of 2Cl-PTH on the binding of BaP and the 6-substituted benzo[a]pyrenes to cytochrome P-450 was investigated by difference spectroscopy. Also, the effect of 2Cl-PTH on the subsequent metabolism of 6-CH3-BaP and 6-CH2OH-BaP was investigated by rapid scan difference spectroscopy and high-performance liquid chromatographic separation of products. The results are consistent with a major mechanism of inhibition for 2Cl-PTH involving a competition for the cytochrome P-450 binding site.
Fibers of the frog glossopharyngeal nerve that are sensitive to water stimulation also respond to Ca, Mg and Na salts. During stimulation with a salt, the cation and the anion are applied together and the anion could influence the response to the cation. We examined this interaction using single unit recordings at the level of fungiform papilla. Nerve impulses of large amplitude were recorded in response to the stimulation of adjacent papillae with increasing concentrations of various Ca, Mg and Na salts. For a given cation, the elicited spike frequency depended on the anion. For example, the responses of single fibers to Ca2+ concentrations above 0.1 mM were maximal with CaSO4 and minimal with Ca(SCN)2. The rank order of efficiency was CaSO4 > CaCl2 = CaBr2 = Ca(NO3)2 > Ca(SCN)2 for Ca2+ ions at 5 mM. The effects of these anions were reversed for Mg and Na salts, the rank orders being Mg(SCN)2 > Mg(NO3)2 > MgBr2 = MgCl2 > MgSO4, for Mg2+ ions at 200 mM, and NaSCN > NaI > NaNO3 > NaBr > NaCl >> NaF = Na2SO4, for Na+ ions at 500 mM. All these sequences correspond to the lyotropic rank order of the anions. In stimulation by a mixture of Ca and Na salts, which have different rank orders with respect to anions, either the response to Ca2+ ions or the response to Na+ ions could be eliminated as a result of mutual antagonism between Ca2+ and Na+ ions. In this case, the rank order of anions was dependent only on the cation that was able to exert a stimulatory effect in the mixture. Threshold concentrations for Ca, Mg and Na salts are influenced by cationic properties, but not by anionic properties. We hypothesize that anions can modulate the efficacy of cation transduction by binding to a membrane element that interacts with each of the three distinct receptors for Ca2+, Mg2+ and Na+ ions without altering the affinities of these receptors for the respective cations. The present results cannot be interpreted in terms of permeability of the apical membrane to anions and changes in surface potential on the apical membrane. The possibility is discussed that an anion-selective paracellular pathway between taste cells is responsible for the effect of anions on the cation-induced response.
We have studied the specificities of two human monoclonal, IgM containing sera, s/IgMMAC and s/IgMFIS, from patients with polyneuropathy. s/IgMMAC precipitates only with chondroitin sulfate C and not with A and B whereas s/IgMFIS is precipitated by chondroitins A, B (dermatan sulfate), and C. Inhibition assays using 2-acetamido-2-deoxy-3-O-(4-deoxy-beta-L-threo-hex-4-enopyranosyluroni c acid)-D-galactose and its 6- and 4-sulfate derivatives showed that the disaccharide 6-sulfate was the best inhibitor of precipitation of s/IgMMAC by chondroitin sulfate C, and the disaccharide 4-sulfate the best inhibitor of precipitation of s/IgMFIS by either chondroitin sulfates C or B. The nonsulfated disaccharide was a good inhibitor in each instance. D-Glucose 6-sulfate, Na2SO4, several sugar phosphates, and phosphate buffer also inhibited but to different extents with the s/IgMMAC and s/IgMFIS. All studies were carried out in 0.15M NaCl. The data indicate that both monoclonal proteins are antibodies comparable to the phosphorylcholine-binding myeloma proteins, and that the reactions show specificities above and beyond charge effects. The relation of various cross-reacting macromolecules to the monoclonal antibody was studied by diffusion in gels.
In an attempt to explain the mechanism of comutagenesis by bisulfite, the extent and accuracy of DNA synthesis by E. coli DNA polymerase I was examined in the presence of sodium bisulfite. Bisulfite concentration of 100 mM caused nearly complete inhibition of dNTP incorporation into activated calf thymus DNA. Other salts (NaCL, Na2SO4) at the same concentration had no effect on enzyme activity. Preincubation of the various DNA synthesis assay components in 100 mM bisulfite showed that only preincubation of DNA polymerase I caused inhibition of DNA synthesis. Exonuclease functions of DNA polymerase I were unaffected by up to 100 mM bisulfite. Accuracy of DNA synthesis in the presence of bisulfite was determined using poly (dA-dT) as a template-primer. Concentrations of bisulfite greater than 50 mM caused a progressive decrease in enzyme accuracy. At 100 mM bisulfite there was an approximate 7.5-fold decrease in the fidelity of DNA synthesis, compared to control values, as measured by the ratio of noncomplementary (dGTP) to complementary (dTTP) nucleotide incorporated. Based on the known chemistry of bisulfite, it is hypothesized that sulfitolysis of the one disulfide group in DNA polymerase I by bisulfite might be responsible for the reduced polymerase activity and accuracy. The exonuclease functions of DNA polymerase I do not seem to require the disulfide linkage. These results suggest that the effects of bisulfite on mutation frequency might be mediated by effects on the fidelity of DNA repair systems.
1. A radiolabelled tetrasaccharide mixture (GlcNS-UA-GlcNS-UOA) containing GlcNSO3-UA-GlcNSO3-L-[6, 3H]idonic acid, GlcNSO3-UA-GlcNSO3-anhydro-L-[6, 3H]idonic acid and GlcNSO3-UA-GlcNSO3-L-[6, 3H]gulonic acid was evaluated together with a radiolabelled disaccharide O-(alpha-2-sulfamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-L-[6, 3 H]idonic acid (GlcNS-IdOA) and a trisaccharide GlcNSO3-UA-D-[1, 3 H]glucosaminitol N-sulfate (GlcNS-UA-GlcitolNS) as diagnostic substrates for sulfamidase present in cultured human skin fibroblasts and leucocytes. 2. Sulfamidase activity assessed with GlcNS-UA-GlcNS-UOA was up to 10 times higher than the value obtained for GlcNS-IdOA and the trisaccharide. These results demonstrate that an adjacent GlcNS-UOA disaccharide residue to the sulfaminoglucosamine under attack may play a role in the mechanism of action or binding of sulfamidase toward its substrates. 3. Sulfamidase activity in fibroblast and leucocyte homogenates with GlcNS-UA-GlcNS-UOA exhibited a pH optimum at pH 5.0, an apparent Km of 27 to 50 mumol/l and inhibition by both NaCl and Na2SO4. 4. No detectable sulfamidase activity toward the tetrasaccharide, trisaccharide and disaccharide substrates could be detected using homogenates of fibroblast cultures from Sanfilippo A patients (sulfamidase deficient). Sulfamidase activity assayed with GlcNS-UA-GlcNS-UOA clearly distinguished Sanfilippo A patients from normal controls, heterozygotes and other mucopolysaccharidosis types. Because of the higher activity of sulfamidase toward the tetrasaccharide substrate, compared to that observed for the other substrates evaluated, we recommend its use for the routine enzymic detection of the Sanfilippo A syndrome.
Cortical granule exocytosis in sea urchins was studied using hyperosmotic and polymer-containing seawater to halt granule matrix dispersal. Addition of Na2SO4-containing seawater (2.5 osmole/kg) to Strongylocentrotus purpuratus eggs 10 to 40 sec after insemination resulted in arrest of the exocytic wave during propagation. EM examination of these eggs revealed that matrix disassembly occurred in distinct stages. In the earliest stage, granule-plasma membrane fusion had occurred, but the matrix remained completely intact. This early stage was observed in hyperosmotic media, either ionic or nonionic, suggesting that matrix hydration is required for disassembly and exocytic pore widening, but not for membrane fusion. Subsequent stages, in which partially disassembled matrices remained within omega-configured pockets, were captured by activating eggs in 30% dextran in seawater. Stability of these intermediates stages required the presence of Ca2+ and Mg2+; in the absence of divalent cations the matrices completely disassembled and the exocytic pockets flattened. Divalent cations appeared to prevent fragmentation of the matrix lamellae. Late stages of matrix disassembly, in which the lamellae fragmented and formed small particles, were inhibited by media of high ionic strength. Hyperosmolality alone, provided by sucrose, was unable to halt these late stages suggesting that water availability does not play an important role once a critical point in matrix dispersal has been reached.
The high density of 'peripheral-type' benzodiazepine receptors (PBR) in the thick ascending loop of Henle (TAL) of the kidney prompted an evaluation of the effects of anions and anion channel blockers on these sites. [3H]Ro 5-4864 binding to rat kidney membranes was inhibited by halides in a concentration dependent fashion. Significant differences were observed in the efficacies (i.e. maximal effect achieved) of these halides to inhibit [3H]Ro 5-4864 binding. The effects of halides to inhibit this binding was manifest as a reduction in the apparent affinity of this radioligand with no effect on the maximum number of binding sites. The concentration necessary to achieve half maximal inhibition was around the physiologic range for Cl- in all extracellular body fluids. A strong correlation (r = 0.95; P less than 0.001) was obtained between the permeability of anions relative to Cl- and their efficacies to inhibit [3H]Ro 5-4864 binding. DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), an ion transport blocker, inhibited [3H]Ro 5-4864 binding to a maximum of 80%. The presence of Na2SO4 enhanced the potency of DIDS up to five-fold giving an IC50 of 43 +/- 6 microM. In contrast, the binding of [3H]PK 11195 was unaffected by DIDS and only slightly inhibited by I-. These data demonstrate that characteristics of radioligand binding to peripheral-type benzodiazepine receptors in the kidney are regulated by anions and anion transport blockers and suggest that the PBR may play an important role in anion transport. Additionally, these data suggest that the sites at which [3H]Ro 5-4864 and [3H]PK 11195 bind on the PBR may be overlapping, but not identical.