Chloromercuri Benzenesulfonic Acid Research Articles

Effects of ABA on the Distribution of Sucrose and Protons Across the Plasmalemma of Pea Mesophyll Protoplasts. — Suggesting a Sucrose/Proton Symport

Summary The effects of abscisic acid (10 −9 −10 −6 mol/L) were studied on light induced CO 2 -dependent O 2 evolution, sucrose synthesis, efflux of sucrose and changes in cytosolic proton concentration in mesophyll protoplasts of Pisum sativum L. cv. Fenomen. Photosynthesis did not change with increased apoplastic ABA or sucrose concentrations although sucrose synthesis decreased in the presence of ABA. In the absence of apoplastic sucrose the sucrose effiux increased and was most pronounced at 10 −7 mol/L ABA. The sucrose efflux also increased in the presence of ABA and sucrose together and was most pronounced at 10 −9 mol/L ABA and 20 mmol/L apoplastic sucrose. The cytosolic proton concentration decreased upon addition of ABA with or without sucrose, but increased upon addition of p -chloromercuribenzenesulfonic acid, a sucrose transport inhibitor. The cytosolic proton concentration decreased in the presence of 10 -9 mol/L ABA in combination with various apoplastic sucrose concentrations. In the presence of 10 -7 -10 -6 mol/L ABA in combination with apoplastic sucrose levels higher than 1 mmol/L, the cyrosolic concentration of proton increases. The results indicate that there is a relationship between the external environment and the sucrose effiux and cytosolic proton concentration in pea mesophyll protoplasts and that there is a sucrose/proton symport at the plasmalemma of pea mesophyll cells. They also show that sucrose increases the sensitivity to ABA in its action on sucrose effiux and cytosolic proton concentration.

Mannitol transport by vacuoles of storage parenchyma of celery petioles operates by facilitated diffusion

Summary The sugar alcohol mannitol is the main water-soluble carbohydrate of the storage parenchyma of celery petioles. Compartmentation studies had indicated that mannitol was about equally distributed between the large central vacuole and the cytosol of these cells (Keller, F. and P. Matile, New Phytol., 113, 291–299 [1989]). Evidence is now presented for a tonoplast-bound mannitol carrier that operates by facilitated diffusion, i.e. protein-mediated and passively. With isolated parenchyma vacuoles it was shown that the uptake rate of mannitol, at 1 mmol/L external concentration, was about twice that of L-glucose and glucitol (sorbitol), both established markers for simple diffusion. Mannitol uptake was a saturable process with apparent K m and V max values of 14 mmol/L and 0.067 nmol/μL vacuoles · min, respectively. It was also quite specific as indicated by competition studies with glucitol, galactitol, glucose, and fructose. The functionality of the mannitol carrier was unaffected by three protein-modifying reagents, the sulfhydryl group modifiers p -chloro mercuribenzenesulfonic acid (PCMBS) and N -ethylmaleimide (NEM) and the histidine modifier diethyl pyrocarbonate (DEPC). Mannitol uptake was neither stimulated by energization with ATP or pyrophosphate nor was it impaired by dissipation of the pH gradient or the membrane potential and is, therefore, passive. The implications of this facilitated diffusion mechanism for the proposed physiological functions of mannitol in celery are discussed.

Covalent crosslinking of human coagulation factor V by activated factor XIII from guinea pig megakaryocytes and human plasma

Coagulation factor V (FV) has been shown to be synthesized in both the liver and megakaryocytes. We now present evidence that FV can be covalently crosslinked by an enzyme originating from megakaryocytes to form polymeric multimers of factor V. The guinea pig megakaryocyte enzyme appears to be factor XIIIa since the FV-crosslinking activity (1) had an absolute requirement for Ca++, (2) was completely inhibited by iodoacetamide, 5,5′-dithiobis- (2-nitrobenzoic acid), p- chloromercuribenzene sulfonic acid, and N-ethylmaleimide, all known alkylators of the thiol group at the active site of the factor XIIIa, (3) was blocked by known pseudoamine donor substrates of factor XIIIa including dansylcadaverine and putrescine, and (4) could be directly demonstrated in the guinea pig megakaryocyte lysate by a specific activity staining procedure. No tranglutaminase was detected in guinea pig megakaryocytes in contrast to red cells and liver. A similar pattern of covalent crosslinking of human FV by purified activated human plasma factor XIII was also demonstrated. Analysis of the crosslinked products of FV formed by the guinea pig enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicates the formation of intermediate as well as higher molecular weight polymers, suggesting that the crosslinking is a stepwise polymerization process.

Selective Solubilization of Membrane Proteins Differentially Labeled by p-Chloromercuribenzenesulfonic Acid in the Presence of Sucrose

Broadbean (Vicia faba L.) leaf discs have been incubated with the slowly permeant thiol reagent [(203)Hg]-para-chloromercuribenzenesulfonic acid (PCMBS) in the presence or in the absence of sucrose, and the release of PCMBS-labeled proteins has been monitored in media containing various concentrations of urea, ethyleneglycol-bis-(beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA), sodium cholate, sodium dodecyl sulfate, Triton X-100, octylglucoside or (3-[3-cholamidopropyl)-dimethylammonio] 1-propane-sulfonate) (CHAPS). The proteins differentially labeled by PCMBS in the presence of sucrose which, on the basis of previous results, are assumed to include the sucrose carrier, were preferentially solubilized by 1% CHAPS, 1% octylglucoside, or 1% Triton X-100. Other PCMBS-labeled proteins (;background' proteins) could be partially removed by EGTA, urea, or 0.1% cholate. Sequential treatment by 10 mm EGTA and 1% CHAPS was found to give a fraction highly enriched in the differentially labeled proteins. Analysis of the specific activity of microsomal pellets suggests that the results obtained with leaf discs give a good account of what is occurring at the plasma membrane level. These data, which suggest that the proteins differentially labeled by PCMBS in the presence of sucrose are intrinsic membrane proteins, can be used to solubilize these proteins from microsomal fractions.

Inhibition of phosphate transport in human erythrocytes by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl)

Treatment of intact human erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) leads to inhibition of anion transport as measured by [ 32P]phosphate exchange for intracellular chloride. Inhibition is rapid at 37°C (80% inhibition, 1.7 mM NBD-Cl, 3 min, pH 6.9) and not reversed by washing the cells with 1% bovine serum albumin in isotonic sucrose citrate buffer. Pretreatment of cells with N- ethylmaleimide and p- chloromercuribenzenesulfonic acid enhanced transport inhibition by NBD-Cl. Transport inhibition caused by brief incubations of erythrocytes with NBD-Cl could be almost completely reversed with dithiothreitol or β-mercaptoethanol. Prolonged incubation (60 min, 37°C, pH 6.4, sucrose-citrate buffer) following NBD-Cl treatment leads to partial reversal of transport inhibition. The residual inhibition is then only partially reversed by dithiothreitol treatment. Reversal of transport inhibition of dithiothreitol or β-mercaptoethanol may be prevented by incubation of the erythrocytes with sodium dithionite. Phosphate transport was readily inhibited by other tyrosine-directed reagents, tetranitromethane (55% inhibition, 1.6 mM, 3 min, 37°C, pH 8.3 in sucrose-citrate medium) and p- nitrobenzene sulfonyl fluoride (31% inhibition, 1.8 mM, 3 min, 37°C, pH 8.1 in sucrose-citrate medium) but not by N- acetylimidazole (10% inhibition, 37.5 mM, 30 min, 37°C, pH 7.5). These results suggest that NBD-Cl inhibits anion exchange by two mechanisms; a rapid inhibition reversible by sulfhydryl reagents, possibly due to modification of a tyrosine residue(s), and a slower irreversible inhibition due to modification of an essential amino group in the transporter.

On the water and proton permeabilities across membranes from erythrocyte ghosts

The diffusional permeability of water across membranes from bovine and human erythrocyte ghosts was measured by a recently developed method which is based on the different indices of refraction of H 2O and 2H 2O. Resealed erythrocyte ghosts were prepared by a gel-filtration technique. P d ( 2H 2O/H 2O) values of 1.2 · 10 −3 cm/s (human) and 1.7 · 10 −3 cm/s (bovine) were calculated at 20°C. The activation energies of the water exchange were 23.5 kJ/mol (human) and 25.4 kJ/mol (bovine). Treatment of the ghosts with p- chloromercuribenzenesulfonic acid (PCMBS) led to a 60–70% inhibition of the diffusional water exchange. The pH equilibration across membranes of erythrocyte ghosts was measured by intracellular carboxyfluorescein. The rates of proton flux after pH-jumps (pH 7.3 to pH 6.1) were about 100-fold lower than those of the water exchange and dependent on the kind of anions present (Cl −, NO 3 −, SO 4 2−). The activation energies of proton flux were 60–70 kJ/mol. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) inhibited the exchange by 97–98% and lowered the activation energy. The inhibitor of water exchange, PCMBS, increased the proton-permeation rate by a factor of 4–5. It is assumed that the rate-limiting step for the proton permeation is determined by the anion exchange. Under this condition our results are not in accord with one channel as a common pathway for both the passive water and anion transport.

Effect of Inhibitors on Solute Efflux from Seed-Coat Halves and Cotyledons of Pisum sativum L., after Uptake from a Bathing Medium. : The Difference between Sucrose and Amino Acids

Summary Net sucrose and amino acid efflux from excised seed-coat halves and cotyledons of developing seeds of Pisum sativum L. into a bathing medium (pH 5.5) was measured over a period of 5 h, after uptake from a solution containing a mixture of 3 H - and 14 C-labelled solutes. Net efflux of amino acids (α-aminoisobutyric acid, valine and asparagine) from seed-coat halves and cotyledons was strongly inhibited by p -chloromercuribenzenesulfonic acid (PCMBS). Net efflux of labelled sucrose was not markedly inhibited by a treatment with PCMBS; under certain conditions it was enhanced by PCMBS. The effect of a low-temperature (0 °C) treatment did not differ much from the effect of a treatment with PCMBS. Net efflux of labelled solutes from seed-coat halves and cotyledons was enhanced by KCN, but this effect was much more pronounced in the case of sucrose than in the case of amino acids. The parallel between seed-coat halves and cotyledons illustrates the point that the seed coat contains tissues with ≪cotyledon characteristics≫ (e.g. intensive resorption of sucrose). After unloading into the seed coat apoplast from the seed-coat unloading sites, amino acids encounter less resorption activity than sucrose.

Effect of KCN and p‐chloromercuribenzenesulfonic acid on the release of sucrose and 2‐amino(1‐14C)isobutyric acid by the seed coat of Pisum sativum

The process of sugar and amino acid release by the seed coat of Pisum sativum L. cv. Marzia was studied. Prior to measuring the release of solutes by the seed coat of developing ovules, the embryo was removed from each ovule studied. After this surgical treatment, each “empty” seed coat was filled with the appropriate solution (pH 5.5) with or without inhibitor. Both KCN and p‐chloromercuribenzenesulfonic acid (PCMBS) strongly inhibited the release of sucrose and p‐aminoisobutyric acid (AIB) by the seed‐coat. These data support the view that phloem unloading is an energy‐dependent process sensitive to the sulfhydryl group modifier PCMBS. In pulse‐labelling experiments, addition of high concentrations of unlabelled sucrose (200 mM) and AIB (25 mM) to the solution filling the seed coat cavity did not diminish the release of labelled solutes by the unloading sites of the seed coat. This observation presents evidence against the view that phloem unloading into a strong sink is related to low sugar concentrations in the apoplast.

Characterization of partially purified [formula omitted] from porcine lens

The partial purification of ( Na + + K +)- ATPase from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in p- nitrophenylphosphatase activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa α subunit of ( Na + + K +)- ATPase which can be phosphorylated by reaction with [γ- 32P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51 000 and thus appears to be the β subunit of the enzyme. The enzyme is sensitive to ouabain with the I 50 for ( Na + + K +)- ATPase and p- nitrophenylphosphatase inhibition being 1.2 and 1.3 μM, respectively. Several agents which inhibit Na + + K +)- ATPase from other tissues such as oligomycin, Ca 2+, vanadate, N- ethylmaleimide , p- chloromercuribenzenesulfonic acid (PCMBS) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K + are partially effective in activating the ( Na + + K + )-ATPase and p- nitrophenylphosphatase activities. The K + congeners were relatively more effective in supporting ( Na + + K +)- ATPase compared to p- nitrophenylphosphatase activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of ( Na + + K +)- ATPase activity, p- nitrophenylphosphatase activity and fluoresence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed.

Purification and some properties of inducible [formula omitted] kinase from Candida albicans

N- Acetylglucosamine kinase (ATP:2-acetamido-2-deoxy- d-glucose 6-phosphotransferase, EC 2.7.1.59) catalyzes the first reaction in the inducible N- acetylglucosamine catabolic pathway of Candida albicans, an obligatory aerobic yeast. As a part of continuing biochemical studies concerning the regulation of gene expression in a simple eukaryote, N- acetylglucosamine kinase has been purified and characterized biochemically. The enzyme has been purified about 300-fold from the crude extract and its molecular weight of 75 000 has been determined by Sephadex G-100 gel filtration. Isolation and analysis procedures are described. The kinase reaction is optimal within a pH range of 7–8. The enzyme is strictly specific for GlcNAc as phosphate acceptor; ATP is the phosphoryl group donor for the kinase reaction and to a lesser extent dATP and CTP. K m values for GlcNAc and ATP are 1.33 mM and 1.82 mM, respectively. The enzyme requires Mg 2+, which may be replaced by other bivalent metal ions such as Mn 2+, Ca 2+, Ba 2+ and Co 2+ for a lesser degree of effectiveness. The purified enzyme is extremely sensitive to thermal denaturation and becomes completely inactive by heating at 65°C for 2 min. The enzyme is also inactivated by sulphydryl reagents such as p- chloromercuribenzene sulfonic acid and N- ethylmaleimide .

The exchange and maximal net flux of glucose across the human erythrocyte. II. The effect of two sulphydryl enzyme inhibitors, chlormerodrin and [formula omitted] sulfonic acid

The exchange and maximal net fluxes of [ 14C]glucose across the membrane of the human red cell were measured. The effects of p- chloromercuribenzene sulfonic acid and chlormerodrin on these two parameters of glucose transport were determined. At low concentrations p- chloromercuribenzene sulfonic acid (a non-penetrating organic mercurial) was found to readily inhibit the exchange flux but not the net efflux. At extremely high concentrations of p- chloromercuribenzne sulfonic acid the maximal net efflux showed some degree of inhibition. Chlormerodrin (a penetrating organic mercurial) inhibited both the exchange and net fluxes in the same manner. The addition of insulin in certain instances reduced the degree of inhibition caused by the organic mercurials. Insulin had no effect on the amount of either p- chloromercuribenzene sulfonic acid or chlormerodrin which bound to the red cell. From the results obtained, it is suggested that there exist glucose-reactive sites on both the outer and inner surfaces of the membrane. The results also suggest a carrier system possessing different sites or molecular arrangements for glucose egrees and for glucose entry.