potassium-thiocyanate and methylamine

N,N'-Dicyclohexylcarbodiimide (DCC) has been previously shown to inhibit the amine transporter from chromaffin granules [Gasnier, B., Scherman, D., & Henry, J.P. (1985) Biochemistry 24, 3660-3667]. A study of the mechanism of inhibition is presented together with the demonstration of covalent modification of the protein. DCC inhibits binding of R1 (reserpine) and R2 (tetrabenazine) types of ligands to the transporter as well as transport. Ligands of the R2 type, but not those of the R1 type, protect against inhibition of all the reactions by DCC, i.e., accumulation of serotonin, binding if reserpine (R1 ligand), and binding of ketanserine (R2 ligand). The ability of a given R2 ligand to protect the transporter correlates well with its binding constant. Water-soluble carbodiimides, such as 1-ethyl-3-[3-(diethylamino)propyl]carbodiimide (EDC), do not have any effect on the catalytic activity of the transporter. A fluorescent hydrophobic analogue of DCC, N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4), inhibits at about the same concentration range as DCC. [14C]DCC labels several polypeptides in the chromaffin granule membranes. Labeling of a polypeptide with an apparent Mr of 80K is inhibited in the presence of R2 ligands. The labeled polypeptide copurifies with the recently identified and isolated transporter [Stern-Bach, Y., Greenberg-Ofrath, N., Flechner, I., & Schuldiner, S. (1990) J. Biol. Chem. 256, 3961-3966].

Topics: Adrenal Medulla; Animals; Carrier Proteins; Cattle; Chromaffin Granules; Dicyclohexylcarbodiimide; Hydrogen-Ion Concentration; Intracellular Membranes; Ketanserin; Kinetics; Membrane Proteins; Methylamines; Molecular Weight; Potassium; Protein Binding; Serotonin; Thiocyanates

The membrane potential (DeltaPsi) and the pH gradient (DeltapH) across the membrane of the insulin-secretory granule were determined in studies in vitro from the uptake of the permeant anion thio[(14)C]cyanate or the permeant base [(14)C]methylamine. Freshly prepared granules incubated in iso-osmotic medium containing sucrose and low concentrations of buffer salts exhibited an acidic internal pH and a DeltaPsi positive inside. Addition of MgATP(2-) under these conditions did not alter the DeltapH, but produced a marked increase in the DeltaPsi. Conversely, when a permeant anion was also included, ATP produced a marked increase in the DeltapH and a lesser increment in the DeltaPsi. NH(4) (+) salts reduced the DeltapH across granule membranes. In the presence of ATP this effect was accompanied by a reciprocal increase in the DeltaPsi. A similar reciprocity was evident when nigericin was added together with K(+) or on decreasing the medium pH, suggesting that these gradients were linked by a common electrogenic process. The effects of ATP were reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(+), and by the Mg(2+)-dependent ATPase inhibitor tributyltin. Uptakes of (14)C-labelled tracer molecules were also markedly reduced by cryogenic disruption of the granule membrane or hypo-osmotic incubation conditions. These results were readily interpreted within a chemiosmotic hypothesis, which proposed that the insulin granules possess an inwardly-directed electrogenic proton-translocating Mg(2+)-dependent ATPase with the additional postulate that the membrane has a low proton permeability. The intragranular pH was estimated as being between 5 and 6 in vivo. Such a value corresponds to optimal conditions for the crystallization of zinc-insulin hexamers. Several other functions related to chemiosmotic processes within insulin granules, however, may be envisaged.

Topics: Adenoma, Islet Cell; Adenosine Triphosphatases; Animals; Ca(2+) Mg(2+)-ATPase; Cytoplasmic Granules; Hydrogen-Ion Concentration; In Vitro Techniques; Insulin; Insulin Secretion; Membrane Potentials; Methylamines; Models, Biological; Pancreatic Neoplasms; Rats; Thermodynamics; Thiocyanates