Subcellualr distribution of protein carboxymethylase and its endogenous substrates in the adrenal medulla: possible role in excitation-secretion coupling.

Abstract

Protein carboxymethylase (S-adenosyl-L-methionine:protein O-methyltransferase, EC 2.1.1.24) transfers a methyl group from S-adenosyl-L-methionine to carboxyl side chains of proteins to form labile protein-methyl esters which, thus, neutralize negative charges. This enzyme was examined for its possible participation in excitation-secretion coupling in the adrenal medulla. Protein carboxymethylase has a specific activity several times higher in the adrenal medulla than in the adrenal cortex; also, the medulla has a higher concentration of methyl-acceptor proteins. In the adrenal medulla, 97% of the enzyme was localized in the cytosol. Of the various subeellular fractions of the medulla, the catecholamine-containing chromaffin vesicles had the highest concentrations of substrate~s) for (4,5). During exocytosis, the vesicle and plasma membrane fuse and the intravesicular products are discharged into the extracellular space (6-10). Membrane fusion is rapidly followed by retrieval of the excess membrane by micropinocytosis (7,11,12). The cytoplasmic surface of the plasma and vesicle membranes have net negative charges (4,5,13) and the electrostatic barrier should be decreased before the two membranes come into contact and fuse (4, 5). The reversal of the change in surface charge may contribute to the retrieval of the empty vesicle membrane.Secretion of catecholamines from the adrenal medulla occurs by exocytosis (6-10) and the negative surface potential of the catecholamine-containing chromaffin vesicle is mainly derived from carboxyl groups of protein in the vesicle membrane (4). For carboxymethylation to be a step in excitation-secretion coupling, the protein(s) in the cytoplasmic surface of the plasma and/or chromafin vesicle membrane should be exposed to, and be substrate(s) for, the enzyme. We report now the predominant localization of protein carboxymethylase in the cytosol of the adrenal medullary cells and of methyl-acceptor proteins on the surface of chromaffin vesicles. Preliminary reports of these findings have been published elsewhere (14, 15). MATERIALS AND METHODS Materials. S-adenosyl-L-[methyl-3H]methionine, 12.6 Ci/ mmol, and [14C]tryptamine, 8.7 mCi/mmol, were purchased from New England Nuclear Corp. Gelatin (swine skin type I) was obtained from Sigma Chemical Co., Sephadex G-100 and QAE-Sephadex A-50 were from Pharmacia Fine Chemicals Inc.; sucrose (crystalline, density gradient grade) was supplied by Schwarz/Mann. Enzyme Purification. Protein carboxymethylase was purified from bovine pituitaries by the procedure previously described (2, 3).Protein Carboxymethylase Assay. Protein carboxymethylase activity was assayed by a modification of the method previously described (2, 3). Protein-methyl esters formed through the transfer of the methyl group from S-adenosyl-L- [methyl-3H] and terminated by the addition of 1 ml of 10% trichloroacetic acid. After centrifugation, the protein-methyl esters were hydrolyzed with 400 Mul of 1.0 M borate buffer at pH 11.0 containing methanol, 0.7% (vol/vol), ...

Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals?

“…There is in fact a history of ideas regarding a role for PIMT (when it was known as PCMT or PCM) in the regulation of neurotransmission and neurotransmitter receptor activity, (reviewed in [84]). Diliberto et al found in 1976 that carboxymethylation by this enzyme led to release of membrane proteins from chromaffin granules of the adrenal medulla and proposed that methylation was important for excitation-secretion coupling [85]. However, this hypothesis was later contested based on studies of protein secretion in the pancreas and parotid gland [86].…”

Prenatal and Postnatal Development of Protein Carboxymethylation Activity in the Mouse

“…In the homogenate assay for protein carboxymethylase a standard incubation time of 15 min has been used elsewhere [9][10][11]. This was used here as well since the rate of incor poration decreased with longer incubation periods ( fig.…”

“…The background value obtained from incubation at 0°C was subtracted from that at 37°C to obtain the level of specific activity. [ 10] Conceptuses were rinsed in PBS+5 mg/ml glucose, suspended in 0.3 M sucrose at 10 mg protein per millilit er, minced, and homogenized in a Dounce homogenizer (5 strokes A pestle and 5 strokes B pestle). Samples were then removed for Lowry protein determinations.…”

Effect of L-Methionine on Contractile Response, Calcium Influx and Calcium Channel Blocking Agents in the Rat Aorta

“…Diliberto et al [20] considered the possi bility that a similar carboxymethylation of protein would by reduction of negative charges modulate cell secretory function. Subsequent investigation of protein carboxymethylation appears to indicate that the protein carboxy methylation is associated only with abnormal amino acids and is not involved in direct reg ulation of eukaryotic cell function [21].…”