Protein carboxymethylase, an enzyme that transfcrs the methyl group of S‐adenosyl‐L‐methionine to carboxyl groups of proteins and endogenous acceptor proteins were examined in nerve and endocrine tissues. The highest protein carboxymethylase activity was found in the brain, followed by the testis, pituitary and heart. On the other hand, the tissue with the highest level of endogenous substrate(s) was the pituitary. The nearly identical specific activity ratio for two different protein substrates in all tissues examined, suggests that one enzyme is responsible for carboxymethylase activity in different tissues. The subcellular distribution of the enzyme in brain showed a high concentration in the soluble fraction, presumably representative of the enzyme in the cytosol of cell bodies. Considerable enzyme activity was also found in brain synaptosomes which was increased by osmotic lysis. Protein carboxymethylase was shown to accumulate proximally to a ligation of the rat sciatic nerve. A possible physiological role for protein carboxymethylase in neuronal function is discussed.

Two methyltransferases involved in the methylation of phosphatidylethanolamine to form phosphatidytcholine were demonstrated in a microsomal fraction of bovine adrenal medulla. The first methyltransferase catalyzes the methylation of phoshatidylethanol'amine to form phosphatidyl-N-monomethylethanolamine. This enzyme has an optimum pH of 6.5, a low Km for S-adenosyl-L-methionine (1.4 ;M), and an absolute requirement for Mg2+. The second methyltransferase catalyzes the two successive methylations of phosphatidyl-N-monomethylethanolamine to phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. In contrast to the first methyltransferase, it has an optimum pH of 10 and a high Km for S-adenosyl-L-methionine (0.1 mM) and does not require MgS+.Several investigations have shown that enzymatic methylations can occur on the amino group of phospholipids to form phosphatidyicholine (1-4). The enzyme(s) catalyzing this sequence of methylation were shown to reside in the microsomes of rat liver and Neurospora. A preparation of rat liver microsomes has been described that catalyzed the stepwise methylation of phosphatidyl-N-monomethylethanolamine to phosphatidylcholine but not of phosphatidylethanolamine (1; 4). The enzyme catalyzing the first methylation step has been suggested to be rate-limiting (1), but its properties have not yet been described. Recently, our laboratory reported on the ability of the enzyme, protein carboxymethylase, to transfer a methyl group from S-adenosyl-L-methionine to carboxy groups of membrane proteins of chromaffin granules in the adrenal medulla (5-7). In studies to examine the effects of cations on this enzyme activity with various membrane fractions, it was observed that methylation of lipids also occurred which depended upon the presence of Mg2+. Since the methylation of phospholipids has not been shown to require Mg2+ (1-4), this led us to search for and characterize the Mg2+ dependent enzyme that methylates lipids. This communication presents evidence that this Mg2+-dependent enzyme is involved in the conversion of phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine and that a second methyltransferase converts the latter compound to phosphatidylcholine. METHODS AND MATERIALSAssay of Phosphatide Methyltransferases. The methylation of phosphatidylethanolamine to phosphatidyl-N-monomethylethanolamine was assayed by measuring incorporation of the methyl group from S-adenosyl-L-[methyl-3H]methionine into phospholipids. The assay medium, in a 6-ml stoppered polyethylene tube, contained 4 ,uM S-adenosyl-L-[methyl-3H]-The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "'advertisement"9 in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1718 methionine (2 ,uCi), 10 mM MgCl2, 0.1 mM sodium EDTA, 50 mM sodium acetate buffer (pH 6.5), and tissue extract (0.1 mg of protein) in a total volume of 50 1l. The reaction was started by the addition of radioactive S-adenosyl-L...

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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), ...

AtT-20 cells comprise a mouse anterior pituitary tumor cell line that synthesizes and secretes adrenocorticotropin hormone (ACTH). beta-Adrenergic receptors were characterized on AtT-20 cells using receptor binding methodology and the ability of beta-receptor agonists to stimulate intracellular cyclic adenosine 3':5'-monophosphate (cAMP) formation and the release of ACTH immunoreactivity. The density of beta-receptors on membrane preparations of these cells is 64 fmol/mg of protein and their affinity constant (KD value) for tritiated dihydroalprenolol is 11 nM. The binding of [3H] dihydroalprenolol to AtT-20 cells is stereoselectively inhibited by propranolol and isoproterenol but is not affected by phentolamine. The beta-receptors on these cells appear to be of the beta 2-receptor subtype since a selective beta 2-receptor agonist, salmefamol, can inhibit [3H]dihydroalprenolol binding, whereas practolol, a beta 1-receptor blocker, is ineffective. (-)-Isoproterenol stimulates cAMP formation in AtT-20 cells and this effect is blocked by dl-propranolol. Both l-epinephrine and l-norepinephrine induce dose-dependent increases in cAMP formation with the former agonist being more potent. Salmefamol also stimulates cAMP formation in these cells. The secretion of ACTH from AtT-20 cells is induced by (-)-isoproterenol as well as by other adrenergic agonists. The isoproterenol effect on ACTH release is stereoselective, calcium dependent, and blocked by dl-propranolol but not by phentolamine or practolol.