Secretion of catecholamines from single bovine chromaffin cells in culture was elicited by brief pressure ejections from a micropipette containing nicotine, carbamoylcholine, or potassium ions or by mechanical stimulation. Release was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. Cyclic voltammetry was used to identify secreted species, whereas constant potential amperometry was used for improved temporal resolution (millisecond range) of catecholamine detection. During secretion, brief current spikes were observed, which were shown to be due to detection of catecholamines by electrooxidation. The spikes have the physical characteristics of multimolecular packets of catecholamines released at random times and locations from the surface of the single cell. The half-width of the spikes was found to increase with an increase in cell-electrode spacing. The properties of the catecholamine spikes correlate well with expectations based on secretion from individual storage vesicles. Spikes do not occur in the absence of Ca2+ in the buffer, and the majority of spikes are found to be distributed between 0.2 and 2 picocoulombs, corresponding to 1-10 attomoles of catecholamine detected. The frequency of the spikes increases with the intensity of the stimulus, but the average quantity of catecholamine in each spike is independent of the stimulus. Thus, these measurements represent timeresolved observation of quantal secretion of catecholamines and provide direct evidence for the exocytotic hypothesis.

Catecholamine secretion has been measured with electrochemical techniques from isolated, single adrenal medullary chromaffin cells with carbon-fiber microelectrodes. The electrode tip, which is of similar dimensions to the cell, is placed adjacent to the cell to enable the measurement of local secretion. Secretion is caused by exposing the cell to nanoliter volumes of solution containing nicotinic receptor agonists or depolarizing agents. The identification of secreted substances is made with cyclic voltammetry at both bare electrodes and electrodes coated with a perfluorinated cation-exchange polymer. Catecholamine secretion is induced by nicotine (10-500 microM), carbamylcholine (1 mM), and K+ (60 mM). All agents that induce secretion lead to a broad envelope of secreted catecholamines on which sharp concentration spikes are superimposed. The concentration spikes can be monitored with a time resolution of tens of milliseconds when the electrodes are used in the amperometric mode. Release induced by nicotine and K+ is inhibited by Cd2+ (0.5 mM), and hexamethonium selectively blocks the nicotine-induced secretion. The actions of nicotine are found to continue for a longer period of time than those of the other secretagogues tested.

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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