Identification and properties of two methyltransferases in conversion of phosphatidylethanolamine to phosphatidylcholine.

Two methyltransferases involved in the methylation of phosphatidylethanolamine to form phosphatidylcholine were demonstrated in a microsomal fraction of bovine adrenal medulla. The first methyltransferase catalyzes the methylation of phosphatidylethanolamine to form phosphatidyl-N-monomethylethanolamine. This enzyme has an optimum pH of 6.5, a low Km for S-adenosyl-L-methionine (1.4 micron), and an absolute requirement for Mg2+. The second methyltransferase catalyzes the two successive methylations of phodphatidyl-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 Mg2+.     

Reconstitution of ATP-dependent aminophospholipid translocation in proteoliposomes.

In addition to ion-pumping ATPases, most plasma membranes of animal cells contain a Mg2+ ATPase activity, the function of which is unknown. This enzyme, of apparent molecular mass 110 kDa, was purified from human erythrocyte membranes by a series of column chromatographic procedures after solubilization in Triton X-100. When reincorporated into artificial bilayers formed from phosphatidylcholine, it was able to transport a spin-labeled phosphatidylserine analogue from the inner to the outer membrane leaflet provided Mg2+ ATP was present in the incubation mixture. The ATP-dependent transport of the phosphatidylethanolamine analogue required the presence of an anionic phospholipid (e.g., phosphatidylinositol) in the outer membrane leaflet. In contrast the transmembrane distribution of spin-labeled phosphatidylcholine was unaffected in the same experimental conditions. This transmembrane movement of aminophospholipid analogues was inhibited by treatment of the proteoliposomes with a sulfhydryl reagent. We conclude that the Mg2+ ATPase is sufficient for the biochemical expression of the aminophospholipid translocase activity, which is responsible for the inward transport of phosphatidylserine and phosphatidylethanolamine within the erythrocyte membrane. The presence of this transport activity in many animal cell plasma membranes provides a function for the Mg2+ ATPase borne by these membranes.     

Insulin stimulation of phospholipid methylation in isolated rat adipocyte plasma membranes.

Partially purified plasma membranes prepared from rat adipocytes contain N-methyltransferase(s) that utilize(s) S-adenosyl-L-methionine to synthesize phosphatidylcholine from phosphatidylethanolamine. The incorporation of [3H]methyl from S-adenosyl-L-[methyl-3H]methionine into plasma membrane phospholipids was linear with incubation time and plasma membrane protein concentration and was inhibited in a dose-dependent manner by both S-adenosyl-L-homocysteine and 3-deazadenosine. The addition of insulin to plasma membranes stimulated the methylation of endogenous phosphatidylethanolamine, as evidenced by an increase in the levels of phosphatidyl-N-monomethylethanolamine, phosphatidyl-N, N-dimethylethanolamine, and phosphatidylcholine. The effect of insulin was rapid and concentration-dependent, with 100 microunits/ml providing near maximal stimulation. The incorporation of [3H]methyl into phospholipids of control and insulin-stimulated plasma membranes was enhanced by the addition of exogenous methyltransferase substrates phosphatidylethanolamine, phosphatidyl-N-monomethylethanolamine, and phosphatidyl-N,N-dimethylethanolamine. The stimulatory effect of insulin on adipocyte plasma membrane phospholipid methylation may have a physiological role in insulin action.     

Glycoproteins of Sendai virus are transmembrane proteins.

Radiolabeled Sendai viral envelope proteins were incorporated into human erythrocyte membranes by the process of fusion of the viral envelope with the erythrocyte membrane. Inside-out (IO) vesicles were prepared from the erythrocyte membranes containing viral proteins, and the presence of viral proteins was assessed by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. Proteolysis with trypsin, chymotrypsin, or Pronase, which digests only the external surface of the IO vesicles (the cytoplasmic surface of the erythrocyte membrane) revealed that the viral nucleocapsid and the nonglycosylated inner-envelope (M) proteins were present on the external surface. In addition, small segments of the viral envelope glycoproteins (HN and F1) were removed by these proteases, while the major portions of the glycoproteins were protected from digestion, indicating that in the erythrocyte membrane they are transmembrane proteins. Results of experiments carried out on right side-out membranes, unsealed membranes, and membranes containing attached virus that was unable to fuse indicated that the results obtained with IO membranes could not be accounted for by contamination with these membrane species. The identify of the proteolysis products was confirmed by peptide mapping. The selective exposure of the cytoplasmic surface, and hence the internal components of the virus, in IO vesicles makes this membrane system an attractive model for studying the interactions involved in virus maturation at host cell membranes.     

Phospholipid N-methylation as a Signal Transduction Mechanism in Normal and Failing Hearts

The phospholipid N-methylation pathway comprises of three successive N-terminal methylations of phosphatidylethanolamine where S-adenosyl-L-methionine acts as the physiological donor. Under optimal conditions in cardiac membranes, the catalytic sites I, II, and III of methyltransferase have been identified which are responsible for the synthesis of the major product, phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine, respectively. The characterization of the phosphatidylethanolamineN-methyltransferase system has shown that each of the catalytic sites exhibits different biochemical properties. The phospholipid N-methylation pathway has also been observed to regulate heart function by inducing localized structural, compositional, and functional changes in cardiac membranes under different pathological conditions of chronic nature. This review deals with the phosphatidylethanolamine N-methylation–mediated signal transduction mechanism involving modification of the Ca2+-transporting activities of the sarcolemmal and sarcoplasmic reticular membranes of the cardiomyocyte. In this regard, special attention is given to the status of this pathway and its relevance for the functioning of membrane-related Ca2+-transport systems in heart dysfunction due to different cardiac pathologies, such as diabetes-induced cardiomyopathy, catecholamine-induced cardiomyopathy, genetically linked cardiomyopathy, and adriamycin-induced cardiomyopathy. In addition, changes in phosphatidylethanolamine N-methylation in heart dysfunction due to cardiac hypertrophy, Ca2+-paradox hearts, and ischemic-reperfused hearts have been described. It is suggested that an increase in phosphatidylethanolamine N-methylation activity may play an adaptive role, whereas a depression may contribute towards contractile dysfunction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Subcellular localization of phosphatidylethanolamine N-methylation activity in rat heart

Synthesis of phosphatidylcholine (PC) by S-adenosyl-L-methionine (AdoMet)-dependent methylation of phosphatidylethanolamine (PE) has been recently characterized in rat heart sarcolemma obtained by hypotonic shock-LiBr treatment method. The present study, employing different procedures for the isolation of purified cardiac sarcolemmal membranes in rat, confirms the existence of three catalytic sites which are specifically involved in the sequential methyl transfer reactions from PE to PC. Other subcellular organelles such as sarcoplasmic reticulum (microsomes) and mitochondria showed methyltransferase activity which was absent in myofibrils and in cytosolic fraction. Experiments with several concentrations of AdoMet revealed that the kinetic pattern of methyltransferase activity in both microsomes and mitochondria was comparable to that obtained in sarcolemma. In addition, the characteristics of three catalytic sites as identified by the synthesis of phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine and PC in these subcellular organelles were similar to those of sarcolemma. The results are consistent with the view that methyltransferase activity is localized in different membrane systems of the myocardium.     

A unique phospholipid organization in bovine erythrocyte membranes

Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies; namely, low or absent phosphatidylcholine (PC) along with high sphingomyelin levels. Here, we report another anomaly in bovine erythrocytes that affects aminophospholipids: phosphatidylethanolamine (PE) shows an extreme asymmetry, with only 2% of the total present in the outer leaflet. Furthermore, we found that phospholipase A(2), an enzyme located on the external surface of the erythrocytes, shows higher activity against PC than against PE. In addition, we observed that acylation of PE is by far the most important biosynthetic event in this system. We propose that deacylation of PE and PC by phospholipase A(2) to generate lysocompounds, followed by selective reacylation of lyso-PE in the inner leaflet, can account for the compositional and architectural peculiarities of bovine erythrocyte membranes.     

The distribution of erythrocyte phospholipids in hereditary spherocytosis demonstrates a minimal role for erythrocyte spectrin on phospholipid diffusion and asymmetry

In the human erythrocyte membrane phosphatidylcholine and sphingomyelin reside mainly in the outer leaflet, whereas the aminophospholipids, phosphatidylethanolamine and phosphatidylserine, are mainly found in the inner leaflet. Maintenance of phospholipid asymmetry has been assumed to involve interactions between the aminophospholipids and the membrane skeleton, in particular spectrin. To investigate whether spectrin contributes to maintaining the phospholipid transbilayer distribution and kinetics of redistribution, we studied erythrocytes from hereditary spherocytosis patients whose spectrin levels ranged from 34% to 82% of normal. The phospholipid composition and the accessibility of membrane phospholipids to hydrolysis by phospholipases were in the normal range. Spin-labeled phosphatidylserine and phosphatidylethanolamine analogues that had been introduced into the outer leaflet were rapidly transported at 37 degrees C to the inner leaflet, whereas the redistribution of spin-labeled phosphatidylcholine was slower. The kinetics of transbilayer movement of these spin-labeled phospholipid in all samples was in the normal range and was not affected by the level of spectrin. Although these erythrocyte membranes contained as little as 34% of the normal level of spectrin and were characterized by several physical abnormalities, the composition, distribution, and transbilayer kinetics of the phospholipids were found to be normal. We therefore conclude that spectrin plays, at best, only a minor role in maintaining the distribution of erythrocyte membrane phospholipid.     

Effect of phospholipid methylation on beta-adrenergic receptors in the normal and hypertrophied rat myocardium

Abdominal aortic constriction in rats results in mild cardiac hypertrophy (20% increase in left ventricular weight compared to sham-operated controls) which is associated with increased numbers of beta-adrenergic receptors (123 +/- 7.3 fmol/mg protein (mean +/- SE) vs. 87 +/- 4.3 fmol/mg in controls, P < 0.01) without changes in their affinities for dihydroalprenolol. In vitro synthesis of phosphatidylcholine through successive methylation of phosphatidylethanolamine by S-adenosyl-L-methionine is enhanced in the hypertrophied myocardium) 0.38 +/- 0.03 nmol/mg per 30 minutes vs. 0.23 +/- 0.03 nmol/mg per 30 minutes in controls, P < 0.01). In both experimental groups, methyltransferase activity has a high affinity for S-adenosyl-L-methionine (Km = 6.8 microM), depends on Mg2+, is optimal around pH 9.0, and is inhibited by S-adenosyl-L-homocysteine (ki = 8.3 microM). The possible relationship between phospholipid methylation and changes in myocardial beta-adrenergic receptors was studied in both normal and hypertrophied hearts. Preincubation of cardiac membranes with S-adenosyl-L-methionine increased the numbers of b eta-adrenergic receptors in proportion to the duration of incubation and the concentration of S-adenosyl-L-methionine. In both groups, S-adenosyl-homocysteine, but not 5'-AMP or L-methionine, attenuated the increase in beta-adrenoreceptors. These results indicate that phospholipid methylation may be an important mechanism for regulation of beta-adrenergic mechanisms in both normal and hypertrophied myocardium.     

Adipocyte and erythrocyte plasma membrane phospholipid composition and hyperinsulinemia: a study in nondiabetic and diabetic obese women

BACKGROUND: The cell functions involved in the action of insulin--receptor binding, enzyme and transporter activities--are controlled by membrane properties. We have previously shown that the fasting plasma insulin (FPI) concentration and the homeostasis model assessment (HOMA) estimate of insulin resistance are associated with the sphingomyelin concentration in the erythrocyte membranes of obese women. OBJECTIVES: (1) To study the distribution of phospholipid classes in the plasma membrane and their association with insulin resistance markers in the adipocyte, an insulin-sensitive cell in obese women. (2) To investigate the influence of diabetes in a small group of obese women treated by diet alone. (3) To compare the distribution of phospholipids in erythrocyte membranes in a subgroup of obese nondiabetic and diabetic women. SUBJECTS: Subcutaneous fat biopsies were taken from the abdominal region of 19 obese non-diabetic and seven obese type 2 diabetic women. Erythrocyte membrane assessment was performed in a subgroup of 10 of the 19 obese nondiabetic and in the seven diabetic patients. METHODS: The phospholipid composition of adipocyte and erythrocyte plasma membranes was analyzed by high performance liquid chromatography. RESULTS: FPI was positively correlated with the adipocyte membrane contents of sphingomyelin (P < 0.001), phosphatidylethanolamine (P < 0.05), and phosphatidylcholine (P < 0.01) in the obese nondiabetic women. Similar correlations were obtained with HOMA. A stepwise multiple regression analysis indicated that sphingomyelin accounted for 45.6 and 43.8% of the variance in FPI and HOMA values as an independent predictor. There was a similar positive independent association between FPI and SM in the erythrocyte membranes of the studied subgroup. Diabetes per se did not influence the independent association between SM membrane contents and FPI in both cell types. CONCLUSION: These results suggest a link between membrane phospholipid composition, especially SM, and hyperinsulinemia in obese women.     

ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 5 degrees C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATP-driven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape.     

Demonstration and partial characterisation of phospholipid methyltransferase activity in bile canalicular membrane from hamster liver

BACKGROUND/AIMS: Methylation of phosphatidylethanolamine to phosphatidylcholine predominantly takes place in mitochondrial-associated membrane and the endoplasmic reticulum of the liver. The transport of the phospholipids from endoplasmic reticulum to the bile canalicular membrane is via vesicular and protein transporters. In the bile canalicular membrane a flippase enzyme helps to transport phosphatidylcholine specifically to the biliary leaflet. The phosphatidylcholine then enters the bile where it accounts for about 95% of the phospholipids. We postulated that the increased proportion of phosphatidylcholine in the bile canalicular membrane and the bile compared to the transport vesicles may be due to a methyltransferase activity in the bile canalicular membrane which, using s-adenosyl methionine as the substrate, converts phosphatidylethanolamine on the cytoplasmic leaflet to phosphatidylcholine, which is transported to the biliary leaflet. The aim of our study was to demonstrate and partially characterise methyltransferase activity in the bile canalicular membrane. METHODS: Organelles were obtained from hamster liver by homogenisation and separation by sucrose gradient ultracentrifugation. These, along with phosphatidylethanolamine, were incubated with radiolabelled s-adenosyl methionine. Phospholipids were separated by thin-layer chromatography and radioactivity was counted by scintigraphy. RESULTS: We demonstrated methyltransferase activity (nmol of SAMe converted/mg of protein/h at 37 degrees C) in the bile canalicular membrane of 0.442 (SEM 0.077, n=8), which is more than twice that found in the microsomes at 0.195 (SEM 0.013, n=8). The Km and pH optimum for the methyltransferase in the bile canalicular membrane and the microsomes were similar (Km 25 and 28 microM, respectively, pH 9.9 for both). The Vmax was different at 0.358 and 0.168 nmol of SAMe converted/mg of protein/h for the bile canalicular membrane and the microsomes, respectively. CONCLUSION: The presence of the methyltransferase activity in the bile canalicular membrane may be amenable to therapeutic manipulation.     

Altered Acylation of Erythrocyte Phospholipids in Alcoholism

The composition and metabolism of erythrocyte lipids were studied in 10 chronic alcoholic patients within 48 hr after discontinuation of alcohol intake and in 10 nonalcoholic control subjects. Chronic alcoholism produced no change in contents of cholesterol, total phospholipids, and proportions of phosphatidylcholine and phosphatidylethanolamine in erythrocyte phospholipids. The mean values of the rates of acylation of phosphatidylcholine and phosphatidylethanolamine with oleic acid were increased respectively by 59% (p less than 0.001) and 38% (p less than 0.05) as compared with the controls. There was no correlation between acylation rates and mean cellular volumes. Increases in acylation rates normalized over several weeks after alcohol withdrawal and were not related to a direct effect of alcohol on the intact erythrocyte, suggesting that these alterations result from ethanol-induced changes in the membrane during erythrocyte formation. The increased rates of acylation might modify the remodeling of the lipid matrix and thereby the membrane function.     

Primary structure and subcellular localization of the knob-associated histidine-rich protein of Plasmodium falciparum.

Plasmodium falciparum-infected erythrocytes bind to venular endothelial cells by means of electron-dense deformations (knobs) on the parasitized erythrocyte surface. The primary structure of a parasite-derived histidine-rich protein associated with the knob structure was deduced from cDNA sequence analysis. The 634 amino acid sequence is rich in lysine and histidine and contains three distinct, tandemly repeated domains. Indirect immunofluorescence, using affinity-purified monospecific antibodies directed against recombinant protein synthesized in Escherichia coli, localized the knob-associated histidine-rich protein to the membrane of knobby infected erythrocytes. Immunoelectron microscopy established that the protein is clustered on the cytoplasmic side of the erythrocyte membrane and is associated with the electron-dense knobs. A role for this histidine-rich protein in knob structure and cytoadherence is suggested based upon these data.     

Sendai virus-induced hemolysis: reduction in heterogeneity of erythrocyte lipid bilayer fluidity.

Hemolysis of human or chicken erythrocytes by Sendai virus causes a change in the structure of the erythrocyte membrane lipid bilayer that can be detected by spin label electron spin resonance. In the intact erythrocyte, the phosphatidylcholine derivative spin label exists in a more rigid environment than the corresponding phosphatidylethanolamine label. Virus-induced hemolysis tends to abolish this difference in fluidity, i.e., the region of the phosphatidylcholine spin label becomes more fluid and that of the phosphatidylethanolamine spin label becomes more rigid. Fatty acid derivative spin labels, which may detect some "average" environment, show no change in fluidity. The fluidity change is detected at several different positions in the fatty acyl chain of the phosphatidylcholine spin label. Sendai virions grown in Madin-Darby bovine kidney (MDBK) cells or grown in eggs and harvested early, which lack hemolytic activity, cause no significant change in bilayer structure. Hemolytic activity and the ability to alter erythrocyte bilayer fluidity can be activated in MDBK-grown Sendai virions by trypsin treatment in vitro and in early-harvest egg-grown Sendai virions by freezing and thawing. Erythrocyte ghosts prepared by osmotic hemolysis and resealed by treatment with Mg2+ or elevated ionic strength exhibit a difference in fluidity between phosphatidylcholine and phosphatidylethanolamine spin labels, although less than that observed in whole cells. Incubation of resealed ghosts with Sendai virus abolishes the difference in fluidity. Unsealed ghosts that have been extensively washed show no heterogeneity in membrane bilayer fluidity, and incubation with Sendai virus causes no further fluidity change. Virus-induced hemolysis as measured by hemoglobin release is more sensitive to inhibition by Ca2+ than is the associated fluidity change in the bilayer.     

Evidence against phospholipid asymmetry in intracellular membranes from liver.

We have studied the distribution of phospholipids across the membrane of microsomal vesicles and Golgi-derived secretory vesicles from rat liver by the use of phospholipases. Model studies on single-bilayer phospholipid vesicles showed that phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) cleaved at least 80% of the lipids on the outer surface of such vesicles without significant attack on the inner surface. In microsomal vesicles approximately 40% of the outer surface phospholipids were cleaved before the enzyme gained access to the interior of the vesicles. The same conclusion was reached for Golgi vesicles. By following the degradation of the three major phospholipids in intact microsomes and in extracted lipids we found that the same fraction of each of these phospholipids was exposed on the outer surface of the microsomal vesicles. Corresponding experiments with Golgi vesicles showed that distinctly different fractions of phosphatidylcholine and phosphatidylethanolamine were present on the surface of these vesicles. However, the difference was accounted for by enrichment of phosphatidylcholine in intravesicular particles rather than by asymmetry across the vesicle membrane. The results from specific hydrolysis of phosphatidylinositol confirmed an essentially symmetric distribution of this phospholipid across the microsomal and the Golgi vesicle membranes.     

Novel high-performance liquid chromatography for determination of membrane phospholipid composition of rat hepatocytes

We have developed a new, quick and efficient method of high-performance liquid chromatography (HPLC) for the isolation and quantitative determination of phospholipids in hepatocyte membranes. A silica gel column was used for the isolation and determination, and an isocratic mixture of acetonitrile, methanol and 85% phosphoric acid (130:5:1.7, v/v/v) was used as a mobile phase. Six kinds of phospholipids, i.e. phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphinogomyelin (SPH), in this order, were completely isolated within 45 min. The phospholipid composition of sinusoidal membrane vesicles (SMV) and canalicular membrane vesicles (CMV) obtained from rat liver, as well as of human erythrocyte ghosts were determined by this HPLC method. The level of SPH in CMV was significantly higher than that in SMV, and the level of PC in CMV was significantly lower than that in SMV. These results were considered attributable to the low fluidity of CMV. The phospholipid composition of human erythrocyte membrane was different from that of rat SMV and CMV. The present technique is suitable for quantitative determination of phospholipids in cell membranes.     

Changes in lipid composition of erythrocyte membranes with administration of S-adenosyl-L-methionine in chronic liver disease.

This study was undertaken to determine whether S-adenosyl-L-methionine (SAMe) changes the lipid composition and fluidity of erythrocyte membranes in chronic liver disease. SAMe was administered intravenously at a daily dose of 600 mg for 2 weeks to 10 patients; 6 patients with cirrhosis and four with primary biliary cirrhosis. The elevated free cholesterol to phospholipid molar (C/PL) ratio of the erythrocyte membranes of the patients (0.857 +/- 0.018) significantly decreased after the administration of SAMe (1 week, 0.823 +/- 0.021; 2 weeks, 0.823 +/- 0.013). In all of the four patients whose erythrocyte membrane fluidity was measured, fluidity improved with the administration of SAMe and correlated with the C/PL ratio of the membranes. These results suggest that SAMe decreases the C/PL ratio of erythrocyte membranes and thus improves membrane fluidity in chronic liver disease.     

Phospholipid methylation stimulates lactogenic binding in mouse mammary gland membranes.

Addition of the methyl donor S-adenosyl-L-methionine to membranes prepared from mammary glands of lactating mice results in increased binding of 25I-labeled human growth hormone to the lactogenic receptors. This stimulation is dose dependent and specific for S-adenosyl-L-methionine and is partially inhibited by simultaneous addition of S-adenosyl-homocysteine to the reaction. Pretreatment of the membranes with S-adenosyl-L-methionine for 30 min at 37 degrees C is sufficient to cause enhanced binding. Scatchard analysis shows that treatment with S-adenosyl-L-methionine results in an increase in the number of lactogenic binding sites without changing the apparent affinity constant for 125I-labeled human growth hormone. The increase in the number of binding sites is believed to be due to alteration in the phospholipid composition of the membrane because methylation of phospholipids is observed under these conditions.     

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

Protein carboxymethylase (S-adenosyl-L-methionine:protein O-methyltransferase, EC 2.1.1.24) transfers a methyl group from S-adenoxyl-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 subcellular fractions of the medulla, the catecholamine-containing chromaffin vesicles had the highest concentrations of substrat(s) for protein carboxymethylase. Carboxymethylation of proteins in intact chromaffin vesicles results in stripping of methylated protein(s) from the membranes. Thus, protein carboxymethylase appears to be involved in the neutralization of charges on the surface of chromaffin vesicles and in the release of surface proteins; both phenomena are likely to be required for exocytosis.