Protein-carboxyl O-methyltransferase Research Articles

Immunoglobulin can be functionally regulated by protein carboxylmethylation in Fc region

Protein carboxylmethylation methylates the free carboxyl groups in various substrate proteins by protein carboxyl O-methyltransferase (PCMT) and is one of the post-translational modifications. There have been many studies on protein carboxylmethylation. However, the precise functional role in mammalian systems is unclear. In this study, immunoglobulin, a specific form of gamma-globulin, which is a well-known substrate for PCMT, was chosen to investigate the regulatory roles of protein carboxylmethylation in the immune system. It was found that the anti-BSA antibody could be carboxylmethylated via spleen PCMT to a level similar to gamma-globulin. This carboxylmethylation increased the hydrophobicity of the anti-BSA antibody up to 11.4%, and enhanced the antigen-binding activity of this antibody up to 24.6%. In particular, the Fc region showed a higher methyl accepting capacity with 80% of the whole structure level. According to the amino acid sequence alignment, indeed, 7 aspartic acids and 5 glutamic acids, as potential carboxylmethylation sites, were found to be conserved in the Fc portion in the human, mouse and rabbit. The carboxylmethylation of the anti-BSA antibody was reversibly demethylated under a higher pH and long incubation time. Therefore, these results suggest that protein carboxylmethylation may reversibly regulate the antibody-mediated immunological events via the Fc region.

Protein carboxylmethylation in porcine spleen is mainly mediated by class I protein carboxyl O-methyltransferase.

The functional role of protein carboxylmethylation (PCM) has not yet been clearly elucidated in the tissue level. The biochemical feature of PCM in porcine spleen was therefore studied by investigating the methyl accepting capacity (MAC) of natural endogenous substrate proteins for protein carboxyl O-methyltransferase (PCMT) in various conditions. Strong acidic and alkaline-conditioned (at pH 11.0) analyses of the MAC indicated that approximately 65% of total protein methylation seemed to be mediated by spleen PCMT. The hydrolytic kinetics of the PCM products, such as carboxylmethylesters (CMEs), under mild alkaline conditions revealed that there may be three different kinds of CMEs [displaying half-times (T1/2) of 1.1 min (82.7% of total CMEs), 13.9 min (4.6%), and 478.0 min (12.7%)], assuming that the majority of CME is base-labile and may be catalyzed by class I PCMT. In agreement with these results, several natural endogenous substrate proteins (14, 31 and 86 kDa) were identified strikingly by acidic-conditioned electrophoresis, and their MAC was lost upon alkaline conditions. On the other hand, other proteins (23 and 62 kDa) weakly appeared under alkaline conditions, indicating that PCM mediated by class II or III PCMT may be a minor reaction. The MAC of an isolated endogenous substrate protein (23-kDa) was also detected upon acidic-conditioned electrophoresis. Therefore, our data suggest that most spleen PCM may be catalyzed by class I PCMT, which participates in repairing aged proteins.

Purification and characterization of protein methylase II from Helicobacter pylori

Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS–PAGE. On non-denaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4×(78+29)=428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a K m value of 5.0×10 −6 M for S-adenosyl- L-methionine and a V max of 205 pmol methyl- 14C transferred min −1 mg −1 protein.

Purification and characterization of protein methylase II from Helicobacter pylori.

Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS-PAGE. On non-denaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4x(78+29)=428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a K(m) value of 5.0x10(-6) M for S-adenosyl-L-methionine and a V(max) of 205 pmol methyl-(14)C transferred min(-1) mg(-1) protein.

A peptide inhibitor for S-adenosyl- l-methionine-dependent transmethylation reactions : Purification and characterization

1. 1. A proteinaceous inhibitor for S-adenosyl- l-methionine (AdoMet)-dependent transmethylation reactions has been purified to apparent homogeneity from rat liver cytosolic fraction. 2. 2. The peptide was made up of 29 amino acid residues with a molecular weight of 2,584. Glycine accounted for 52% of the total amino acids. 3. 3. Employing AdoMet: protein-carboxyl O-methyltransferase (Protein methylase II) and bovine serum γ-globulin as in vitro substrate, the mode of inhibition was found to be non-competitive with K i value of 1.9 × 10 −8 M. 4. 4. When the inhibitor was present in the reaction mixture together with S-adenosyl- l-homocysteine (AdoHcy), which is a competitive inhibitor for AdoMet, the extent of inhibition exceeded that exerted by each individual inhibitor alone, suggesting that the sites of the inhibitors on the enzyme molecule are different. 5. 5. Almost a stoichiometric relationship exists between the enzyme and the inhibitor molecule, the ratio being approx one.

Identification of the S-adenosyl-L-methionine binding site of protein-carboxyl O-methyltransferase using 8-azido-S-adenosyl-L-methionine

Protein-carboxyl O-methyltransferase (protein methylase II) transfers the methyl group from S-adenosyl-L-methionine (AdoMet) to the carboxyl side chains of the amino acids in the proteins. We have used the radiolabeled analogue of AdoMet, 8-azido-S-adenosyl-L-[methyl-3H]methionine (8-N3-Ado[methyl-3H]Met), to investigate the AdoMet binding site of protein methylase II. The incorporation of the photoaffinity label in the enzyme upon UV irradiation is highly specific. In the absence of UV irradiation or if the photoprobe is irradiated prior to its addition to the reaction mixture, no photoinsertion of the label occurs. Moreover, the presence of a competitive inhibitor of protein methylase II, S-adenosyl-L-homocysteine (AdoHcy), or the unlabeled AdoMet itself in the reaction mixture diminished labeling of the enzyme. Sequential digestion of the labeled enzyme with trypsin, chymotrypsin, and endoproteinase Glu-C yielded a modified and radiolabeled decapeptide. When compared with the reported primary amino acid sequence of protein methylase II from rat brain, the amino acid composition of the decapeptide matched residues 113-121. This segment forms the midpoint region of the enzyme (234 amino acid residues). An important characteristic of the sequence is the presence of two adjacent aspartic acid residues (Asp117-Asp118) which most likely provide the negative charge environment for the sulfonium moiety of the AdoMet molecule.

Age-related decline in S-adenosylmethionine and protein methyl esterification levels in bovine lenses

The enzymatic methyl esterification of proteins, catalyzed by the enzyme S-adenosylmethionine: protein carboxyl-O-methyl transferase (PCMT), has been investigated in two age-related fractions of bovine lenses. The eukaryotic PCMT methyl esterifies peptides and proteins containing altered aspartyl residues, such as D-aspartyls and L-isoaspartyls, which can arise from the age-dependent deamidation of labile Asn residues. When intact bovine lenses were incubated in a cell culture medium under physiological conditions (pH 7.4, 37 degrees C), in the presence of L-(methyl-(14)C)methionine, the in vivo precursor of the methyl donor substrate S-adenosylmethionine (AdoMet), a significant decrease in the level of protein methyl esterification was observed in the oldest cell fraction compared to the youngest one; in contrast, the in vitro activity of PCMT does not change during cell aging. It has also been demonstrated that the cell aging in the lens is accompanied by a reduction of the AdoMet synthetase activity and, consequently, of the endogenous level of the methyl donor AdoMet. Furthermore, the activity of the enzyme S-adenosylhomocysteine (AdoHcy) hydrolase, which cleaves the methylation inhibitor AdoHcy, also appears dramatically reduced in the oldest cells, probably leading to an increase in the AdoHcy cellular level. Finally, the possible correlation between the decrease in the efficiency of the overall methyl esterification process during aging and the accumulation of aberrant forms of proteins observed in aged tissues is also discussed.

Repair of isopeptide bonds by protein carboxyl O-methyltransferase: seminal ribonuclease as a model system.

Previous work has shown that in the peptide segment 62-76 of naturally deamidated alpha subunit of bovine seminal ribonuclease (BS-RNase) the alpha-carboxyl group of iso-Asp67 is selectively methylated by S-adenosylmethionine:protein carboxyl O-methyltransferase [Di Donato, A., Galletti, P., & D'Alessio, G. (1986) Biochemistry 25, 8361-8368]. In the present study this reaction has been characterized, by using the tryptic segment 62-76 of the protein chain (peptide alpha 16). The peptide is stoichiometrically methyl esterified with a Km of 6.17 microM and a Vmax of 19.56 nmol min-1 mg-1, and the product of demethylation has been identified as the cyclic succinimidyl derivative of iso-Asp67-Gly68. The cleavage of the succinimidyl ring yields two isomeric peptides containing an aspartyl residue (peptide alpha 17) and an isoaspartyl residue (peptide alpha 16). On the basis of these results conditions were defined in which repeated cycles of methylation-demethylation led to an effective conversion of peptide alpha 16 into peptide alpha 17, a process that can be interpreted as the repair of an altered isopeptide bond. When the methyl esterification reaction was studied on the native dimeric isoenzymes of seminal RNase and on catalytically active monomeric derivatives, including a stabilized alpha-type subunit, the results of these experiments showed that none of the protein forms were substrates for the methyltransferase. Only the unfolded alpha-type subunit was methylated to a stoichiometric extent. These results indicate that the repair of altered isopeptide bonds is chemically feasible in peptides but is hindered in the case of seminal RNase by its three-dimensional structure.

Human placenta S-adenosylmethionine: Protein carboxyl O-methyltransferase (protein methylase II). Purification and characterization

1. 1. Protein methylase II was purified from human placenta approx. 8700-fold with a yield of 14%. 2. 2. Unlike protein methylase II from other sources, the activity of human placenta enzyme was completely inhibited by 2 mM Cu 2+. Other divalent ions were without effect. 3. 3. Human chorionic gonadotropin (HCG), immunoglobulin A and calf thymus histones served as good in vitro substrates for the enzyme, particularly HCG. 4. 4. The K m for S-adenosyl- l-methionine and K i for S-adenosyl- l-homocysteine were 2.08 × 10 −6 and 5.8 × 10 −7 M, respectively. 5. 5. The protein methylase II activity in human placenta changed with gestational age, the activity at 1st and 2nd trimester being approximately twice that of term placenta.

Mechanism of protein carboxyl methyl transfer reactions: structural requirements of methyl accepting substrates.

The enzyme S-adenosylmethionine: protein carboxyl-O-methyltransfe-rase (PCMT) was first identified in 1965 by Axelrod and Daly as “methanol forming “ enzyme, since it was thought to catalyze the hydrolysis of S-adenosylmethionine (AdoMet) yielding methanol and S-adenosylhomocysteine (AdoHcy) (1). A few years later Liss et al. (2) and Kim and Paik (3) described an AdoMet-dependent methyl transferase which methylates free carboxyl groups in protein substrates and leads to the formation of unstable methyl esters. The two enzymes shared methanol as a common end product and were found to be identical on the basis of their properties and tissue distribution (4). During the following fifteen years the enzyme was purified from eukaryotic and prokaryotic sources, extensively characterized, and a number of different regulatory roles have been attributed to it, as reviewed by Paik and Kim in 1980 (5). However, in eukaryotes none of the hypothesized regulatory functions has been unequivocally demonstrated. More recently the eukaryotic and prokaryotic enzymes were found to differ in terms of methylation site, substrate specificity and functional role (6,7); therefore a new systematic name and classification number have been assigned to the eukaryotic PCMT. This enzyme indeed has been shown to recognize and selectively modify proteins at D-aspartyl and L-isoaspartyl residue levels (6,7).KeywordsDisulfide BridgeMethyl EsterificationIsopeptide BondErythrocyte Membrane ProteinNormal PeptideThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

ReducedS-adenosylmethionine: Protein-lysineN-methyltransferase activity (protein methylase III) in shiverer mutant mouse brain

Mice with the dysmyelinating mutation shiverer were studied by measuring the activity of two protein methylases and myelin marker enzymes in the brain. It was observed that S-adenosylmethionine:protein-lysine N-methyltransferase (protein methylase III, EC. 2.1.1.43) activity is significantly reduced in phenotypically affected homozygous shiverer (shi/shi) mutant mouse brain compared to the unaffected heterozygous littermate brain. This reduction in enzyme activity is manifested mainly by reduced formation of trimethyllysine during the in vitro methylation of histone. In contrast, myelin marker enzymes such as 2',3'-cyclic nucleotide 3'-phosphohydrolase and 5'-nucleotidase as well as S-adenosyl-methionine:protein-carboxyl O-methyltransferase (protein methylase II, EC. 2.1.1.24) activities were not significantly affected in these strains of mice.

Stoichiometric methylation of calcineurin by protein carboxyl O-methyltransferase and its effects on calmodulin-stimulated phosphatase activity.

Calcineurin, a calmodulin-stimulated protein phosphatase, was a substrate for purified bovine brain protein carboxyl O-methyltransferase (protein O-methyltransferase; EC 2.1.1.24) and incorporated up to 2 mol of CH3 per mol of calcineurin. Carboxyl methylation was dependent on the concentrations of S-adenosyl-L-[methyl-3H]methionine and was prevented by addition of the carboxyl methylation inhibitor S-adenosylhomocysteine. The stoichiometry of methyl group incorporation was related to the ratio of methyltransferase/calcineurin. The rate of spontaneous hydrolysis of carboxyl methylester groups on calcineurin increased rapidly above pH 6.5 with those on native carboxyl-methylated calcineurin substantially more labile than for trichloracetic acid-precipitated calcineurin. Polyacrylamide gel electrophoresis in the presence of NaDodSO4 (pH 2.4) confirmed that the A subunit of calcineurin (Mr = 61,000) was the primary site of carboxyl methylation with little, if any, modification of the B subunit (Mr = 18,000). When carboxyl-methylated calcineurin (approximately 1-2 mol of CH3 per mol of protein) was assayed for p-nitrophenyl phosphatase activity at pH 6.5, a marked inhibition of calmodulin-stimulated activity was observed while there was little effect on Mn2+-stimulated phosphatase activity. Thus, calcineurin appears to be an excellent substrate for protein carboxyl O-methylation and this modification, which impairs calmodulin stimulation of phosphatase activity, may be of functional significance.

Abnormal membrane protein methylation and merocyanine 540 fluorescence in sickle erythrocyte membranes

Sickle cell erythrocytes exhibit reduced carboxyl methylation of membrane proteins compared to normal erythrocytes. This altered methylation in sickle membrane proteins is also observable when extracted membranes, both intact and alkali treated, were used as substrates for the homologous protein methylase II (S-adenosylmethionine: protein-carboxyl O-methyltransferase, EC. 2.1.1.24). However, when glycophorin A, one of the major methyl acceptors in both membranes, was extracted by lithium diiodosalicylate and used as the methyl acceptor, the proteins from both membranes were methylated equally, suggesting an involvement of membrane structure in membrane-bound protein methylation. Merocyanine 540 (MC-540), a fluorescent probe, was used to determine if the membranes differed in organization. Incubation of both normal and sickle erythrocytes membranes with MC-540 produced a marked increase in extrinsic fluorescence, reflecting a relatively nonpolar environment for the dye bound to the membranes. The fluorescence from sickle cell ghosts was only 87% as intense as that from normal ghosts, while the actual amount of MC-540 associated with sickle cell membranes was only 62% of normal. These data suggest that differences exist in the distribution of surface charges on these plasma membranes. These results are consistent with the hypothesis that abnormal levels of membrane protein methylation observed in sickle erythrocytes may be a result of abnormal membrane organization characteristic to sickle cell anemia.

Differential membrane protein carboxyl-methylation of intact human erythrocytes by exogenous methyl donors.

The patterns of membrane protein carboxyl-methylation by protein methylase II (S-adenosylmethionine: protein-carboxyl O-methyltransferase, EC 2.1.1.24) in intact human erythrocytes were shown to differ markedly whether the methyl donor, S-adenosyl-L-[methyl-3H]methionine, was supplied exogenously or formed intracellularly via exogenously added L-[methyl-3H]methionine. The differences include the following. (1) The methylation of cytoskeletal components (band 2.1 and 4.1) occurs only in the case of the L-[methyl-3H]methionine-labelled cells. (2) The methionine-mediated methylation was much less sensitive to S-adenosyl-L-homocysteine inhibition than the adenosylmethionine-mediated methylation (22% versus 95% inhibition at 10 microM). (3) The membrane protein methylation mediated by exogenous adenosylmethionine and methionine differed markedly in their alkali labilities; at pH 6.0, 30% of the adenosylmethionine-mediated protein methyl esters were hydrolysed after 30 min (37 degrees C) while the methionine-mediated esters were stable. At pH 7.4, the respective labilities were 60% and 30% for the 30 min incubation. To explain these results, a possible involvement of cytoskeletal structure associated with the intact erythrocyte is discussed.

28] S-adenosylmethionine: Protein-carboxyl O-Methyltransferase (protein methylase II)

This chapter illustrates assay, purification, and properties of S-adenosylmethionine : protein-carboxyl O-methyltransferase or protein methylase II. It is ubiquitous in nature and has been purified from various mammalian organs, wheat germ, and bacteria. The substrate specificity and molecular properties of the enzyme vary greatly depending on the origin. The enzyme transfers the methyl group of S-adenosyl-L-methionine (SAM) to free carboxyl groups of protein or polypeptide substrates, yielding protein-carboxylmethyl esters. Protein methylase II activity can be determined by two different approaches utilizing radiomethyl-labeled SAM. The first approach is to directly determine the amount of radioactivity transferred from [methyl14C] SAM into trichloroacetic acid (TCA)-precipitable substrate protein. The second approach takes advantage of the alkali lability of the enzymatically formed ester bond; the reaction product is hydrolyzed in an alkaline solution and the liberated radioactive methanol is extracted using isoamyl alcohol or collected by distillation under reduced pressure and moderate temperature. The enzyme in the crude form may be stored at -20 ° for a few years without appreciable loss of the activity. The activity of purified enzyme however is very susceptible toward acidic pH, air oxidation, high temperature, and low protein concentration.

Purification and properties of protein methylase II from wheat germ.

Protein methylase II (S-adenosylmethionine: protein-carboxyl O-methyltransferase, EC 2.1.1.24) was purified from wheat germ approximately 1200-fold with a yield of 4.6% by employing gel filtration. The enzyme, using S-adenosyl-L-methionine, methyl-esterifies the free carboxyl group of protein. Since the enzymatic product was unstable and hydrolyzed non-enzymatically to yield methanol, methanol was identified from the hydrolysate by the formation of the methyl ester of 3.5-dinitrobenzoate. The pH optimum for the wheat germ enzyme was 7.0 in contrast to the 6.0 for the methylase II of calf brain. The wheat germ enzyme had a molecular weight of 41 000 compared to 25 000 for the corresponding rat erythrocyte enzyme [S. Kim (1975) Arch. Biochem. Biophys. 161, 652-657]. The enzyme also differed with the mammalian enzyme in protein substrate preference: the mammalian enzyme showed equal preference to histone and immunoglobulin G while the wheat germ enzyme transferred a methyl group 4.5 times more to histone than to immunoglobulin G. The Km values for histone and S-adenosyl-L-methionine were 0.2 mM and 5 microM. S-Adenosyl-L-homocysteine and its analogues, sinefungin and A9145C, were competitive inhibitors with Ki values 1.5 microM, 0.4 microM and 0.1 microM, respectively. To investigate the identity of endogenous substrate(s) for protein methylase II, the crude wheat germ extract was incubated with S-adenosyl-L-[methyl-3H]methionine and the methylated proteins were analyzed by acid/urea and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Among other methyl acceptors in the wheat germ, histone polypeptides were the major endogenous substrates.

Reduced erythrocyte membrane protein methylation in sickle cell anemia.

The methylation of erythrocyte membrane components in sickle cell anemia has been studied and found to differ considerably from that of normal erythrocytes. When sickle erythrocytes were incubated under physiological conditions (pH = 7.4, 37 degrees C) in the presence of L-[methyl-3H]methionine or S-adenosyl-L-[methyl-3H] methionine, a 50% decrease in the protein-carboxyl methylation was observed compared to the normal erythrocyte. This reduction in degree of methylation was reflected in all of the major methylated protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since this methylation is catalyzed by the cytosolic protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC 2.1.1.24), the in vitro substrate capability of sickle erythrocyte ghosts and pH 11 treated ghosts were tested by incubation with purified protein methylase II and S-adenosyl-L-[methyl-14C]methionine. Both types of sickle cell ghost preparations showed the same 50% decrease in methylation as was seen in the intact cells. Since it was also shown that the protein methylase II and methyl acceptor membrane protein levels in the sickle erythrocytes are the same as the normal control, the data suggest that the observed reduced methylation may be due to an altered membrane conformation. The methylation of phospholipid was also studied and found to be decreased in sickle cell erythrocytes. However, this methylation was extremely minor in comparison to protein-carboxyl methylation which represents the bulk of the membrane methylation.

Enzymatic methyl esterification of specific glutamyl residue in corticotropin.

Ovine corticotropin (alpha s-ACTH) was enzymatically methylated with purified calf brain protein methylase II (protein O-methyltransferase; S-adenosyl-L-methionine: protein-carboxyl O-methyltransferase, EC 2.1.1.24) and S-adenosyl-L-[methyl-14C]methionine. After incubation for 60 min at 37 degrees C, 30 mol % of the hormone was methylated on the basis of the [14C]methyl incorporation. In order to assess the location of methylation, the modified peptide was digested with pepsin. Analytical results derived from studies on the peptic digest led to the suggestion that the alpha s-ACTH-(6--28) peptide fragment was esterified. Because there is only one available methylation site at Glu-28, these results indicate that Glu-28 of alpha s-ACTH was specifically methyl esterified to yield [Glu(OMe)28]-alpha s-ACTH.

Methyl acceptors for protein methylase II from human-erythrocyte membrane.

Membrane proteins from human erythrocytes were methylated with purified protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC.2.1.1.24). The methylated proteins were analyzed by dodecyl sulfate/polyacrylamide gel electrophoresis. Monomeric and dimeric glycophorin A (NaIO4/Schiff-2 and NaIO4/Schiff-1 positive bands) and 'band 4.5' were identified as two major classes of methyl-acceptor polypeptides for protein methylase II. In rabbit erythrocyte membrane where glycophorin A is absent, 'band 4.5' was the only major methyl-acceptor protein component. Extracted and purified glycophorin A from human erythrocytes was also found to be an excellent substrate for protein methylase II with a Km of 35.7 microM. The role of erythrocyte membrane protein methylation is discussed with regard to membrane function.

ENZYMATIC METHYL ESTERIFICATION OF PITUITARY POLYPEPTIDES

Methyl esterification of pituitary polypeptides by protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC. 2.1.1.24) has been investigated. Ovine lutropin and adrenocorticotropin (alpha1-39-ACTH) were found to be good methyl acceptor substrates, followed by beta-lipotropin. While the alpha-subunit of lutropin had nearly equal the methyl accepting activity of lutropin, the beta-subunit was devoid of accepting activity. The maximum amount of esterification occurred between 15 and 30 min at 37 degrees C) depending on the methyl acceptor molecule. The rate of the methyl esterification of adrenocorticotropin fragments was also studied. While alpha7-38-ACTH had less than half of alpha1-37-ACTH methyl accepting capacity, alpha1-17-ACTH did not serve as methyl acceptor. However, when a mixture of the two fragments was preincubated, the resulting mixture had full alpha1-39-ACTH activity.