Amplification and detection of substrates for protein carboxyl methyltransferases in PC12 cells.

A strategy that facilitates the identification of substrates for protein carboxyl methyltransferases that form "stable" methyl esters, i.e., those that remain largely intact during conventional polyacrylamide gel electrophoresis is described. Rat PC12 cells were cultured in the presence of adenosine dialdehyde (a methylation inhibitor) to promote the accumulation of hypomethylated proteins. Nonidet P-40 cell extracts were then incubated in the presence of S-[methyl-3H]adenosyl-L-methionine to label methyl-accepting sites via endogenous methyltransferases. After labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel slices were incubated in 4 N methanesulfonic acid or 6 N HCl to hydrolyze methyl esters. The resulting [3H]methanol was detected by trapping in liquid scintillation fluid. Seven carboxyl methylated proteins were observed with masses ranging from 18 to 96 kDa. Detection of five of these proteins required prior treatment of cells with adenosine dialdehyde, while methyl incorporation into one protein at 18 kDa was substantially enhanced by the treatment. The use of acidic conditions for methyl ester hydrolysis has an important advantage over assays that utilize alkaline hydrolysis conditions. In PC12 cells, and possibly other cell types where there are significant levels of arginine methylation, the methanol signal becomes obscured by high levels of volatile methylamines generated under the alkaline conditions. Carrying out diffusion assays under acidic conditions eliminates this interference. Adenosine dialdehyde, by virtue of increasing the methyl-accepting capacity of substrates for protein carboxyl methyltransferases, in combination with a more selective assay for carboxyl methylation, should prove useful in the isolation and characterization of new protein carboxyl methyltransferases and their substrates.     

Increased methyl esterification of membrane proteins in aged red-blood cells. Preferential esterification of ankyrin and band-4.1 cytoskeletal proteins

The enzymatic carboxyl methyl esterification of erythrocyte membrane proteins has been investigated in three different age-related fractions of human erythrocytes. When erythrocytes of different mean age, separated by density gradient centrifugation, were incubated under physiological conditions (pH 7.4, 37 degrees C) in the presence of L-[methyl-3H]methionine, the precursor in vivo of the methyl donor S-adenosylmethionine, a fourfold increase in membrane-protein carboxyl methylation was observed in the oldest cells compared with the youngest ones. The identification of methylated species, based on comigration of radioactivity with proteins stained with Coomassie blue, analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, shows, in all cell fractions, a pattern similar to that reported for unfractionated erythrocytes. However in the membrane of the oldest erythrocytes the increase in methylation of the cytoskeletal proteins, bands 2.1 and 4.1, appears to be significantly more marked compared with that observed in the other methylated polypeptides. Furthermore the turnover rate of incorporated [3H]methyl groups in the membrane proteins of the oldest cells markedly increases during cell ageing. Particularly in band 4.1 the age-related increase in methyl esterification is accompanied by a significant reduction of the half-life of methyl esters. The activity of cytoplasmic protein methylase II does not change during cell ageing, while the isolated ghosts from erythrocytes of different age show an age-related increased ability to act as methyl-accepting substrates, when incubated in presence of purified protein methylase II and methyl-labelled S-adenosylmethionine, therefore the relevance of membrane structure in determining membrane protein methylation levels can be postulated. Finally the possible correlation of this posttranslational protein modification with erythrocyte ageing is discussed.     

Kinetic and electrophoretic analysis of transmethylation reactions in intact Xenopus laevis oocytes

Transmethylation reactions in fully grown Xenopus oocytes were analyzed following the microinjection of S-adenosyl-L-[methyl-3H]methionine (AdoMet). The size of the endogenous AdoMet pool, measured by cation exchange high pressure liquid chromatography is 5.91 pmol/oocyte. The AdoMet pool turns over with a half-time of 2 h, at a rate of 2.07 pmol/h/oocyte. Fractionation experiments indicate that approximately one-third of the AdoMet in oocytes is utilized for protein carboxylmethylation reactions and another third is metabolized into small molecules which are secreted. The remainder of the intracellular AdoMet is used primarily for protein N-methylation reactions, although some methylation of phospholipids and nucleic acids also occurs. Polyacrylamide gel electrophoresis of 3H-methylated proteins at pH 2.4 in the presence of sodium dodecyl sulfate demonstrated that methyl esters are associated with a heterogeneous group of proteins in both the nucleus and cytoplasm of oocytes, coincident with the subcellular distribution of the protein D-aspartyl, L-isoaspartyl methyl transferase (O'Connor, C. M. (1987) J. Biol. Chem. 262, 10398-10403). The protein methyl esters associated with oocyte proteins turn over rapidly, as evidenced from the presence of [3H]methanol in the medium. The calculated rate of protein carboxyl methylation, 0.7 pmol/h/oocyte, is similar to that of protein synthesis in oocytes, suggesting that the modification of derivatized aspartyl residues represents a major pathway in oocyte protein metabolism. Since the formation of protein methyl esters is unaffected by cycloheximide, it is unlikely that methyl-accepting sites on oocyte proteins arise primarily from errors in protein synthesis. Unlike protein carboxyl methylation reactions, protein N-methylation reactions are closely linked to protein synthesis, and the methyl group linkages are stable over a period of at least 4 h. Numerous protein acceptors for N-methylation reactions were identified by polyacrylamide gel electrophoresis.     

Analysis of erythrocyte protein methyl esters by two-dimensional gel electrophoresis under acidic separating conditions

A two-dimensional polyacrylamide gel electrophoresis system which is suitable for the analysis of protein methylation reactions in cells incubated with L-[methyl-3H]methionine is described. The procedure separates proteins under primarily acidic conditions by isoelectric focusing in the first dimension and by sodium dodecyl sulfate electrophoresis at pH 2.4 in the second dimension. The low pH is essential for preserving protein [3H]methyl esters, but it limits the effective separating range of this system to proteins with isoelectric points between 4 and 8. With this system, we have shown that most, if not all, erythrocyte membrane and cytosolic proteins can act as substoichiometric methyl acceptors for an intracellular S-adenosylmethionine-dependent carboxyl methyltransferase and that protein carboxyl methylation reactions may be the major methyl transfer reaction in erythrocytes. These results are most consistent with the generation of protein substrate sites for the carboxyl methyltransferase by spontaneous deamidation and racemization reactions.     

Identification and Topography of Substrates for Protein Carboxyl Methyltransferase in Synaptic Membrane and Myelin-Enriched Fractions of Bovine and Rat Brain

The major components of crude brain synaptosomes (synaptic membranes, mitochondria, and myelin) have been separated and analyzed by polyacrylamide gel electrophoresis for the presence of proteins that serve as substrates for protein carboxyl methyltransferase. Of the three fractions, synaptic membranes contain the largest number of individual methyl acceptors (at least seven), while mitochondria contain no well-defined methyl acceptors. Undisrupted myelin contains a single major methyl acceptor with a very low apparent molecular weight. The patterns of protein methylation in synaptic membranes prepared from cerebral cortex, hippocampus, striatum, thalamus, and tectum showed marked differences; however, these differences could largely be explained by differential degrees of myelin contamination in synaptic membranes from the different regions. The effect of trypsin pretreatment on the carboxyl methylation of intact and lysed synaptosomes was studied to estimate the sidedness of the major methylation sites on synaptic membranes. One of the methyl acceptors (Mr 48K) appears to be facing the intracellular surface of the synaptosome, but most sites appear to be outward facing.     

Tubulin and high molecular weight microtubule-associated proteins as endogenous substrates for protein carboxymethyltransferase in brain

The endogenous substrate for protein carboxymethyltransferase in brain was examined. Several polypeptides were methylated when brain slices were incubated with L-methionine or when subcellular fractions of brain, such as the cytosolic fraction, were incubated with S-adenosyl L-methionine. Two methyl-accepting proteins in the cytoplasm were identified as tubulin and high molecular weight microtubule-associated proteins (300 kDa), which are components of microtubules. Tubulin behaved as a 43 kDa protein in acidic polyacrylamide gel electrophoresis, but as a 55 kDa protein in SDS-polyacrylamide gel electrophoresis. The methyl moiety transferred to these proteins from L-methionine was labile at alkaline pH. The high molecular weight microtubule-associated proteins showed higher methyl-accepting activity than tubulin or ovalbumin, which was used as a standard substrate: about 20 mmol of high molecular weight microtubule-associated proteins, 2 mmol of tubulin and 10 mmol of ovalbumin were methylated per mol of each protein in 30 min under the experimental conditions used.     

RNase treatment of yeast and mammalian cell extracts affects in vitro substrate methylation by type I protein arginine N-methyltransferases.

Type I protein arginine N-methyltransferases catalyze the formation of omega-NG-monomethylarginine and asymmetric omega-NG, NG-dimethylarginine residues using S-adenosyl-l-methionine as the methyl donor. In vitro these enzymes can modify a number of soluble methyl-accepting substrates in yeast and mammalian cell extracts including several species that interact with RNA. We treated normal and hypomethylated Saccharomyces cerevisiae and RAT1 cell extracts with RNase prior to in vitro methylation by recombinant protein N-arginine methyltransferases and found that the methylation of certain polypeptides is enhanced up to 12-fold whereas that of others is diminished. 2-D gel electrophoresis of RNase-treated yeast extracts allowed us to tentatively identify the glycine- and arginine-rich (GAR) domain-containing proteins Gar1, Nop1, Sbp1, and Npl3 as major methyl-acceptors based on their known isoelectric points and apparent molecular weights. These results suggest that the methylation and RNA-binding of GAR domain-containing proteins in vivo may regulate protein-nucleic acid or protein-protein interactions.     

The localization of protein carboxyl-methylase in sperm tails

Protein carboxyl-methylase (PCM), an enzyme known to be involved in exocytotic secretion and chemotaxis, has been studied in rat and rabbit spermatozoa. PCM activity and its substrate methyl acceptor protein(s) (MAP) were demonstrated in the supernate after solubilization of the sperm cell membrane by detergent (Triton X-100). A protein methylesterase that hydrolyzes methyl ester bonds created by PCM was demonstrated in rabbit but not in rat spermatozoa. This enzyme was not solubilized by nonionic detergent. The specific activities of PCM in rat spermatozoa from caput and cauda epididymis were similar and lower than that found in testis. By contrast, MAP substrates were low in testis and increased in parallel with sperm maturation in the epididymis. Multiple MAP were demonstrated in spermatozoa by polyacrylamide gel electrophoresis. The pattern of these proteins was similar in spermatozoa from different portions of the reproductive tract. Fractionation of heads and tails of rat spermatozoa on sucrose gradients indicated that PCM was found exclusively in the tail fraction, whereas MAP was detected both in head and tail fractions. The presence of all the components of the protein carboxyl-methylation system in spermatozoa and the localization of PCM and some of its substrates in the sperm tail are consistent with their involvement in sperm cell motility.     

Protein carboxyl methylation-demethylation system in developing rat livers

Protein carboxyl methyltransferase and protein methylesterase activity was assayed in various cell fractions prepared from rat livers. Significant amounts of protein carboxyl methyltransferase were detected in the cytosol and nucleoplasm. The cellular concentration of this enzyme paralleled development, activity being highest in the liver from young animals. If methylation was inhibited at any point during the reaction with S-adenosylhomocysteine, protein methylesterase activity was evident by a rapid decrease in carboxyl-methylated proteins. Protein methylesterase activity could be assessed by measuring the amount of [3H]methanol present in reaction filtrates. After a 10-min lag, the rate of demethylation was equivalent to the rate of methylation. The turnover of methyl groups was primarily enzymatic, since little or no methanol was generated when adrenocorticotropic hormone was incubated with purified protein carboxyl methyltransferase. Assessment of protein methylesterase activity as a function of the amount of methanol in the reaction filtrates represents minimal values, since the resultant [3H]methanol was metabolized rapidly via an alcohol dehydrogenase and/or oxidase. The rapid turnover of the protein methyl esters makes it difficult to assess the endogenous methyl acceptor proteins. Protein methyl esters were not detectable in any significant amounts in hepatic cell fractions in vivo; however, the nuclei contained measurable amounts of carboxyl-methylated proteins in vitro. These proteins are firmly bound to DNA but are not an integral part of the nucleosome. Analysis of the proteins, after fractionation on hydroxylapatite and sodium dodecyl sulfate-acrylamide gel electrophoresis, revealed that several non-histone chromosomal proteins were carboxyl methylated. The approximate molecular weights of these proteins were 172K, 106K, 98K, 81K, 66K, 62K, 52K, and 38K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein N-arginine methylation in subcellular fractions of lymphoblastoid cells

Arginine methylation in RNA-binding proteins containing arginine- and glycine-rich RGG motifs is catalyzed by specific protein arginine N-methyltransferase in cells. We previously showed that lymphoblastoid cells grown in the presence of an indirect methyltransferase inhibitor, adenosine dialdehyde (AdOx), accumulated high level of hypomethylated protein substrates for the endogenous protein methyltransferases or recombinant yeast arginine methyltransferase [Li, C. et al. (1998) Arch. Biochem. Biophys. 351, 53-59]. In this study we fractionated the lymphoblastoid cells to locate the methyltransferases and the substrates in cells. Different sets of hypomethylated methyl-accepting polypeptides with wide range of molecular masses were present in cytosolic, ribosomal, and nucleus fractions. The methylated amino acid residues of the methyl-accepting proteins in these fractions were determined. In all three fractions, dimethylarginine was the most abundant methylated amino acid. The protein-arginine methyltransferase activities in the three fractions were analyzed using recombinant fibrillarin (a nucleolar RGG protein) as the methyl-accepting substrate. Fibrillarin methylation was strongest in the presence of the cytosolic fraction, followed by the ribosomal and then the nucleus fractions. The results demonstrated that protein-arginine methyltransferases as well as their methyl-accepting substrates were widely distributed in different subcellular fractions of lymphoblastoid cells.     

Purification and Characterization of Two Distinct Isozymes of Protein Carboxymethylase from Bovine Brain

Protein carboxymethylase (EC 2.1.1.24) from cytosol of bovine brain was found to exist as two apparent isozymes that could be separated by chromatography on DEAE-cellulose at pH 8.O. Rechromatography of the two forms, designated PCM I and PCM II, indicated that they are not interconvertible. Both enzymes have a molecular weight of 24,300 by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. PCM I consists mainly of one isoelectric form, pI 6.5, whereas PCM II resolves into two forms of pI 5.6 and 5.7. The relative amounts of PCM I and PCM II show a marked tissue dependence. Brain has approximately twice as much PCM I as II, whereas liver contains only the type II enzyme. The two enzymes were found to have similar substrate specificities when tested with five different methyl-accepting proteins. Synapsin I, a basic protein associated with synaptic vesicles, was found to be an excellent methyl-accepting protein with regard to its K_m (1.2 μM), but it exhibited a low stoichiome-try of methyl incorporation.     

The methyl-accepting chemotaxis proteins of Escherichia coli. Identification of the multiple methylation sites on methyl-accepting chemotaxis protein I

The methyl-accepting chemotaxis proteins (MCPs) are integral membrane proteins that undergo reversible methylation during adaptation of bacterial cells to environmental attractants and repellents. The numerous methylated forms of each MCP are seen as a pattern of multiple bands on polyacrylamide gels. We have characterized the methylation sites in MCPI by analyzing methyl-accepting tryptic peptides. At least two different tryptic peptides accept methyl esters; one methyl-accepting peptide contains methionine and lysine and may be methylated a maximum of four times. The second methyl-accepting tryptic peptide contains arginine and may be methylated twice. Base-catalyzed demethylations of tryptic peptides and analysis of the charge differences between the different methylated forms of MCPI show that MCPI molecules may be methylated a total of six times. The two methyl esters on the methyl-accepting arginine peptide appear to be preferentially methylated in most of the forms of MCPI in attractant-stimulated cells. The ability to acquire six methylations on MCPI allows the bacterial cells to adapt to a broad range of attractant and repellent concentrations.     

Carboxyl methylation of cytosolic proteins in intact human erythrocytes. Identification of numerous methyl-accepting proteins including hemoglobin and carbonic anhydrase

Intact human erythrocytes incubated with L-[methyl-3H]methionine incorporated radioactivity into base-labile linkages with membrane and cytosolic proteins which are characteristic of protein methyl esters. Kinetic analysis of the methylation reactions in intact cells shows that individual erythrocytes contain approximately 38,000 and 115,000 protein methyl esters with biological half-lives of 150 min or less in the membrane and cytosolic protein fractions, respectively. Fractionation of the methylated cytosolic species by gel filtration chromatography at pH 6.5 followed by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 reveals that many different cytosolic proteins serve as methyl acceptors and that the degree of modification varies widely for individual proteins. For example, hemoglobin is modified to the extent of 3 methyl groups/10(6) polypeptide chains, while carbonic anhydrase contains 1 methyl group/approximately 16,500 polypeptide chains at steady state. Aspartic acid beta-[3H]methyl ester (Asp beta-[3H]Me) can be isolated from carboxypeptidase Y digests of cytosol proteins. By synthesizing and separating diastereomeric L-Leu-L-Asp beta Me and L-Leu-D-Asp beta Me dipeptides, we show that all of the Asp beta-[3H]Me recovered from cytosolic proteins is in the D-stereoconfiguration. Based on these data and on previous observations that erythrocytes contain a single methyltransferase which also methylates red cell membrane proteins at D-aspartyl residues both in vivo (McFadden, P. N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2460-2464) and in vitro (O'Connor, C. M., and Clarke, S. (1983) J. Biol. Chem. 258, 8485-8492), we propose that protein carboxyl methylation is part of a generalized mechanism for metabolizing damaged proteins. The infrequent and spontaneous occurrence of D-aspartyl residues in proteins adequately explains the broad substrate specificity and limited stoichiometries of protein carboxyl methylation reactions.     

SUBCELLULAR LOCALIZATION OF PROTEIN CARBOXYL-METHYLASE AND ITS SUBSTRATES IN RAT PITUITARY LOBES

Subcellular distribution of protein carboxyl-methylase (PCM) and its endogenous substrates the methyl acceptor proteins (MAP) have been studied in the anterior, the posterior and the intermediate lobes of the rat pituitary gland. In all three lobes, PCM was found to be a cytosolic enzyme whereas the highest MAP specific capacity was observed in the lysate of the granular fractions. The methylated proteins from the lysates of the granular fractions and the cytosolic fractions were analyzed with a novel electrophoretic system which prevents the hydrolysis of the protein-methyl esters. Gel electrophoretic profiles from the lysates of the granular fractions were different from those of the cytosolic fractions. In the lysatcs of the granular fractions. the MAP had a molecular weight of less than 25,000 suggesting that the methylated polypeptides in these fractions were the pituitary peptide hormones (and their subunits) and neurophysins.     

Protein Carboxylmethyltransferase Activity in Intact, Differentiated Neuroblastoma Cells: Quantitation by S[3H]Adlenosylmethionine Prelabeling

Abstract: Protein carboxylmethyltransferase has been proposed to play a role in the regulation of neuroblastoma differentiation (Kloog et al., 1983). When we investigated this hypothesis further, different results for methyl ester formation were obtained when measured in acid-precipitated proteins and in proteins separated by acidic polyacrylamide gel electrophoresis, following the incubation of intact neuroblastoma cells with [³H]methionine. These unexpected findings led to the development of a modified assay using S -[³H]-adenosylmethionine as the radiolabeled precursor for quan-titating carboxyl methylation in intact cells. Data obtained from either acid-precipitated proteins or those separated on an electrophoresis gel following S -[³H]adenosylmethionine incubation directly correlated with data obtained from proteins separated by electrophoresis following [³H]methionine incubation. Using each of the three methods, an approxi-mately twofold increase in the carboxyl methylation of cellular proteins was detected in neuroblastoma cells differentiated by reducing the serum concentration from 10 to 0.5%, but not in those cells differentiated with either 5 m M hexa-methylene bisacetamide or 2% dimethyl sulfoxide. The finding that all detectable methyl acceptor proteins are increasingly methylated following 0.5% serum treatment and that this modification is substoichiometric suggests that protein carboxyl methylation is not an essential component of the differentiation process in neuroblastoma cells.     

Protein carboxyl methylation in kidney brush-border membranes.

Protein carboxyl methylation activity was detected in the cytosol and in purified brush-border membranes (BBM) from the kidney cortex. The protein carboxyl methyltransferase (PCMT) activity associated with the BBM was specific for endogenous membrane-bound protein substrates, while the cytosolic PCMT methylated exogenous substrates (ovalbumin and gelatin) as well as endogenous proteins. The apparent Km for S-adenosyl-L-methionine with endogenous proteins as substrates were 30 microM and 4 microM for the cytosolic and BBM enzymes, respectively. These activities were sensitive to S-adenosyl-L-homocysteine, a well known competitor of methyltransferase-catalyzed reactions, but were not affected by the presence of chymostatin and E-64, two protein methylesterase inhibitors. The activity of both cytosolic and BBM PCMT was maximal at pH 7.5, while BBM-phospholipid methylation was predominant at pH 10.0. Separation of the = methylated proteins by acidic gel electrophoresis in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride revealed distinct methyl accepting proteins in the cytosol (14, 17, 21, 27, 31, 48, 61 and 168 kDa) and in the BBM (14, 60, 66, 82, and 105 kDa). Most of the labelling was lost following electrophoresis under moderately alkaline conditions, except for a 21 kDa protein in the cytosol and a 23 kDa protein in the BBM fraction. These results suggest the existence of two distinct PCMT in the kidney cortex: a cytosolic enzyme with low selectivity and affinity, methylating endogenous and exogenous protein substrates, and a high-affinity BBM-associated methylating activity.     

Multiple methylation of methyl-accepting chemotaxis proteins during adaptation of E. coli to chemical stimuli

In bacterial chemotaxis, adaptation is correlated with methylation or demethylation of methyl-accepting chemotaxis proteins (MCPs). Each protein migrates as a characteristic set of multiple bands in sodium dodecylsulfate polyacrylamide gel electrophoresis. The changes in MCP methylation that accompany adaptation are not the same for all bands of a set. Adaptation to a type II repellent stimulus results in an overall decrease in MCP II methylation, but also in an increase in the amount of radioactive methyl groups in the upper band of the set. We demonstrate that this increase is not due to new methylation, but rather to reduced electrophoretic mobility of previously methylated molecules that have lost some but not all of their methyl groups. We suggest that the pattern of multiple bands is a direct reflection of multiple sites for methylation on MCP molecules, and that the distribution of radiolabel among the bands is determined by the total extent of methylation. The patterns of methylated peptides produced by limited proteolysis of different MCP bands imply that methylation of the multiple sites on a molecule may occur in a specific order.     

Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.     

In vitro methylation of bacterial chemotaxis proteins: Characterization of protein methyltransferase activity in crude extracts of salmonella typhimurium

A specific in vitro assay was developed for the protein carboxyl methyltransferase that is involved in the chemotactic behaviour of Salmonella typhimurium. This cytosolic enzyme catalyzes an S-adenosyl-L-methionine-dependent methyl esterification of glutamyl residues on a class of 60,000-dalton inner-membrane proteins. The activity was found to display a pH optimum of 6.5 and be sensitive to the concentration of salts in the assay medium. No detectable activity was found towards a variety of other proteins which serve as substrates for mammalian and other bacterial carboxyl methyltransferases. This assay was used to quantitate the methylation of the 60,000-dalton methyl-accepting proteins in response to chemoeffectors. Small but reproducible concentration-dependent changes in the initial rates of in vitro methylation were observed with chemotactic attractants and repellents. The specific methyltransferase activity was found to be absent in several mutants in flagellar synthesis (fla^?), suggesting that the synthesis of this enzyme is coordinately regulated with that of flagellin and basal bodies. The hydrodynamic properties of the enzyme in crude extracts were determined by gel filtration and sucrose velocity gradient centrifugation, and a native molecular weight of 41,000 was calculated from these data.     

Methylation of erythrocyte membrane proteins at extracellular and intracellular D-aspartyl sites in vitro. Saturation of intracellular sites in vivo

A cytosolic protein carboxyl methyltransferase (S-adenosyl-L-methionine:protein O-methyltransferase, E.C. 2.1.1.24) purified from human erythrocytes catalyzes the methylation of erythrocyte membrane proteins in vitro using S-adenosyl-L-[methyl-3H]methionine as the methyl group donor. The principal methyl-accepting proteins have been identified by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 and fluorography as the anion transport protein (band 3), ankyrin (band 2.1), and integral membrane proteins with molecular weights of 45,000, 28,000, and 21,000. Many of the methylation sites associated with intrinsic membrane proteins may reside in their extracellular portions, since these same proteins are methylated when intact cells are used as the substrate. The maximal number of methyl groups transferred in these experiments is approximately 30 pmol/mg of membrane protein, a value which represents less than one methyl group/50 polypeptide chains of any methyl-accepting species. The number of methylation sites associated with the membranes is increased, but not to stoichiometric levels, by prior demethylation of the membranes. The additional sites are associated primarily with bands 2.1 and 4.1, the principal methyl acceptors in vivo, suggesting that most methylation sites are fully modified in vivo. Extracellular methylation sites are not increased by demethylation of membranes. The aspartic acid beta-methyl ester which can be isolated from carboxypeptidase Y digests of [3H]methylated membranes is in the unusual D-stereoconfiguration. Similar results have been obtained with [3H]methylated membranes isolated from intact cells (McFadden, P.N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464). It is proposed that the methyltransferase recognizes D-aspartyl residues in proteins and is involved with the metabolism of damaged proteins in vivo.