Substrates For Protein Carboxyl Methyltransferase Research Articles

Evidence for the in vivo deamidation and isomerization of an asparaginyl residue in cytosolic serine hydroxymethyltransferase.

Rabbit liver cytosolic serine hydroxymethyltransferase exists in several subforms which have different isoelectric points. Incubation of the purified enzyme with chymotrypsin cleaves the enzyme at Trp14. The released amino-terminal 14-mer peptide was shown to exist in three forms of equal concentration. The peptides differ in structure only at the asparaginyl residue at position 5. In addition to asparagine at this position we found both aspartyl and isoaspartyl residues. The deamidation of Asn5 does not appear to occur during the purification of the enzyme. The in vitro rate of deamidation of Asn5 in the enzyme is more than 5-fold slower than the rate of deamidation of this residue in the free 14-mer peptide. The isoaspartyl residue at position 5 serves as a substrate for protein carboxyl methyltransferase both in the free 14-mer peptide and the native enzyme. The enzyme which has had the amino-terminal 14 residues removed by digestion with chymotrypsin still exists in several forms with different isoelectric points. Reaction of peptides from this enzyme with carboxyl methyltransferase suggests that there is at least one more asparaginyl residue in this enzyme other than Asn5 which has undergone deamidation with the formation of isoaspartyl bonds.

Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.

Posttranslational modification of calmodulin in rat brain and pituitary.

The posttranslational modification of calmodulin has been studied in six brain regions and the anterior pituitary. Carboxylmethylation, calmodulin converting enzyme, and calmodulin (lysine) N-methyltransferase activities were determined. Incubation of calmodulin with cytosolic extracts of these tissues in the presence of the methyl donor [methyl-3H]-S-adenosyl-L-methionine and identification of labeled proteins by gel electrophoresis and fluorography indicated that calmodulin is a substrate for protein carboxylmethyltransferase in all tissues tested. In hippocampus, caudate nucleus, cerebral cortex, and anterior pituitary, but not in cerebellum, superior colliculus, brainstem, or diencephalon, a second methylated protein was found when calmodulin was added to incubation mixtures. This protein was shown to be identical to the previously described product of calmodulin converting enzyme. Converted calmodulin was isolated by fast protein liquid chromatography and shown to be des(Lys)calmodulin, lacking the carboxy terminal lysine residue of calmodulin. The anterior pituitary had by far the highest levels of calmodulin converting enzyme; this enzyme, in turn, was identified as a cobalt-stimulated carboxylpeptidase B. In contrast to the regional differences in these parameters, the levels of calmodulin (lysine) N-methyltransferase did not differ greatly among brain regions, although regional differences in the activity of this enzyme were statistically significant.

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.

The effects of etorphine on the carboxylmethylation of synaptosomal proteins of rat striatum

The stimulation of protein carboxylmethyl transferase (PCMT) activity in rat striatal synaptosomes by the dopamine agonist, apomorphine, and a PCMT subtrate, calmodulin, was measured in normal and opioid-treated rats to see if inactivation of calmodulin by methylation is a factor in opioid action. Total carboxyl methyl acceptors were measured in preparations from alkaline homogenates, while those already occupied in vivo were measured in acidic homogenates, since the carboylmethyl group is stable in acid. The administration of etorphine acutely increased the number of already occupied acceptors while chronic morphine treatment decreased this number. Apomorphine stimulation of PCMT activity was significant only when tested for direction of change from control values. Calmodulin was a substrate for PCMT in all preparations. We conclude that the method of estimating total and occupied acceptors was partially successful. Perhaps a more alkaline homogenate would produce a synaptosomal prepration with a maximal PCMT activity. The importance of calmodulin carboxylmethylation cannot be assessed from these experiments, although the results shown in figure 3 suggest that opioid treatments have different effects on the methylation of calmodulin.

Endogenous substrates for protein carboxyl methyltransferase in cytosolic fractions of bovine brain.

A method of polyacrylamide gel electrophoresis utilizing the discontinuous pH-stacking gel format, the cationic detergent cetylpyridinium chloride, and an acidic buffer system has been applied to detection of specific substrates for protein carboxyl methyltransferase (PCM, EC 2.1.1.24) in cytosol fractions of bovine cerebral cortex. This electrophoresis system produces a high-resolution separation of proteins while preventing spontaneous hydrolysis of protein carboxyl methyl esters. Separation occurs largely on the basis of molecular weight. By running polyacrylamide gels at 4 degrees C or 25 degrees C, it was possible to demonstrate that any specific methyl-accepting protein is modified to form a labile methyl ester rather than the more stable N-derivative. Using this system, we have found that partially purified fractions of PCM contain a variety of endogenous methyl-accepting proteins. The apparent specificity of these substrates varies widely; some apparently abundant proteins show little or no methylation, while other apparently less abundant proteins exhibit a relatively high degree of methylation. One protein, with an apparent Mr of 46,000, exhibited an exceptional degree of methylation. Two distinct classes of protein carboxyl methyl esters could be distinguished by their differing susceptibility to nonenzymatic hydrolysis. The possible relevance of our findings to the recent suggestion that PCM specifically methylates abnormal D-aspartyl residues in age-racemized proteins is considered.

15] Posttranslational modification of calmodulin

Publisher Summary Calmodulin is a substrate for three known post-translational modification reactions. Trimethyllysine occurs as the result of posttranslational N-methylation catalyzed by S -adenosylmethionine: calmodulin (lysine) N -methyltransferase. Calmodulin is also a substrate for protein carboxylmethyltransferase. This covalent modification of calmodulin is reversed by the action of protein carboxylmethylesterase. Whereas the N -methylation of calmodulin occurs at stoichiometric levels, the carboxylmethylation of calmodulin is substoichiometric, as it is for other substrates of protein carboxylmethyltransferase. Last, calmodulin can be modified by the action of a third enzyme, calmodulin converting enzyme, which clips the carboxyl terminal lysine from calmodulin, causing a change in its electrophoretic mobility on sodium dodecyl sulphate (SDS) and native polyacrylamide gels. The activity of these four enzymes may be assayed using in vitro systems. For the estimation of N -methylation or the carboxylmethylation of calmodulin, appropriate enzymes and substrates are incubated with S -adenosyl-L-[methyl- 3 H]methionine. The assays of methyl transfer to calmodulin may be accomplished by polyacrylamide gel electrophoresis and fluorography or by radiometric assay. The assay for calmodulin converting enzyme is based upon changes in electrophoretic properties exhibited by the modified calmodulin.