Structural elements affecting the recognition of L-isoaspartyl residues by the L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.

We have synthesized a series of L-isoaspartyl-containing (isoD) peptides and characterized their interaction with the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77). Our findings indicate that this enzyme interacts with 6 residues extending from the isoD-2 to isoD+3 positions in peptide substrates. Although peptides as simple as G-isoD-G are methylated with low affinity (Km = 17.8 mM), a wide variety of L-isoaspartyl-containing sequences in larger peptides are recognized with high affinity (Km less than 20 microM), the best yet discovered being VYP-isoD-HA, with a Km of 0.29 microM. Only two sequence elements have been found that can interfere with the high affinity binding of peptides of 4 or more residues, these being a prolyl residue in the isoD+1 position and negatively charged residues in the isoD+1, isoD+2, and/or isoD+3 positions. We investigated the effect of higher order structure on binding affinity using several L-isoaspartyl-containing proteins. Although conformation did, in some cases, lower the affinity of the methyltransferase for L-isoaspartyl residues, the range of kinetic constants for the methylation of these proteins was similar to that observed with the synthetic peptides. The L-isoaspartyl/D-aspartyl methyltransferase has been proposed to function in vivo to prevent the accumulation of L-isoaspartyl residues that arise spontaneously as proteins age. To examine whether such a mechanism is feasible given the wide range of substrate Km values observed in vitro, we set up a computer simulation to model the degradation and methylation reactions in aging human erythrocytes. Our results suggest that enough methyltransferase activity exists in these cells to significantly lower the expected number of L-isoaspartyl residues, even when these residues have millimolar Km values for methylation.     

Methylation of microinjected isoaspartyl peptides in Xenopus oocytes. Competition with protein carboxyl methylation reactions

Xenopus oocytes possess a highly conserved protein carboxyl methyltransferase postulated to function in the repair or metabolism of age-damaged protein aspartyl residues (O'Connor, C. M. (1987) J. Biol. Chem. 262, 10398-10403). Three hexapeptides of the general sequence Val-Tyr-Pro-isoAsp-X-Ala, in which isoAsp represents an L-isoaspartyl residue and X represents Gly, Ser, or Ala, are methylated with the same order of preference following their microinjection into oocytes as in a purified system containing bovine brain protein carboxyl methyltransferase and S-adenosyl-L-[methyl-3H]methionine. The affinities of the enzyme for the glycyl, seryl, and alanyl variants of the peptides in vitro are 4.25, 3.04, and 1.67 microM, respectively. A nonapeptide of the sequence Lys-Ala-Ser-Ala-isoAsp-Leu-Ala-Lys-Tyr is a higher affinity substrate for the methyltransferase in vitro, characterized by a Km of 0.88 microM, but it is modified to a lesser extent in oocytes, partially because of its reduced stability in cytoplasm. The hexapeptide Val-Tyr-Pro-Asp-Gly-Ala, which contains an aspartyl residue in the usual stereoconfiguration, is not methylated either in vitro or in intact oocytes. Microinjection of any of the four isoaspartyl-containing peptides greatly stimulates total carboxyl methylation in oocytes, with rate increases ranging from 19- to 51-fold after the injection of 30 pmol of peptide. The protein ovalbumin is also modified following its microinjection into oocytes to near its calculated methyl-accepting capacity. Each of the isoaspartyl peptides can act as a competitive inhibitor of ovalbumin methylation both in vitro and in microinjected oocytes. The inhibitory potencies of the peptides parallel their specific methyl-accepting activities. The results demonstrate that the oocyte may be a useful model for studying the significance of protein carboxyl methylation because of the large functional excess of methylation capacity and the fidelity of the reactions compared to those observed in purified systems. This excess capability may have physiological significance when structurally abnormal proteins accumulate as a result of cellular stress and or aging.     

Enzymatic methylation of L-isoaspartyl residues derived from aspartyl residues in affinity-purified calmodulin. The role of conformational flexibility in spontaneous isoaspartyl formation

We have investigated the formation of D-aspartyl and L-isoaspartyl (beta-aspartyl) residues and their subsequent methylation in bovine brain calmodulin by the type II protein carboxyl methyltransferase. Based on the results of studies with unstructured peptides and denatured proteins, it has been proposed that the major sites of carboxyl methylation in calmodulin are at L-isoaspartyl residues that originate from two Asn-Gly sequences. To test this hypothesis, we directly identified the sites of methylation in affinity-purified preparations of calmodulin by peptide mapping using the proteases trypsin, endoproteinase Lys-C, clostripain, chymotrypsin, and Staphylococcus aureus V8 protease. We found, however, that the major high-affinity sites of methylation originate from aspartyl residues at position 2 and at positions 78 and/or 80. The methylatable residue in the first case was shown to be L-isoaspartate by comparison of the properties of a synthetic peptide corresponding to the N-terminal 13 residues substituted with an L-iso-Asp residue at position 2. The second methylatable residue, probably derived from Asp78, also appears to be an L-isoaspartyl residue. These sites appear to be readily accessible to the methyltransferase and are present in relatively flexible regions of calmodulin that may allow the spontaneous degradation reactions to occur that generate L-isoaspartyl residues via succinimide intermediates. Interestingly, the four calcium binding regions, each containing 3-4 aspartyl and asparaginyl residues (including the two Asn-Gly sequences), do not appear to contribute to the high-affinity methyl acceptor sites, even when calcium is removed prior to the methylation reaction. We propose that methylatable residues do not form at these sites because of the inflexibility of these regions when calcium is bound.     

Synthetic peptide substrates for the erythrocyte protein carboxyl methyltransferase. Detection of a new site of methylation at isomerized L-aspartyl residues

Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.     

Modification of synthetic peptides related to lactate dehydrogenase (231-242) by protein carboxyl methyltransferase and tyrosine protein kinase: effects of introducing an isopeptide bond between aspartic acid-235 and serine-236

The possibility that isoaspartyl residues contribute to the substrate specificity of eucaryotic protein carboxyl methyltransferases and/or tyrosine protein kinases has been investigated with two synthetic oligopeptides, Lys-Gln-Val-Val-Asp/isoAsp-Ser-Ala-Tyr-Glu-Val-Ile-Lys, which correspond to amino acids 231-242 of lactate dehydrogenase. One version of the peptide contains the normal amino acid sequence of the chicken muscle M4 isozyme. The other version contains an isoaspartyl residue in position 235 in place of the normal aspartyl residue; i.e., Asp-235 is linked to Ser-236 via its side-chain beta-carboxyl group, rather than via the usual alpha-carboxyl linkage. The normal peptide corresponds to the sequence around Tyr-238 that is phosphorylated in Rous sarcoma virus infected chick embryo fibroblasts [Cooper, J. A., Esch, F. S., Taylor, S. S., & Hunter, T. (1984) J. Biol Chem. 259, 7835]. Using protein carboxyl methyltransferase purified from bovine brain, we found that the normal peptide did not serve as a methyl-accepting substrate but that the isopeptide served as an excellent substrate, exhibiting a stoichiometry of one methyl group per peptide and Km of 0.54 microM. With tyrosine protein kinase partially purified from normal rat spleen both peptides were found to serve as phosphate acceptors at Tyr-238, exhibiting Km values of 4.7 and 8.9 mM for the normal and isopeptide versions, respectively. These results support the idea that protein carboxyl methyltransferase selectively methylates the alpha-carboxyl group of atypical isoaspartyl residues. In contrast, the presence of isoaspartate had a modest negative effect on substrate activity for a tyrosine protein kinase from rat spleen.     

Recognition of D-aspartyl residues in polypeptides by the erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase. Implications for the repair hypothesis.

We provide here the first direct evidence that D-aspartyl residues in peptides are substrates for the L-isoaspartyl/D-aspartyl protein carboxyl methyltransferase (EC 2.1.1.77). We do this by showing that D-aspartic acid beta-methyl ester can be isolated from carboxypeptidase Y digests of enzymatically methylated D-aspartyl-containing synthetic peptides. The specificity of this reaction is supported by the lack of methylation of L-aspartyl-containing peptides under similar conditions. Methylation of D-aspartyl residues in synthetic peptides was not observed previously because with Km values ranging from 2.5 to 4.8 mM, these peptides are recognized by the methyltransferase with 700-10,000-fold lower affinity than are their L-isoaspartyl-containing counterparts. The physiological significance of D-aspartyl methylation was investigated in two ways. First, analysis of in situ methylated human erythrocyte proteins showed that at least 22% of the methyl groups associated with the proteins ankyrin and band 4.1 are on D-aspartyl residues, suggesting that D-aspartyl methylation is an important function of the methyltransferase in vivo. Second, mathematical modeling of the protein aging and methylation reactions occurring in intact erythrocytes indicated that the accumulation of D-aspartyl residues can be reduced as much as 2-5-fold by the methyltransferase activity. Although this reduction is much less than that predicted for L-isoaspartyl residues, it may be significant in maintaining functional proteins throughout the 120-day life span of these cells.     

Replacement of a labile aspartyl residue increases the stability of human epidermal growth factor

Long-term storage of recombinant human epidermal growth factor (EGF), an important promoter of cell division, results in its conversion to a new species that elutes later than native EGF on a reverse-phase column. This new species, called EGF-X, has only 20% of the biological activity of native EGF. Peptide mapping indicated that the primary structure of EGF-X differs from that of native EGF solely within the first 13 residues. N-Terminal sequencing of EGF-X revealed that about 30% of the polypeptides have been cleaved at the Asp-3/Ser-4 bond. In addition, the yields after the His residue at position 10 were extremely low, indicating that a chemical modification occurs at residue 11 that is incompatible with Edman degradation. We hypothesized that aspartic acid 11 had been converted to an isoaspartyl residue, and this was confirmed with L-isoaspartyl/D-aspartyl methyltransferase, an enzyme that methylates the side-chain carboxyl group of L-isoaspartyl residues but does not recognize normal L-aspartyl residues. EGF-X, but not EGF, was found to be a substrate of this enzyme, and proteolytic digestion of EGF-X with thermolysin localized the site of methylation to a nine-residue peptide containing position 11. We did not observe formation of the isoaspartyl derivative in EGF that had been denatured by reduction of its disulfide bonds. In addition, replacement of the aspartyl residue at position 11 with glutamic acid resulted in a fully active EGF derivative that does not form detectable amounts of EGF-X. We propose that conversion of this aspartyl residue to isoaspartate is a significant nonenzymatic degradation reaction affecting this growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Enzymatic protein carboxyl methylation at physiological pH: cyclic imide formation explains rapid methyl turnover

At pH 7.4, 37 degrees C, bovine brain protein carboxyl methyltransferase transiently methylates deamidated adrenocorticotropin. The methylation occurs at the alpha-carboxyl group of an atypical beta-carboxyl-linked isoaspartyl residue (position 25). Several lines of evidence indicate that the immediate product of demethylation is an aspartyl cyclic imide involving positions 25 and 26. The evidence includes (1) the rapid rate of methyl ester hydrolysis, which is consistent with intramolecular catalysis, (2) the inability of the demethylated product to be remethylated, (3) the charge of this product, and (4) its rate of breakdown. The eventual hydrolysis of the cyclic imide produces a 30/70 mixture of peptides containing either alpha- or beta-carboxyl-linked aspartyl residues, respectively. Cyclic imide formation is nonenzymatic and can explain the unusual lability of mammalian protein methyl esters in general. These findings suggest that protein carboxyl methylation in mammalian tissues is not a simple on/off reversible modification as it apparently is in chemotactic bacteria. Carboxyl methylation may serve to activate selected protein carboxyl groups for subsequent longer lasting modifications, possibly subserving a role in protein repair, degradation, cross-linking, or some other as yet undiscovered alteration of protein structure.     

Chemical conversion of aspartyl peptides to isoaspartyl peptides. A method for generating new methyl-accepting substrates for the erythrocyte D-aspartyl/L-isoaspartyl protein methyltransferase

Mammalian protein carboxyl methyltransferases have recently been proposed to recognize atypical configurations of aspartic acid and may possibly function in the metabolism of covalently altered cellular proteins. Consistent with this proposal, the tetrapeptide tetragastrin, containing a single "normal" L-aspartyl residue (L-Trp-L-Met-L-Asp-L-Phe-NH2) was found here not to be an in vitro substrate for erythrocyte carboxyl methyltransferase activity. However, chemical treatment of tetragastrin by methyl esterification and then de-esterification of the aspartic acid residue yielded a mixture of peptide products, the major one of which could now be enzymatically methylated. We show here that this new peptide species is the isomeric beta-aspartyl form of tetragastrin (L-iso-tetragastrin; L-Trp-L-Met-L-Asp-L-Phe-NH2), and it appears that isomerization proceeds via an intramolecular succinimide intermediate during the de-esterification procedure. L-iso-Tetragastrin is stoichiometrically methylated (up to 90% in these experiments) with a Km for the enzyme of 5.0 microM. Similar chemical treatment of several other L-aspartyl peptides also resulted in the formation of new methyltransferase substrates. This general method for converting normal aspartyl peptides to isoaspartyl peptides may have application in the reverse process as well.     

Specific recognition of altered polypeptides by widely distributed methyltransferases

Protein carboxyl methyltransferase activity has been detected in extracts prepared from bacterial cells (Salmonella typhimurium), amphibian (Xenopus laevis) oocytes, and transformed mammalian cell lines. This activity appears to specifically recognize altered aspartyl residues based on the observation that the synthetic peptide L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala is a good methyl-accepting substrate for the methyltransferase activity, but that the corresponding peptide containing a normal L-aspartyl residue is not. These activities are similar to those of the previously described human erythrocyte and bovine brain enzymes which catalyze the formation of polypeptide D-aspartyl beta-methyl esters and L-isoaspartyl alpha-methyl esters. The wide distribution of these enzymatic activites suggest that the methylation of atypical proteins is an essential function in cells.     

New proteomic developments to analyze protein isomerization and their biological significance in plants

Spontaneous isoaspartyl formation from aspartyl dehydration or asparaginyl deamidation is a major source of modifications in protein structures. In cells, these conformational changes could be reverted by the protein L-isoaspartyl methyltransferase (PIMT) repair enzyme that converts the isoaspartyl residues into aspartyl. The physiological importance of this metabolism has been recently illustrated in plants. Recent developments allowing peptide isomer identification and quantification at the proteome scale are portrayed. The relevance of these new proteomic approaches based on 2-D electrophoresis or electron capture dissociation analysis methods was initially documented in mammals. Extended use to Arabidopsis model systems is promising for the discovery of controlling mechanisms induced by these particular post-translational modifications and their biological role in plants.     

Identification of a site for carboxyl methylation in human alpha-globin

The human erythrocyte protein carboxyl methyltransferase modifies unusual protein D-aspartyl and L-isoaspartyl residues which arise spontaneously from internal rearrangements accompanying asparaginyl deamidation and aspartyl isomerization. A site of methylation associated with alpha-globin in intact cells has been identified by peptide mapping of radiolabeled globin isolated from human erythrocytes previously incubated with L-[methyl-3H]methionine. The site is located in a Staphylococcus V8 peptide containing residues 1-30 of alpha-globin. Two potential sources of methylation sites are present in this sequence at Asp-t and Asn-9.     

Metabolism of a synthetic L-isoaspartyl-containing hexapeptide in erythrocyte extracts. Enzymatic methyl esterification is followed by nonenzymatic succinimide formation

The synthetic peptide, L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala, is a substrate for the erythrocyte and brain protein carboxyl methyltransferases. These enzymes catalyze the methyl esterification of the free alpha-carboxyl group of the isoaspartyl residue, to which the glycyl residue is linked through the side chain beta-carboxyl group. In this work, we show that the alpha-methyl ester of this peptide was rapidly demethylated (t1/2 = 4 min at 37 degrees C, pH 7.4) in erythrocyte cytosolic extracts and that the product of this reaction appears to be the succinimide ring derivative of the peptide. The rate of demethylation, measured at either pH 6.0 or 7.4, was the same in buffer and erythrocyte extracts, suggesting that succinimide formation was a nonenzymatic reaction. The L-succinimide is more stable than the ester, but can be hydrolyzed in buffer at pH 7.4 (t1/2 = 180 min at 37 degrees C) to give a mixture of about 75% isoaspartyl peptide and 25% normal aspartyl peptide. The metabolism of the succinimide hexapeptide in erythrocyte extracts appears to be more complex, however. The implications of this work for the methylation and demethylation of cellular proteins containing structurally altered aspartyl residues are discussed.     

Methylation at specific altered aspartyl and asparaginyl residues in glucagon by the erythrocyte protein carboxyl methyltransferase

Protein carboxyl methyltransferases from erythrocytes and brain appear to catalyze the esterification of L-isoaspartyl and/or D-aspartyl residues but not of normal L-aspartyl residues. In order to identify the origin of these unusual residues which occur in subpopulations of a variety of cellular proteins, we studied the in vitro methylation by the erythrocyte enzyme of glucagon, a peptide hormone of 29 amino acids containing 3 aspartyl residues and a single asparagine residue. Methylated glucagon was digested with either trypsin, chymotrypsin, pepsin, or endoproteinase Arg C, and the labeled fragments were separated by high-performance liquid chromatography and identified. In separate experiments, methyl acceptor sites were determined by digesting glucagon first with proteases and then assaying purified glucagon fragments for methyl acceptor activity. Using both approaches, we found that the major site of methylation, accounting for about 62% of the total, was at the position of Asp-9. Chemical analysis of fragments containing this residue indicated that this site represents an L-isoaspartyl residue. A second site of methylation, representing about 23% of the total, was detected at the position of Asn-28 and was also shown to represent an L-isoaspartyl residue. Methyl acceptor sites were not detected at the positions of Asp-15 or Asp-21. Preincubation of glucagon under basic conditions (0.1 M NH4OH, 3 h, 37 degrees C) increased methylation at the Asn-28 site by 4-8-fold while methylation at the Asp-9 site remained unchanged. These results suggest that methylation sites can originate from both aspartyl and asparaginyl residues and that these sites may be distinguished by the effect of base treatment.     

Stoichiometric methylation of porcine adrenocorticotropin by protein carboxyl methyltransferase requires deamidation of asparagine 25. Evidence for methylation at the alpha-carboxyl group of atypical L-isoaspartyl residues

The enzymatic methylation of porcine adrenocorticotropin (ACTH) in both its native form and a form which is deamidated at asparagine 25 has been compared using purified protein carboxyl methyltransferase from bovine brain. Incubation of deamidated ACTH with high concentrations of methyltransferase resulted in near stoichiometric levels of methyl incorporation (78 mol %), while the methylation of native ACTH was highly substoichiometric (3-12 mol %). The Km and Vmax for deamidated ACTH were 1.9 microM and 11,200 pmol/min/mg, respectively, making this peptide the most specific substrate known for the mammalian methyltransferase. Deamidation of asparagine 25 leads to the formation of an atypical isopeptide bond in which the resulting aspartyl residue is linked to the adjacent glycine 26 via its side-chain beta-carboxyl group rather than the usual alpha-carboxyl linkage (Gráf, L., Bajusz, S., Patthy A., Barát, E., and Cseh, G. (1971) Acta Biochim. Biophys. Acad. Sci. Hung. 6, 415-418; Bornstein, P., and Balian, G. (1977) Methods Enzymol. 47, 132-145). A synthetic isopeptide (beta-linked) analog of deamidated ACTH serves as a highly effective substrate for the methyltransferase, but the corresponding normal (alpha-linked) peptide does not, indicating that this enzyme selectively recognizes the alpha-carboxyl group of atypical beta-linked L-aspartyl residues (see also accompanying paper (Murray, E.D., Jr., and Clarke, S. (1984) J. Biol. Chem. 259, 10722-10732]. Methylation of atypical beta-linked L-aspartyl residues resulting from deamidation can account for previous observations that in vitro protein carboxyl methylation in mammalian systems almost always occurs with a low stoichiometry and that these protein methyl esters are considerably less stable than most chemically formed protein methyl esters.     

Spontaneous peptide bond cleavage in aging alpha-crystallin through a succinimide intermediate

Cleavage of specific peptide bonds occurs with aging in the alpha A subunit of bovine alpha-crystallin. One of the breaks occurs at residue Asn-101. This same residue undergoes in vivo deamidation, isomerization, and racemization. Deamidation and isomerization are known to occur via succinimide ring formation of labile asparagine residues. Model studies on peptides have shown that imide formation can also lead to peptide bond cleavage (Geiger, T., and Clarke, S. (1987) J. Biol. Chem. 262, 785-794). In that case, both asparagine and aspartic acid amide would be expected as C termini of the truncated polypeptide, and this is indeed the case in the alpha A-(1-101)-chain. This thus represents a first example of nonenzymatic in vivo peptide bond cleavage in an aging protein through the formation of a succinimide intermediate. In addition, we found that in bovine lens no detectable conversion (through the action of protein-carboxyl methyltransferase) of isoaspartyl to normal aspartyl residues occurs in vivo after deamidation of Asn-101.     

Inhibition of GTPgammaS-dependent L-isoaspartyl protein methylation by tyrosine kinase inhibitors in kidney

Protein carboxyl methylation in rat kidney cytosol is increased by the addition of guanosine 5'-O-[gamma-thio]triphosphate (GTPgammaS), a non-hydrolysable analogue of GTP. GTPgammaS-stimulated methyl ester group incorporation takes place on isoaspartyl residues, as attested by the alkaline sensitivity of the labelling and its competitive inhibition by L-isoaspartyl-containing peptides. GTPgammaS was the most potent nucleotide tested, whereas GDPbetaS and ATPgammaS also stimulated methylation but to a lesser extent. Maximal stimulation (5-fold) of protein L-isoaspartyl methytransferase (PIMT) activity by GTPgammaS was reached at a physiological pH in the presence of 10 mM MgCl2. Other divalent cations, such as Cu2+, Zn2+ and Co2+ (100 microM), totally inhibited GTPgammaS-dependent carboxyl methylation. The phosphotyrosine phosphatase inhibitor vanadate potentiated the GTPgammaS stimulation of PIMT activity in the kidney cytosol at a concentration lower than 40 microM, but increasing the vanadate concentration to more than 40 microM resulted in a dose-dependent inhibition of the GTPgammaS effect. The tyrosine kinase inhibitors genistein (IC50 = 4 microM) and tyrphostin (IC50 = 1 microM) abolished GTPgammaS-dependent PIMT activity by different mechanisms, as was revealed by acidic gel analysis of methylated proteins. Whereas tyrphostin stabilised the methyl ester groups, genistein acted by blocking a crucial step required for the activation of PIMT activity by GTPgammaS. The results obtained with vanadate and genistein suggest that tyrosine phosphorylation regulates GTPgammaS-stimulated PIMT activity in the kidney cytosol.     

The carboxyl methylation of aspartyl residues in eledoisin and tetragastrin by rabbit erythrocyte aspartyl β-carboxyl methyltransferase

Rabbit erythrocytes contain a soluble aspartyl β-carboxyl methyltransferase capable of specifically carboxyl methylating the β-carboxyl group of an internal aspartyl residue in the synthetic polypeptide eledoisin, a hypotensively active peptide from the cephalopodsEledone moschata andE. aldrovandi, and tetragastrin, the biologically active C-terminal tetrapeptide of human gastrin. However, the aspartyl residue in delta sleep-inducing peptide (DSIP) could not be carboxyl methylated, nor could glutamyl residues in any polypeptide tested.