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

Aswad, Dana W.; Deight, Elizabeth A.

doi:10.1111/j.1471-4159.1983.tb08147.x

Cited by 94 publications

(61 citation statements)

References 32 publications

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“…1). The efficient repair is also a reflection of the high affinity (K m Ϸ 0.1 M) with which isoaspartyl synapsin binds to PIMT (19). Isoaspartate formation has been shown to markedly reduce the function of proteins such as calmodulin (10), the HPr phosphocarrier protein of E. coli (11), and others (49).…”

Section: Discussionmentioning

confidence: 99%

“…Mice-We chose to study the status of synapsin I in PIMT-deficient mice because of its high affinity for PIMT (19), its tendency to form isoaspartate rapidly in vitro at physiological pH and temperature (20), and its important role in modulating synaptic transmission (17). Multiple smallscale synapsin I purifications from each of three PIMT genotypes (ϩ/ϩ, ϩ/Ϫ, and Ϫ/Ϫ) were performed to achieve an accurate representation of isoaspartate levels.…”

Section: Synapsin I Is a Major Isoaspartate-forming Protein In The Brmentioning

confidence: 99%

“…Synapsin I appears to play an important role in controlling the availability or "ready state" of small synaptic vesicles in response to regulatory signals mediated by several protein kinases, including protein kinase A and Ca 2ϩ /calmodulin-dependent protein kinase type II. Before it was established that PIMT has a specificity for isoaspartyl sites in proteins (a time when this enzyme was known simply as an abundant brain enzyme catalyzing formation of protein carboxyl methyl esters), we reported that purified bovine synapsin I serves as a highly efficacious substrate for this methyltransferase in vitro, exhibiting an apparent K m in the low M range (19). Subsequently, we found that purified bovine synapsin I is unusually susceptible to spontaneous formation of isoaspartyl sites during incubation at pH 7.4 and 37°C, conditions designed to model in vitro protein "aging" (20,21).…”

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Synapsin I Is a Major Endogenous Substrate for Protein L-Isoaspartyl Methyltransferase in Mammalian Brain

Reissner

Paranandi

Luc

et al. 2006

Journal of Biological Chemistry

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The accumulation of potentially deleterious L-isoaspartyl linkages in proteins is prevented by the action of protein L-isoaspartyl O-methyltransferase, a widely distributed enzyme that is particularly active in mammalian brain. Methyltransferase-deficient (knock-out) mice exhibit greatly increased levels of isoaspartate and typically succumb to fatal epileptic seizures at 4 -10 weeks of age. The link between isoaspartate accumulation and the neurological abnormalities of these mice is poorly understood. Here, we demonstrate that synapsin I from knock-out mice contains 0.9 ؎ 0.3 mol of isoaspartate/mol of synapsin, whereas the levels in wild-type and heterozygous mice are undetectable. Transgenic mice that selectively express methyltransferase only in neurons show reduced levels of synapsin damage, and the degree of reduction correlates with the phenotype of these mice. Isoaspartate levels in synapsin from the knock-out mice are five to seven times greater than those in the average protein from brain cytosol or from a synaptic vesicle-enriched fraction. The isoaspartyl sites in synapsin from knock-out mice are efficiently repaired in vitro by incubation with purified methyltransferase and S-adenosyl-L-methionine. These findings demonstrate that synapsin I is a major substrate for the isoaspartyl methyltransferase in neurons and suggest that isoaspartate-related alterations in the function of presynaptic proteins may contribute to the neurological abnormalities of mice deficient in this enzyme. Fig. 1, dashed arrows) constitutes a major pathway for spontaneous protein damage at physiological pH and temperature (1-3). The internal ␣-carboxyl characteristic of isoaspartyl sites is selectively methylated by a widely distributed protein L-isoaspartyl O-methyltransferase (PIMT) 2 (4 -6). Considerable evidence suggests that PIMT functions to repair this damage in vivo by the mechanism illustrated in Fig. 1 (solid arrows). PIMT selectively methylates the atypical ␣-carboxyl of the isoAsp site. This is followed immediately by a spontaneous demethylation to form a metastable succinimide. Spontaneous hydrolysis of the succinimide over a time course of 2-4 h generates a normal L-aspartyl site with an efficiency of 15-30%. Purified PIMT catalyzes conversion of isoaspartyl sites to normal Asp-Xaa linkages in synthetic peptides (7-9) and has been shown to restore function to isoaspartate-containing forms of proteins such as calmodulin (10) and the Escherichia coli phosphocarrier protein HPr (11). Pharmacological inhibition of PIMT activity in rat PC12 cells leads to reversible accumulation of isoaspartyl sites in a variety of proteins, including tubulin (12) and histone H2B (13). Formation of isoaspartyl sites (Studies with PIMT-deficient mice further support a repair function for PIMT (14,15). Whereas proteins in extracts from wild-type or heterozygous PIMT mice contain only trace levels of isoaspartate, proteins in extracts from knock-out mice show greatly increased isoaspartate levels in extracts from all tissues examined, w...

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Section: Discussionmentioning

confidence: 99%

Section: Synapsin I Is a Major Isoaspartate-forming Protein In The Brmentioning

confidence: 99%

mentioning

confidence: 99%

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Synapsin I Is a Major Endogenous Substrate for Protein L-Isoaspartyl Methyltransferase in Mammalian Brain

Reissner

Paranandi

Luc

et al. 2006

Journal of Biological Chemistry

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“…Bovine brain protein carboxyl 0-methyltransferase was purified to apparent homogeneity by using minor modifications of previously published techniques (26,27). Bovine brain calcineurin was purified to homogeneity (28) (33,300 dpm/pmol; using 15 Ci/mmol of Ado[3H]Met).…”

Section: Materials S-adenosyl-l-[methyl-3h]methionine (Ado[3h]-mentioning

confidence: 99%

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

Billingsley¹,

Kincaid²,

Lovenberg³

1985

Proc. Natl. Acad. Sci. U.S.A.

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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 tricholoracetic 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, ifany, modification of the B subunit (Mr = 18,000). When carboxyl-methylated calcineurin (r1-2 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.Carboxyl methylation of glutamic and/or aspartic acid residues of proteins by the enzyme protein carboxyl 0-methyltransferase (protein O-methyltransferase; EC 2.1.1.24) is well documented (1-3), although the functional significance of this reversible modification of proteins remains an enigma (4). It has been suggested that carboxyl methylation can influence neurotransmitter synthesis and release (5-7), leukocyte chemotaxis (8, 9), and repair/recognition of Daspartate residues in protein (10)(11)(12)(13)(14)(15) METHODS Materials. S-Adenosyl-L-[methyl-3H]methionine (Ado[3H]-Met) (62 Ci/mmol and 15 Ci/mmol; Ci = 37 GBq) was purchased from Amersham; 2-(N-morpholino)ethanesulfonic acid (Mes) was from Calbiochem; AdoMet and Sadenosylhomocysteine (AdoHcy) were from Sigma; AdoHcy-agarose was from Bethesda Research Laboratories; electrophoresis supplies were from Bio-Rad; and frozen bovine brains were from Hazelton Labs (Denver, PA). Allreagents were of the highest available purity. CalmodulinSepharose was prepared according to the method of Kincaid and Vaughan (25).Enzyme Preparations. Bovine brain protein carboxyl 0-methyltransferase was purified to apparent homogeneity by using minor modifications of previously published techniques (26,27). Bovine brain calcineurin was purified to homogeneity (28) and contained subunits of Mr 61,000 and Mr 18,000 in approximately equal proportions, which accounted for -95% of Coomassie-stained protein after NaDodSO4 gel electrophoresis.Carboxyl Methylation Assays. Reactions were carried out in a fi...

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“…In vitro, these enzymes methylate a large variety of both heterologous and endogenous proteins in a substoichiometric fashion (1,(4)(5)(6). The "nonspecific" methyltransferase is widely distributed in mammalian tissues (1) and has been purified from both mammalian brain (1,(5)(6)(7) and erythrocyte (8,9) tissues. In brain, there are at least two functionally similar isozymes that can be separated by DEAE-cellulose chromatography (5).…”

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confidence: 99%

Mammalian brain and erythrocyte carboxyl methyltransferases are similar enzymes that recognize both D-aspartyl and L-isoaspartyl residues in structurally altered protein substrates.

O’Connor¹,

Aswad²,

Clarke³

1984

Proc. Natl. Acad. Sci. U.S.A.

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Two purified isozymes of protein carboxyl methyltransferase from bovine brain catalyze the substoichiometric transfer of methyl groups in vitro from S-adenosyl-L-[methyl-3H]methionine to several erythrocyte membrane proteins, which include bands 2.1, 3, and 4.1, as well as several integral membrane polypeptides. D-Aspartic acid f3-[3H]methyl ester has been isolated from proteolytic digests of these methylated proteins, suggesting that protein D-aspartyl residues can serve as methyl-acceptor sites for the two brain enzymes. This formation of D-aspartic acid 3-[3H]methyl ester is competitively inhibited by the peptide L-Val-L-Tyr-L-Pro-LisoAsp-Gly-L-Ala, which contains an L-aspartyl residue in an unusual /-peptide linkage. Since this peptide is a stoichiometric substrate for the brain methyltransferases, it appears that one enzymatic activity can catalyze methyl ester formation at both D-aspartyl and L-isoaspartyl sites. In these respects, the activity of both brain isozymes closely resembles those previously described for the erythrocyte enzyme. The results are discussed in terms of a model in which derivatized aspartyl residues in proteins, arising by either racemization or isomerization, are recognized by the methyltransferase; the enzyme may function in either the metabolism or correction of the altered structures. The presence of a similar enzyme in both translationally active (brain) and inactive (erythrocyte) tissues suggests that the reactions are of general importance to cellular integrity.Protein carboxyl methyltransferases are widely distributed in both bacteria and eukaryotic tissues (1). In bacteria, very specific carboxyl methyltransferases regulate the bacterial chemotactic response by the methylation of several glutamyl residues in membrane chemoreceptor proteins (2, 3). The eukaryotic enzymes, on the other hand, have very different characteristics. In vitro, these enzymes methylate a large variety of both heterologous and endogenous proteins in a substoichiometric fashion (1, 4-6). The "nonspecific" methyltransferase is widely distributed in mammalian tissues (1) and has been purified from both mammalian brain (1, 5-7) and erythrocyte (8,9) tissues. In brain, there are at least two functionally similar isozymes that can be separated by DEAE-cellulose chromatography (5).The precise function of eukaryotic protein carboxyl methylation reactions has not been determined. It has been proposed that the brain enzyme regulates several cellular processes, including, among others, the storage and release of neuroendocrine substances (10, 11), the modulation of receptor function (12, 13), and the regulation of calmodulin activity (14, 15). However, the data supporting such roles have not been compelling (4,16,17). In particular, the broad substrate specificity and substoichiometric nature of the reactions catalyzed by the enzyme are unusual features for regulatory post-translational modifications. Alternative roles for eukaryotic methylation reactions in the repair or metabolism of damaged proteins ha...

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

Cited by 94 publications

References 32 publications

Synapsin I Is a Major Endogenous Substrate for Protein L-Isoaspartyl Methyltransferase in Mammalian Brain

Synapsin I Is a Major Endogenous Substrate for Protein L-Isoaspartyl Methyltransferase in Mammalian Brain

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

Mammalian brain and erythrocyte carboxyl methyltransferases are similar enzymes that recognize both D-aspartyl and L-isoaspartyl residues in structurally altered protein substrates.

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