Purification and some molecular properties of protein methylase II from equine erythrocytes

Polastro, Enrico; Deconinck, Marc Melchior; M, Devogel; Mailier, E.L.; Looza, Y.B.; Schnek, Arthur G.; Léonis, J

doi:10.1016/0006-291x(78)91439-0

Cited by 26 publications

(6 citation statements)

References 28 publications

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“…The Torpedo electric organ PCM is strongly inhibited by sulfhydryl reagents (Table II); AdoHcy can protect against this inactivation, suggesting that the enzyme has at least one cysteine residue that is important for its catalytic activity. In this respect the enzyme resembles PCMs from human Polastro et al, 1978) and horse erythrocytes and from rat brain , all of which contain a cysteine residue. Polastro et al (1978) have shown that the equine erythrocyte PCM is inhibited by sulfhydryl reagents, although they found that iodoacetamide and /V-ethylmaleimide do not inhibit the enzyme unless previously denatured.…”

Section: Discussionmentioning

confidence: 95%

“…In this respect the enzyme resembles PCMs from human Polastro et al, 1978) and horse erythrocytes and from rat brain , all of which contain a cysteine residue. Polastro et al (1978) have shown that the equine erythrocyte PCM is inhibited by sulfhydryl reagents, although they found that iodoacetamide and /V-ethylmaleimide do not inhibit the enzyme unless previously denatured. In line with this report, we found that partially purified human erythrocyte PCM in a nondenatured state is hardly affected by iodoacetic acid or by Nethylmaleimide.…”

Section: Discussionmentioning

confidence: 95%

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Purification and characterization of protein carboxyl methyltransferase from Torpedo ocellata electric organ

Haklai¹,

Kloog²

1987

Biochemistry

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Posttranslational modification of proteins by the enzyme protein carboxyl methyltransferase (PCM) has been associated with a variety of cellular functions. A prerequisite for the understanding of cellular mechanisms associated with PCM is the characterization of purified PCMs from different tissues. We describe here the purification and characterization of PCM from the electric organ of Torpedo ocellata. The enzyme was purified to homogeneity by ion-exchange chromatography and ammonium sulfate precipitation, followed by chromatography on Sephadex G-100 and hydroxylapatite columns. When visualized by silver staining, the 700-fold-purified PCM exhibited a single band on sodium dodecyl sulfate-polyacrylamide gels, corresponding to a polypeptide of Mr 29,000. The molecular weight of the nondenatured enzyme (as determined by rechromatography on Sephadex G-100 column) was also 29,000, suggesting that the enzyme is a monomer. Two isoelectric forms of PCM (pI = 6.1 and pI = 6.4) were detected in the purified enzyme preparation. The enzyme methylates various exogenous and endogenous proteins, including the acetylcholine receptor. Of the four different polypeptides of the acetylcholine receptor, the gamma and beta polypeptides were selectively methylated by the purified PCM. Purified Torpedo PCM is highly sensitive to sulfhydryl reagents. The competitive inhibitor of PCM S-adenosyl-L-homocysteine (AdoHcy) protected the enzyme from inactivation by sulfhydryl reagents, suggesting the existence of a cysteine residue at the active site of the enzyme. The purified PCM has a low affinity toward DEAE-cellulose and toward AdoHcy-agarose. This property, as well as the relatively high molecular weight and the marked sensitivity to sulfhydryl reagents, distinguishes between the electric organ PCM and analogous enzymes of mammalian tissues.

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 95%

Purification and characterization of protein carboxyl methyltransferase from Torpedo ocellata electric organ

Haklai¹,

Kloog²

1987

Biochemistry

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“…The sum of the type I and type I1 activities is always less than the total cytosol activity because of losses that occur in the purification. Kim et al, 1978;Polastro et al, 1978). Table 4 shows a comparison of initial rates obtained using five well-characterized pure proteins as methylaccepting substrates.…”

Section: Substrate Specificity Of Pcm I and Pcm I1mentioning

confidence: 99%

“…In mammals, PCM has a ubiquitous tissue distribution (Diliberto and Axelrod, 1976;Paik and Kim, 1980), with highest levels in the testis and brain. The enzyme has been substantially purified from brain (Iqbal and Steenson, 1976;Kim et al, 1978), thymus (Kim, 1973), and erythrocytes (Kim, 1974;Polastro et al, 1978). The molecular weight of mammalian PCM is believed to be 24,000-26,000 in all tissues, although there is one report that the ox brain enzyme is 34,000 (Iqbal and Steenson, 1976).…”

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confidence: 99%

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

Aswad

Deight

1983

Journal of Neurochemistry

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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 8.0. 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-polyacrylamide 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 Km (1.2 microM), but it exhibited a low stoichiometry of methyl incorporation.

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“…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).…”

Section: Introductionmentioning

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 some molecular properties of protein methylase II from equine erythrocytes

Cited by 26 publications

References 28 publications

Purification and characterization of protein carboxyl methyltransferase from Torpedo ocellata electric organ

Purification and characterization of protein carboxyl methyltransferase from Torpedo ocellata electric organ

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

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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