Stimulation of protein methylase II from Torpedo marmorata by cholinergic effectors

Nuske, Joachim H.

doi:10.1016/0005-2736(86)90072-6

Cited by 5 publications

(2 citation statements)

References 33 publications

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“…In chemotactic bacteria, PCM-dependent methylations of 7-glutamyl residues on membrane chemoreceptors participate in the regulation of the chemotactic response (Springer et al, 1979;Koshland, 1979;Kleene et al, 1977). Putative functions of PCM in eucaryotic cells are associated with the secretion of hormones and neurotransmitters (Heisler et al, 1983;Borchardt et al, 1978;Diliberto et al, 1976;Gagnon et al, 1984), sperm motility (Gagnon et al, 1982), leucocyte chemotaxis (O'Dea et al, 1978), the processing of exportable proteins (Diliberto, 1982), modulation of calmodulin-dependent enzymes , repair of damaged proteins (Clarke, 1985), cell differentiation (Duerre & Fetters, 1985; Zukerman et al, 1982;O'Dea et al, 1983), and modulation of photoreceptors (Swanson & Applebury, 1983) and of the acetylcholine receptor from Torpedo electric organ (Kloog et al, 1980;Flynn et al, 1982;Yee & McNamee, 1985;Nuske, 1986).…”

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

Haklai¹,

Kloog²

1987

Biochemistry

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“…The ubiquitous enzyme protein-carboxyl methylase (PCM; S-adenosylmethionine: protein-L-glutamate 0methyltransferase, EC 2.1.1.24) esterifies free carboxy groups of proteins, resulting in the neutralization of their negative charges (Liss et al, 1969;Kim & Paik, 1970, 1971; Morin & Liss, 1973). Several laboratories have provided evidence implicating eukaryotic PCM as a regulatory agent in a wide variety of cellular processes, including storage and release of secretory proteins (Diliberto et al, 1976;Borchardt et al, 1978;Gagnon et al, 1978;Heisler et al, 1983;Kloog & Saavedra, 1983), modulation of acetylcholine receptors (Kloog et al, 1980;Flynn et al, 1982;Nuske, 1986) and dopamine receptors (Billingsley & Roth, 1982), regulation of calmodulin activity (Gagnon et al, 1981;Billingsley et al, 1983), sperm motility (Bouchard et al, 1980), chemotaxis of leucocytes (O'Dea et al, 1978b;Pike et al, 1978) and slime mouds (Mato & Marin-Cao, 1979), platelet function (O'Dea et al, 1978a), development of membrane excitability in neurons and sensory transduction in the vertebrate rod outer segment (Swanson & Applebury, 1983). However, by contrast with PCM involvement in bacterial chemotaxis, strong and direct evidence for a specific regulatory function of protein carboxy-group methylation in eukaryotic cells is still lacking (Clarke & O'Connor, 1983;Clarke, 1985).…”

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confidence: 99%

Mammalian protein methylesterase. Physical and enzymic properties

Veeraragavan

Gagnon

1989

Biochemical Journal

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Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.

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Interaction of protein methylase II and annexin proteins in invertebrate phototransduction

1989

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Stimulation of protein methylase II from Torpedo marmorata by cholinergic effectors

Cited by 5 publications

References 33 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

Mammalian protein methylesterase. Physical and enzymic properties

Interaction of protein methylase II and annexin proteins in invertebrate phototransduction

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