Carboxylmethylation of calmodulin inhibits calmodulin-dependent phosphorylation in rat brain membranes and cytosol

M. L. Billingsley, P. A. Velletri, R. H. Roth, R. J. DeLorenzo

Research output: Contribution to journalArticle

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Abstract

Protein carboxylmethyltransferase, using S-adenosylmethionine (AdoMet) as a methyl donor, forms baselabile carboxylmethylesters on acidic amino acids of various protein substrates. Calmodulin (CaM), an acidic protein, is an excellent substrate for protein carboxylmethyltransferase. Using purified rat brain protein carboxylmethyltransferase, we have investigated the effects of carboxylmethylation of CaM on its subsequent ability to stimulate Ca+2-CaM-dependent phosphorylations in rat brain cytosol and membrane preparations. Incubation of CaM, purified protein carboxylmethyltransferase, and AdoMet resulted in the time- and temperature-dependent formation of carboxylmethylesters, with CaM showing greater methylation than other protein substrates. Acidic gel electrophoresis of CaM after incubation with protein carboxylmethyltransferase and AdoMet revealed a single radioactive band that co-migrated with native CaM. The functional consequences of carboxylmethylation were investigated by preincubating CaM with protein carboxylmethyltransferase, followed by determination of Ca+2-CaM-stimulated phosphorylation in rat brain membranes depleted of CaM. Carboxylmethylated CaM was less able to stimulate Ca+2-CaM-dependent phosphorylation in membrane proteins. This inhibition of Ca+2-CaM-dependent phosphorylation was reversed by including S-adenosylhomocysteine, an inhibitor of protein carboxylmethyltransferase, in the preincubation with protein carboxylmethyltransferase and AdoMet. Similar results were obtained by preincubating rat brain cytosol with purified protein carboxylmethyltransferase and AdoMet. In this preparation, Ca2+-stimulated phosphorylations were inhibited, with maximal inhibition noted at 100 μM AdoMet. These results suggest that carboxylmethylation of CaM may provide an important biochemical mechanism for regulation of Ca2+-CaM-dependent reactions in nervous tissue.

Original languageEnglish (US)
Pages (from-to)5352-5357
Number of pages6
JournalJournal of Biological Chemistry
Volume258
Issue number9
StatePublished - 1983

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Phosphorylation
Calmodulin
Protein O-Methyltransferase
Cytosol
Rats
Brain
Membranes
Substrates
S-Adenosylhomocysteine
Acidic Amino Acids
Nerve Tissue
S-Adenosylmethionine
Proteins
Methylation
Electrophoresis

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Billingsley, M. L., Velletri, P. A., Roth, R. H., & DeLorenzo, R. J. (1983). Carboxylmethylation of calmodulin inhibits calmodulin-dependent phosphorylation in rat brain membranes and cytosol. Journal of Biological Chemistry, 258(9), 5352-5357.
Billingsley, M. L. ; Velletri, P. A. ; Roth, R. H. ; DeLorenzo, R. J. / Carboxylmethylation of calmodulin inhibits calmodulin-dependent phosphorylation in rat brain membranes and cytosol. In: Journal of Biological Chemistry. 1983 ; Vol. 258, No. 9. pp. 5352-5357.
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abstract = "Protein carboxylmethyltransferase, using S-adenosylmethionine (AdoMet) as a methyl donor, forms baselabile carboxylmethylesters on acidic amino acids of various protein substrates. Calmodulin (CaM), an acidic protein, is an excellent substrate for protein carboxylmethyltransferase. Using purified rat brain protein carboxylmethyltransferase, we have investigated the effects of carboxylmethylation of CaM on its subsequent ability to stimulate Ca+2-CaM-dependent phosphorylations in rat brain cytosol and membrane preparations. Incubation of CaM, purified protein carboxylmethyltransferase, and AdoMet resulted in the time- and temperature-dependent formation of carboxylmethylesters, with CaM showing greater methylation than other protein substrates. Acidic gel electrophoresis of CaM after incubation with protein carboxylmethyltransferase and AdoMet revealed a single radioactive band that co-migrated with native CaM. The functional consequences of carboxylmethylation were investigated by preincubating CaM with protein carboxylmethyltransferase, followed by determination of Ca+2-CaM-stimulated phosphorylation in rat brain membranes depleted of CaM. Carboxylmethylated CaM was less able to stimulate Ca+2-CaM-dependent phosphorylation in membrane proteins. This inhibition of Ca+2-CaM-dependent phosphorylation was reversed by including S-adenosylhomocysteine, an inhibitor of protein carboxylmethyltransferase, in the preincubation with protein carboxylmethyltransferase and AdoMet. Similar results were obtained by preincubating rat brain cytosol with purified protein carboxylmethyltransferase and AdoMet. In this preparation, Ca2+-stimulated phosphorylations were inhibited, with maximal inhibition noted at 100 μM AdoMet. These results suggest that carboxylmethylation of CaM may provide an important biochemical mechanism for regulation of Ca2+-CaM-dependent reactions in nervous tissue.",
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Billingsley, ML, Velletri, PA, Roth, RH & DeLorenzo, RJ 1983, 'Carboxylmethylation of calmodulin inhibits calmodulin-dependent phosphorylation in rat brain membranes and cytosol', Journal of Biological Chemistry, vol. 258, no. 9, pp. 5352-5357.

Carboxylmethylation of calmodulin inhibits calmodulin-dependent phosphorylation in rat brain membranes and cytosol. / Billingsley, M. L.; Velletri, P. A.; Roth, R. H.; DeLorenzo, R. J.

In: Journal of Biological Chemistry, Vol. 258, No. 9, 1983, p. 5352-5357.

Research output: Contribution to journalArticle

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T1 - Carboxylmethylation of calmodulin inhibits calmodulin-dependent phosphorylation in rat brain membranes and cytosol

AU - Billingsley, M. L.

AU - Velletri, P. A.

AU - Roth, R. H.

AU - DeLorenzo, R. J.

PY - 1983

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N2 - Protein carboxylmethyltransferase, using S-adenosylmethionine (AdoMet) as a methyl donor, forms baselabile carboxylmethylesters on acidic amino acids of various protein substrates. Calmodulin (CaM), an acidic protein, is an excellent substrate for protein carboxylmethyltransferase. Using purified rat brain protein carboxylmethyltransferase, we have investigated the effects of carboxylmethylation of CaM on its subsequent ability to stimulate Ca+2-CaM-dependent phosphorylations in rat brain cytosol and membrane preparations. Incubation of CaM, purified protein carboxylmethyltransferase, and AdoMet resulted in the time- and temperature-dependent formation of carboxylmethylesters, with CaM showing greater methylation than other protein substrates. Acidic gel electrophoresis of CaM after incubation with protein carboxylmethyltransferase and AdoMet revealed a single radioactive band that co-migrated with native CaM. The functional consequences of carboxylmethylation were investigated by preincubating CaM with protein carboxylmethyltransferase, followed by determination of Ca+2-CaM-stimulated phosphorylation in rat brain membranes depleted of CaM. Carboxylmethylated CaM was less able to stimulate Ca+2-CaM-dependent phosphorylation in membrane proteins. This inhibition of Ca+2-CaM-dependent phosphorylation was reversed by including S-adenosylhomocysteine, an inhibitor of protein carboxylmethyltransferase, in the preincubation with protein carboxylmethyltransferase and AdoMet. Similar results were obtained by preincubating rat brain cytosol with purified protein carboxylmethyltransferase and AdoMet. In this preparation, Ca2+-stimulated phosphorylations were inhibited, with maximal inhibition noted at 100 μM AdoMet. These results suggest that carboxylmethylation of CaM may provide an important biochemical mechanism for regulation of Ca2+-CaM-dependent reactions in nervous tissue.

AB - Protein carboxylmethyltransferase, using S-adenosylmethionine (AdoMet) as a methyl donor, forms baselabile carboxylmethylesters on acidic amino acids of various protein substrates. Calmodulin (CaM), an acidic protein, is an excellent substrate for protein carboxylmethyltransferase. Using purified rat brain protein carboxylmethyltransferase, we have investigated the effects of carboxylmethylation of CaM on its subsequent ability to stimulate Ca+2-CaM-dependent phosphorylations in rat brain cytosol and membrane preparations. Incubation of CaM, purified protein carboxylmethyltransferase, and AdoMet resulted in the time- and temperature-dependent formation of carboxylmethylesters, with CaM showing greater methylation than other protein substrates. Acidic gel electrophoresis of CaM after incubation with protein carboxylmethyltransferase and AdoMet revealed a single radioactive band that co-migrated with native CaM. The functional consequences of carboxylmethylation were investigated by preincubating CaM with protein carboxylmethyltransferase, followed by determination of Ca+2-CaM-stimulated phosphorylation in rat brain membranes depleted of CaM. Carboxylmethylated CaM was less able to stimulate Ca+2-CaM-dependent phosphorylation in membrane proteins. This inhibition of Ca+2-CaM-dependent phosphorylation was reversed by including S-adenosylhomocysteine, an inhibitor of protein carboxylmethyltransferase, in the preincubation with protein carboxylmethyltransferase and AdoMet. Similar results were obtained by preincubating rat brain cytosol with purified protein carboxylmethyltransferase and AdoMet. In this preparation, Ca2+-stimulated phosphorylations were inhibited, with maximal inhibition noted at 100 μM AdoMet. These results suggest that carboxylmethylation of CaM may provide an important biochemical mechanism for regulation of Ca2+-CaM-dependent reactions in nervous tissue.

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