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Isolation and characterization of calmodulin from an insulin-secreting tumour.

ABSTRACT: A major protein constituent of a rat islet cell tumour that exhibited Ca2+-dependent changes in electrophoretic mobility has been purified to homogeneity and compared in its physicochemical and biological properties with bovine brain and rat brain calmodulin (synonymous with phosphodiesterase activator protein, calcium-dependent regulator, troponin C-like protein and modulator protein). The protein, like these calmodulins, contained trimethyl-lysine, exhibited a blocked N-terminus and had an identical amino-acid composition and molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Peptide "maps' prepared after digestion of the three proteins with trypsin, papain or Staphylococcus V-8 proteinase were virtually superimposable. Ca2+ altered the electrophoretic mobilities the enhanced the native protein fluorescence in an equivalent manner with all three proteins. Equilibrium dialysis experiments demonstrated in each case the binding of 4g-atoms of calcium/mol of protein; the binding sites were equivalent and showed Kd 0.8 microM. Tumour and brain proteins were equipotent as Ca2+-dependent activators of partially purified rat brain cyclic nucleotide phosphodiesterase, and in this action were inhibited in an identical manner by trifluoperazine. The proteins also exhibited the common property of Ca2+-dependent binding to troponin I, histone H2B and myelin basic protein. The estimated tumour content of calmodulin was 450 mg/kg fresh wt., a value similar to that reported in islets of Langerhans. These results further document the validity of the islet cell tumour as an experimental model of Ca2+-mediated molecular events associated with insulin secretion. They also suggest that brain calmodulin may be substituted for endogenous calmodulin in experimental investigations into the mechanism of insulin secretion.

SUBMITTER: Hutton JC

PROVIDER: S-EPMC1162680 | biostudies-other | 1981 Mar

REPOSITORIES: biostudies-other

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Project description:Prenylcysteine carboxymethyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, was characterized in insulin-secreting INS-1 cells and normal rat pancreatic islets. The activity of this enzyme was monitored by the methylation of an artificial substrate (a prenylated cysteine analogue) with S-adenosy1[methyl-3H]methionine as methyl donor. More than 95% of the methyltransferase activity was associated with the membranes, and high-salt treatment only partially extracted the enzyme from the membranes. The highest specific activity was in the insulin-granule-enriched 25000 g pellet obtained by differential centrifugation. However, a highly purified insulin-enriched fraction obtained by density centrifugation in Percoll did not exhibit methyltransferase activity. The analyses of marker enzymes for cellular organelles revealed that the methyltransferase was co-localized, with the plasma membrane and probably the endoplasmic reticulum, but not with the mitochondria or lysosomes. Guanosine 5'-[gamma-thio]-triphosphate failed to increase methyltransferase activity directly, although it promotes the methylation of GTP-binding proteins. Mastoparan, Ca2+, cAMP and the protein kinase C activator phorbol 12-myristate 13-acetate did not alter enzyme activity. In addition, methyltransferase activity was not stably modified by stimulation of intact cells using glucose or other agents. However, the carboxymethylation of certain low-molecular-mass G-proteins is increased by glucose stimulation; conversely, treatment of cells with N-acetyl-S-trans,trans-farnesyl-L-cysteine inhibited glucose- and forskolin-induced insulin secretion. These results suggest that the membrane-associated prenylcysteine carboxymethyltransferase may be constitutively active and that the methylation of target proteins in vivo is regulated by the access of these proteins to the methyltransferase, as well as by their active (GTP-liganded) configuration.

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Isolation and characterization of calmodulin from an insulin-secreting tumour.

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