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PubMed · 3567141

Dityrosine formation in calmodulin.

D A Malencik·S R Anderson
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Abstract

Ultraviolet (280-nm) irradiation of bovine brain calmodulin results in calcium-dependent changes in its fluorescence emission spectrum. These consist of a decline in the intrinsic tyrosine fluorescence of the protein and the appearance of a new emission maximum at 400 nm. Chromatography of irradiated calmodulin, using Ultrogel AcA 54 and phenyl-agarose columns, yields several distinctive fractions. One of these, representing 2.8% of the total recovered protein and 53% of the total fluorescence emission at 400 nm, was selected for detailed characterization. Analyses performed on acid hydrolysates reveal the presence of dityrosine, a derivative of tyrosine known for its fluorescence near 400 nm, at the level of 0.59-0.89 mol per 16,700 g of protein. Sodium dodecyl sulfate gel electrophoresis experiments demonstrate two components of apparent molecular weights 14,000 (80%) and 16,000 (20%). Observations on the effects of UV irradiation on the thrombic fragments of calmodulin and on related calcium binding proteins (rabbit skeletal muscle troponin C, bovine cardiac troponin C, and parvalbumin) support the interpretation that dityrosine formation in calmodulin results from the intramolecular cross-linking of Tyr-99 and Tyr-138. The dityrosine-containing photoproduct of calmodulin is unable to stimulate the p-nitrophenyl phosphatase activity of calcineurin under standard assay conditions. Fluorescence titrations show a generally weakened interaction with calcium ion occurring in two stages. The pKa of the derivative is considerably higher than that of free dityrosine and is calcium dependent, decreasing from 7.88 to 7.59 on the addition of 3 mM CaCl2. Smooth muscle myosin light chain kinase binds the derivative about 280-fold less effectively than it binds native calmodulin. Of several metal ions tested, only Cd2+ approaches Ca2+ in its ability to promote the appearance of the 400-nm emission band during UV irradiation of calmodulin. Mn2+ and Cu2+ appear to inhibit dityrosine formation. Ascorbic acid, dithiothreitol, and glutathione are also inhibitory.

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Amino AcidsAnimalsBrainCalmodulinCattleHydrogen-Ion ConcentrationProtein ConformationProteinsSpectrometry, FluorescenceTyrosineUltraviolet RaysComparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.

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D A Malencik, S R Anderson. 1987-02-10. Dityrosine formation in calmodulin.. https://doi.org/10.1021/bi00377a006

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