Ubiquitination of endogenous calmodulin in rabbit tissue extracts

FEBS Letters

Jennissen, Herbert P.¹  Laub, Markus¹ 

[1] Institut für Physiologische Chemie, Universität-GHS-Essen, Hufelandstr. 55, D-1300 Essen 1, Germany

关键词: Calmodulin;    Ubiquitin;    Calmodulin-ubiquitin conjugate;    Protein-ubiquitination;    ATP-dependent proteolysis;    Trimethyl lysine;   

DOI  :  10.1016/0014-5793(91)81436-C

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Previously we were able to show that purified calmodulins from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca²⁺-dependent manner. It was therefore pertinent to answer the question if a tissue extract contains all the components necessary for the endogenous synthesis of ubiquityl calmodulin (uCaM). Therefore [¹²⁵I]ubiquitin, ATP/Mg²⁺ and Ca²⁺ were added to tissue extracts enriched by a single ion exchange step. In such extracts of red blood cells, skeletel muscle and testis a novel ubiquitin conjugate of 27–29 kDa is formed. This novel band could be identified as ubiquityl-calmodulin by the following methods: (i) identical R ₁-value of novel conjugate and standard uCaM in SDS-PAGE; (ii) Ca²⁺-dependent conjugate formation; (iii) Ca²⁺-dependent adsorption to fluphenazine-Sepharose; (iv) Ca²⁺-dependent mobility change of the novel conjugate during SDS-PAGE; and (v) inhibition of conjugate band formation by phosphorylate kinase. These experiments clearly demonstrate that ubiquityl calmodulin can be endogenously generated in enriched cellular extracts and strongly indicate that this reaction is of importance in vivo.

【 授权许可】

Unknown