Preparation, characterization and biological properties of biotinylated derivatives of calmodulin

J. W. Polli, M. L. Billingsley

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Biotinylated derivatives of calmodulin (CaM) were prepared and their biological properties characterized by using enzyme assays, affinity and hydrophobic-interaction chromatography. Several N-hydroxysuccinimidobiotin derivatives [sulphosuccinimidobiotin (sulpho-NHS) and sulphosuccinimido-6-(biotinamido)hexanoate (BNHS-LC)]differing in spacer arm length were used to modify CaM. The shorter-spacer-arm CaM derivative (sulpho-CaM) activated CaM-dependent cyclic nucleotide phosphodiesterase and CaM-dependent protein kinase II; preincubation with avidin blocked its ability to activate these enzymes. The extended-spacer-arm derivative (BNHS-LC-CaM) activated CaM-dependent enzymes both in the presence and in the absence of avidin, suggesting that the longer spacer arm diminished steric effects from avidin preincubation. Other biotinylated CaM derivatives were prepared with biotinylated tyrosine and/or histidine residues (diazobenzoylbiocytin; DBB-CaM) or nucleophilic sites (photobiotin acetate; photo-CaM). These derivatives activated CaM-dependent enzymes in the presence and in the absence of avidin. Oriented affinity columns were constructed with covalently immobilized avidin complexed to each biotinylated CaM derivative. The chromatographic profiles obtained revealed that each column interacted with a specific subset of CaM-binding proteins. Elution profiles of biotinyl CaM derivates on phenyl-Sepharose hydrophobic-interaction chromatography suggested that several derivatives displayed diminished binding to the matrix in the presence of Ca2+. Development and characterization of a series of biotinylated CaM molecules can be used to identify domains of CaM that interact with specific CaM-dependent enzymes.

Original languageEnglish (US)
Pages (from-to)733-743
Number of pages11
JournalBiochemical Journal
Volume275
Issue number3
DOIs
StatePublished - Jan 1 1991

Fingerprint

Calmodulin
Derivatives
Avidin
Enzymes
Chromatography
Hydrophobic and Hydrophilic Interactions
Calmodulin-Binding Proteins
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Cyclic Nucleotides
Phosphoric Diester Hydrolases
Enzyme Assays
Histidine
Tyrosine
Assays

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Polli, J. W. ; Billingsley, M. L. / Preparation, characterization and biological properties of biotinylated derivatives of calmodulin. In: Biochemical Journal. 1991 ; Vol. 275, No. 3. pp. 733-743.
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Preparation, characterization and biological properties of biotinylated derivatives of calmodulin. / Polli, J. W.; Billingsley, M. L.

In: Biochemical Journal, Vol. 275, No. 3, 01.01.1991, p. 733-743.

Research output: Contribution to journalArticle

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