Preparation, characterization and biological properties of biotinylated derivatives of calmodulin

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 derivatives 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.

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High-performance hydrophobic interaction chromatography of estrogen receptors and magnesium dependent protein kinase(S): Detection of two molecular forms of estrogen receptors in the presence and absence of sodium molybdate

Journal of Steroid Biochemistry ◽

10.1016/0022-4731(88)90266-x ◽

1988 ◽

Vol 29 (2) ◽

pp. 197-206 ◽

Cited By ~ 9

Author(s):

Salman M. Hyder ◽

Nobuko Sato ◽

William Hogancamp ◽

James L. Wittliff

Keyword(s):

Protein Kinase ◽

Estrogen Receptors ◽

Hydrophobic Interaction ◽

High Performance ◽

Sodium Molybdate ◽

Hydrophobic Interaction Chromatography ◽

Molecular Forms ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Presence And Absence

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Ca2+-dependent hydrophobic-interaction chromatography. Isolation of a novel Ca2+-binding protein and protein kinase C from bovine brain

Biochemical Journal ◽

10.1042/bj2240117 ◽

1984 ◽

Vol 224 (1) ◽

pp. 117-127 ◽

Cited By ~ 156

Author(s):

M P Walsh ◽

K A Valentine ◽

P K Ngai ◽

C A Carruthers ◽

M D Hollenberg

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Hydrophobic Interaction ◽

Binding Proteins ◽

Binding Protein ◽

Hydrophobic Interaction Chromatography ◽

Bovine Brain ◽

Dependent Protein Kinase ◽

Kinase C ◽

Dependent Protein

Several bovine brain proteins have been found to interact with a hydrophobic chromatography resin (phenyl-Sepharose CL-4B) in a Ca2+-dependent manner. These include calmodulin, the Ca2+/phospholipid-dependent protein kinase (protein kinase C) and a novel Ca2+-binding protein that has now been purified to electrophoretic homogeneity. This latter protein is acidic (pI 5.1) and, like calmodulin and some other high-affinity Ca2+-binding proteins, exhibits a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with an apparent Mr of 22 000 in the absence of Ca2+ and Mr 21 000 in the presence of Ca2+. This novel calciprotein is distinct from known Ca2+-binding proteins on the basis of Mr under denaturing conditions, Cleveland peptide mapping and amino acid composition analysis. It may be a member of the calmodulin superfamily of Ca2+-binding proteins. This calciprotein does not activate two calmodulin-dependent enzymes, namely cyclic nucleotide phosphodiesterase and myosin light-chain kinase, nor does it have any effect on protein kinase C. It may be a Ca2+-dependent regulatory protein of an as-yet-undefined enzymic activity. The Ca2+/phospholipid-dependent protein kinase is also readily purified by Ca2+-dependent hydrophobic-interaction chromatography followed by ion-exchange chromatography, during which it is easily separated from calmodulin. A preparation of protein kinase C that lacks contaminating kinase or phosphatase activities is thereby obtained rapidly and simply. Such a preparation is ideal for the study of phosphorylation reactions catalysed in vitro by protein kinase C.

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