Partial Purification and Characterization of the Calmodulin Sensitive Bovine Brain Adenylate Cyclase

The calmodulin (CaM) sensitive bovine brain adenylate cyclase was resolved, in a solubilized form, from calmodulin and a CaM-insensitive form of the enzyme. Affi-Gel Blue chromatography, incorporating EGTA-containing buffer washes and elution with 8 mM ATP/l M KCl, produced partially purified adenylate cyclase free from calmodulin. Upon application of this preparation to CaM-Sepharose, the adenylate cyclase was fractionated into two forms. The major form of the enzyme, 77% of the recovered activity, did not bind to CaM-Sepharose in the presence of Ca('2+) and was insensitive to calmodulin. The remaining 23% adsorbed to CaM-Sepharose in the presence of Ca('2+), was eluted with an EGTA-containing buffer and was stimulated by Ca('2+) and calmodulin. The CaM-sensitive adenylate cyclase was purified 105-fold by these procedures (specific activity: 288,000 pmol cAMP formed/mg/10 min). The enzyme was further purified on ATP-agarose to 1,028,000 pmol cAMP formed/mg/10 min.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Westcott, Keith Rich

• Chemistry, Biochemistry

Language dc:language

eng

Identifier

(UMI)AAI8108702

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/67408