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Title:Partial Purification and Characterization of the Calmodulin Sensitive Bovine Brain Adenylate Cyclase
Author(s):Westcott, Keith Rich
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Abstract: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.
The resolution of the CaM-sensitive bovine brain adenylate cyclase allowed the study of various properties of CaM-stimulation of the enzyme. In the presence of calmodulin and 0.2 mM EGTA, the CaM-sensitive adenylate cyclase was stimulated 4-fold upon addition of Ca('2+) between 0 and 0.2 mM and inhibited at higher Ca('2+) concentrations. The activity was stimulated 1.6-fold upon addition of Mn('2+) between 0 and 10 mM in the presence of calmodulin. The apparent concentration of calmodulin required for half-maximal stimulation of the adenylate cyclase was approximately 8 x 10('-4) mg/ml. The apparent affinity of calmodulin for the enzyme was observed to decrease in the presence of NaCl or KCl but was found to be independent of ATP concentration. The K(,m) for the substrate, ATP, was found to be (TURN)77 (mu)M with 5 mM Mg('2+) and (TURN)31 (mu)M with 10 mM Mn('2+) but was independent of the presence of calmodulin. Limited proteolysis of the CaM-sensitive adenylate cyclase by trypsin abolished CaM-sensitivity without affecting the basal activity.
The CaM-insensitive adenylate cyclase derived by CaM-Sepharose chromatography was found to be insensitive to NaF and 5'-guanylyl imidodiphosphate (Gpp(NH)p) as well. Sensitivity to all three effectors was restored by incubation with a detergent-solubilized extract of bovine brain. Restoration of CaM-sensitivity required Gpp(NH)p. The factor necessary for reconstitution was sensitive to heat, trypsin and N-ethylmaleimide. These observations suggest that this adenylate cyclase form requires the presence of one or more guanyl nucleotide binding proteins for the restoration of calmodulin responsiveness.
The periodate oxidized analog of ATP, 2',3' dial ATP, competitively inhibited bovine brain adenylate cyclase. The apparent K(,I) for 2',3' dial ATP was 196 (mu)M with 5 mM Mg('2+) and 37 (mu)M with 10 mM Mn('2+). Adenylate cyclase was irreversibly inactivated by 2',3' dial ATP in the presence of NaCNBH(,3). The kinetics for loss of enzyme activity were pseudo-first order. Both ATP and Tris protected the enzyme from irreversible inactivation. It is proposed that 2',3' dial ATP forms a Schiff's base with an amino group at the active site of the enzyme and that reduction of this Schiff's base causes irreversible modification of the catalytic subunit. The K(,m) for 2',3' dial ATP-dependent inactivation, the maximal rate constant of inactivation and protection of the enzyme by ATP were all unaffected by the presence or absence of Mg('2+). These observations indicate that a divalent cation is not required for binding of 2',3' dial ATP to the active site of adenylate cyclase.
Issue Date:1980
Description:170 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.
Other Identifier(s):(UMI)AAI8108702
Date Available in IDEALS:2014-12-14
Date Deposited:1980

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