Studies on the Mechanism of Action of Calmodulin

Laporte, David Coleman

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Description

Title

Studies on the Mechanism of Action of Calmodulin

Author(s)

Laporte, David Coleman

Issue Date

1980

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

Chemistry, Biochemistry

Language

eng

Abstract

• The major goal of this research was to cast some light on the fundamental principles underlying the mechanism of action of calmodulin (CaM). This was approached through the study of the interactions of CaM with Ca('2+)+CaM sensitive cyclic nucleotide phosphodiesterase (PDE) and with troponin I. Because CaM has been found to behave in a similar manner in most, if not all, of the systems in which it is a participant, information gathered with these systems may be of general relevance. The principal conclusions drawn from this research are summarized below.
• CaM was radioiodinated with retention of its ability to stimulate PDE and to form Ca('2+)-dependent complexes with CaM-binding proteins. A technique was described for the detection of CaM-binding components by exploiting their ability to form complexes with {('125)I}CaM. This derivative has also proven to be most useful for determination of the quaternary structure of PDE.
• A procedure was developed which resulted in a 14,000 fold purification of PDE, with a 10% yield. When this preparation was submitted to SDS polyacrylamide gel electrophoresis, a single protein band was obtained. The apparent molecular weight of this polypeptide was found to be 57,000 daltons. The specific activity of the enzyme, determined at nearly saturating cAMP, was 275 (mu)mol cAMP hydrolyzed/mg/min. This is equivalent to a turnover number of ca. 16,000 min('-1). Crosslinking of {('125)I}CaM to the purified PDE indicated that the enzyme consisted of a dimer of 57,000 dalton catalytic subunits, each of which bound one CaM.
• Investigation of the Ca('2+)-sensitive binding of small, amphiphilic ligands to CaM provided evidence that Ca('2+) induced exposure of a hydrophobic surface on the protein. Interaction of these ligands with this surface domain was found to antagonize CaM(.)protein interactions. Furthermore, one of these ligands was selected as an example and it was demonstrated that formation of the CaM(.)troponin I complex inhibited its Ca('2+)-sensitive binding to CaM. It was proposed that this surface may represent the interface site for CaM(.)protein interactions and that exposure of a hydrophobic surface may be a key step in formation of the complex.
• The free energy coupling between the binding of Ca('2+) and troponin I to CaM was determined by measuring the Ca('2+) binding characteristics of CaM in the presence and absence of troponin I. Under both circumstances, CaM exhibited four noninteracting sites with equal affinities for Ca('2+). In the absence of troponin I, the dissociation constant (K(,d)) associated with this equilibrium was 12 (mu)M. The k(,d) was decreased to 2.4 (mu)M when the measurements were made in the presence of troponin I. From these values obtained for the troponin I independent and dependent dissociation constants for Ca('2+), the free energy coupling was calculated to be -950 cal/mol Ca('2+).
• A fluorescent derivative of CaM, AEDANS(.)CaM, has been prepared which retained its ability to form Ca('2+)-sensitive complexes with troponin I and PDE. This preparation also stimulated the PDE, although the activation constant was increased from 120 to 200 pM. Investigation of the rotational diffusion of this derivative has provided support for a model which represents the shape of CaM as approximating a prolate elipsoid.
• Troponin I formed a Ca('2+)-sensitive complex with AEDANS(.)CaM and this equlibrium was monitored by measurement of fluorescence anisotropy. This equilibrium was characterized by a K(,d) of 6 x 10('-8) M. Within the limits of experimental error, AEDANS(.)CaM appeared to represent a reasonably homogeneous class of sites.

Graduation Semester

1980

Type of Resource

text

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