Mapping of the microvillar 110K-calmodulin complex (brush border myosin I). Identification of fragments containing the catalytic and E-actin-binding sites and demonstration of a calcium ion dependent conformational change

Journal Article · Sat Dec 01 00:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00502a011· OSTI ID:5736468

Coluccio, L M ^[1]; Bretscher, A ^[2]

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1. Emory Univ. School of Medicine, Atlanta, GA (USA)
2. Cornell Univ., Ithaca, NY (USA)

In intestinal microvilli, the 110K-calmodulin complex is the major component of the cross-bridges which connect the core bundle of actin filaments to the membrane. The authors previous work showed that the 110-kDa polypeptide can be divided into three functional domains: a 78-dDa fragment that contains the ATPase activity and the ATP-reversible F-actin-binding site, a 12-kDa fragment required for binding calmodulin molecules, and a terminal 20-kDa domain of unkown function. By analysis of limited {alpha}-chymotryptic cleavage products, they now show that the molecular organization is very similar to that described for the S{sub 1} fragment of myosin. The catalytic site was identified by photoaffinity labeling with (5,6-{sup 3}H)UTP, and fragments binding F-actin were identified by cosedimentation assays. Cleavage of the 78-kDa fragment yielded major fragments of 32 and 45 kDa, followed by cleavage of the 45-kDa fragment to a 40-kDa fragment. Of these, only the 32-kDa fragment was labeled by (5,6-{sup 3}H)UTP. They conclude that the catalytic site is located in the 32-kDa fragment and the F-actin-binding site is present in the 45-kDa fragment; the ability to bind actin is lost upon further cleavage of the 45-kDa fragment to 40 kDa. Peptide sequence analysis revealed that the 45-kDa fragment lies within the molecule and suggests that the 32-kDa fragment is the amino terminus. These results suggest that the 12-kDa calmodulin-binding region confers a Ca{sup 2{plus}}-dependent accessibility to the F-actin-protectable cleavage site in the S{sub 1}-like domain. This is the first evidence for a Ca{sup 2{plus}}-regulated structural change in the S{sub 1}-like domain of the 110K-calmodulin complex.

• OSTI ID: 5736468
• Journal Information: Biochemistry; (USA), Vol. 29:50; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ACTIN
RADIORECEPTOR ASSAY
CALMODULIN
CONFORMATIONAL CHANGES
ELECTROPHORESIS
INTESTINAL ABSORPTION
MYOSIN
PROTEIN STRUCTURE
SEDIMENTATION
TRITIUM COMPOUNDS
ABSORPTION
GLOBULINS
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
ORGANIC COMPOUNDS
PROTEINS
TRACER TECHNIQUES
UPTAKE
550201* - Biochemistry- Tracer Techniques