The calmodulin and F-actin binding sites of smooth muscle caldesmon lie in the carboxyl-terminal domain whereas the molecular weight heterogeneity lies in the middle of the molecule.
Caldesmons are major Ca2+-calmodulin regulated F-actin binding proteins of smooth and non-muscle cells that have been implicated as components of a thin filament regulatory system. Chicken gizzard caldesmons are monomeric proteins of Mr 140,000 and 135,000. We have employed enzymatic and chemical cleavage methods in order to dissect the protein to locate the Ca2+-calmodulin and F-actin binding domain and the site of molecular weight heterogeneity. Using a novel mapping procedure that employs partial chemical cleavage at cysteine residues, we show that both caldesmon polypeptides contain 2 cysteine residues located approximately 28,000 from the protein's amino terminus and the second approximately 25,000 from the carboxyl terminus. Identification of the composition of partial cleavage products with region-specific antibodies is consistent with this derived map. The apparent molecular weight heterogeneity was found to lie in the approximately 80,000 region between the 2 cysteine residues and therefore is not due to proteolytic processing. Digestion with alpha-chymotrypsin yields a relatively stable basic Mr 40,000 Ca2+-calmodulin and F-actin binding fragment that we have purified and characterized. The chymotryptic 40,000 fragment contains the 25,000 carboxyl-terminal fragment and therefore is derived from the carboxyl-terminal region of caldesmon. The 25,000 fragment obtained after chemical cleavage at cysteine under native conditions has also been purified and shown to bind F-actin and Ca2+-calmodulin. Surprisingly, the purified carboxyl 25,000 fragment, unlike the reduced intact monomer, cross-links F-actin into tightly ordered bundles in which the filaments are in register.
Riseman
V M
VM
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Lynch
W P
WP
Nefsky
B
B
Bretscher
A
A
D000199:Actins / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping
D000199:Actins; D000596:Amino Acids; D000906:Antibodies; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; C034037:F-actin-binding proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments