Localization and topography of antigenic domains within the heavy chain of smooth muscle myosin.
We have produced and characterized monoclonal antibodies that label antigenic determinants distributed among three distinct, nonoverlapping peptide domains of the 200-kD heavy chain of avian smooth muscle myosin. Mice were immunized with a partially phosphorylated chymotryptic digest of adult turkey gizzard myosin. Hybridoma antibody specificities were determined by solid-phase indirect radioimmunoassay and immunoreplica techniques. Electron microscopy of rotary-shadowed samples was used to directly visualize the topography of individual [antibody.antigen] complexes. Antibody TGM-1 bound to a 50-kD peptide of subfragment-1 (S-1) previously found to be associated with actin binding and was localized by immunoelectron microscopy to the distal aspect of the myosin head. However, there was no antibody-dependent inhibition of the actin-activated heavy meromyosin ATPase, nor was antibody TGM-1 binding to actin-S-1 complexes inhibited. Antibody TGM-2 detected an epitope of the subfragment-2 (S-2) domain of heavy meromyosin but not the S-2 domain of intact myosin or rod, consistent with recognition of a site exposed by chymotryptic cleavage of the S-2:light meromyosin junction. Localization of TGM-2 to the carboxy-terminus of S-2 was substantiated by immunoelectron microscopy. Antibody TGM-3 recognized an epitope found in the light meromyosin portion of myosin. All three antibodies were specific for avian smooth muscle myosin. Of particular interest is that antibody TGM-1, unlike TGM-3, bound poorly to homogenates of 19-d embryonic smooth muscles. This indicates the expression of different myosin heavy chain epitopes during smooth muscle development.
Schneider
M D
MD
Sellers
J R
JR
Vahey
M
M
Preston
Y A
YA
Adelstein
R S
RS
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001665:Binding Sites; D002417:Cattle; D002645:Chickens; D000939:Epitopes; D008854:Microscopy, Electron; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000276:immunology / Q000648:ultrastructure*; D009218:Myosins / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology; D010750:Phosphoproteins / Q000276:immunology; D014422:Turkeys
D000199:Actins; D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D010750:Phosphoproteins; D000251:Adenosine Triphosphatases; D009218:Myosins