Parallel modulation of brush border myosin conformation and enzyme activity induced by monoclonal antibodies.
Monoclonal antibodies binding to distinct epitopes on the tail of brush border myosin were used to modulate the conformation and state of assembly of this myosin. BM1 binds 1:3 of the distance from the tip of the tail to the head and prevents the extended-tail (6S) monomer from folding into the assembly-incompetent folded-tail (10S) state, whereas BM4 binds to the tip of the myosin tail, and induces the myosin to fold into the 10S state. Thus, at physiological ionic strength BM1 promotes and BM4 blocks the assembly of the myosin into filaments. Using BM1 and BM4 together, we were able to prevent both folding and filament assembly, thus locking myosin molecules in the extended-tail 6S monomer conformation at low ionic strength where they normally assemble into filaments. Using these myosin-antibody complexes, we were able to investigate independently the effects of folding of the myosin tail and assembly into filaments on the myosin MgATPase. The enzymatic activities were measured from the fluorescent profiles during the turnover of the ATP analogue formycin triphosphate (FTP). Extended-tail (6S) myosin molecules had an FTPase activity of 1-5 X 10(-3) s-1, either at high ionic strength as a monomer alone or when complexed with antibody, or at low ionic strength as filaments or when maintained as extended-tail monomers by the binding of BM1 and BM4. Folding of the molecules into the 10S state reduced this rate by an order of magnitude, effectively trapping the products of FTP hydrolysis in the active sites.
Citi
S
S
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
Cross
R A
RA
Bagshaw
C R
CR
Kendrick-Jones
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D002645:Chickens; D004848:Epithelium / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure; D000939:Epitopes / Q000276:immunology; D005573:Formycins / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000032:analysis / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008871:Microvilli / Q000201:enzymology / Q000378:metabolism*; D009218:Myosins / Q000032:analysis / Q000378:metabolism*; D011487:Protein Conformation; D012265:Ribonucleotides / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005573:Formycins; D012265:Ribonucleotides; C032552:formycin triphosphate; D009218:Myosins