Reaction of thiol groups of gizzard myosin heavy chains with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.
Chicken gizzard myosin treated with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in a 65% inhibition of the K(+)-ATPase (myosin ATP phosphohydrolase (actin translocating), EC 3.6.1.32) activity and 3.5 mol of the reagent was bound per 4.7 x 10(5) g protein. The labeling was limited to the heavy chain region and none of the light chains were lost. MgATP had no effect on the inactivation or labeling pattern. Thiolysis of NBD-myosin with dithiothreitol restored the K(+)-ATPase activity and concurrently, 1 mol of the NBD group was removed from the heavy chain region. Cysteine residues were modified in NBD-myosin at sites other than the active site when the enzyme activity was inhibited. There was a difference in the extent of NBD-Cl modification of gizzard myosin at 0.6 M KCl (6 S elongated state) when compared to that at 0.15 M KCl (10 S folded state). This was also seen in the heavy meromyosin-like chymotryptic fragments and tryptic fragments of NBD-myosin. The reagent NBD-Cl can detect changes in the conformation of gizzard myosin by way of its reaction with thiol groups of the heavy chain region.
Bailin
G
G
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Piscataway 08854-5635.
Shah
T
T
Huang
J R
JR
D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003545:Cysteine / Q000378:metabolism*; D015879:Myosin Subfragments / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009218:Myosins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010069:Oxadiazoles / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D015879:Myosin Subfragments; D010069:Oxadiazoles; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D009218:Myosins; D009327:4-Chloro-7-nitrobenzofurazan; D003545:Cysteine