Actomyosin kinetics and in vitro motility of wild-type Drosophila actin and the effects of two mutations in the Act88F gene.
Two missense mutations of the flight muscle-specific actin gene of Drosophila melanogaster, Act88F, assemble into normally structured myofibrils but affect the flight ability of flies and the mechanical kinetics of isolated muscle fibers. We describe the isolation of actin from different homozygous Act88F strains, including wild-type, an Act88F null mutant (KM88), and two Act88F single point mutations (E316K and G368E), their biochemical interactions with rabbit myosin subfragment 1 (S1), and behavior with rabbit myosin and heavy meromyosin in in vitro motility assays. The rabbit and wild-type Drosophila actins have different association rate constants with S1 (2.64 and 1.77 microM-1 s-1, respectively) and in vitro motilities (2.51, 1.60 microns s-1) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increasing from 0.11 to 0.17 microM) and a reduced velocity in vitro (reduced by 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with respect to the recently published atomic models for the actomyosin structure and our findings that G368E fibers show a reduced rate constant for delayed tension development and increased fiber stiffness. We interpret these results as possibly caused either by effects on A1 myosin light chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed with respect to its likely position within the tropomyosin binding site of actin.
Anson
M
M
National Institute for Medical Research, Mill Hill, London, United Kingdom.
Drummond
D R
DR
Geeves
M A
MA
Hennessey
E S
ES
Ritchie
M D
MD
Sparrow
J C
JC
D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000205:Actomyosin / Q000737:chemistry* / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D006720:Homozygote; D007700:Kinetics; D008958:Models, Molecular; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; D000251:Adenosine Triphosphatases