Expression and characterization of a functional myosin head fragment in Dictyostelium discoideum.
The isolated head fragment of myosin is a motor protein that is able to use energy liberated from the hydrolysis of adenosine triphosphate to cause sliding movement of actin filaments. Expression of a myosin fragment nearly equivalent to the amino-terminal globular head domain, generally referred to as subfragment 1, has been achieved by transforming the eukaryotic organism Dictyostelium discoideum with a plasmid that carries a 2.6-kilobase fragment of the cloned Dictyostelium myosin heavy chain gene under the control of the Dictyostelium actin-15 promoter. The recombinant fragment of the myosin heavy chain was purified 2400-fold from one of the resulting cell lines and was found to be functional by the following criteria: the myosin head fragment copurified with the essential and regulatory myosin light chains, decorated actin filaments, and displayed actin-activated adenosine triphosphatase activity. In addition, motility assays in vitro showed that the recombinant myosin fragment is capable of supporting sliding movement of actin filaments.
Manstein
D J
DJ
Department of Cell Biology, Stanford University School of Medicine, CA 94305.
Ruppel
K M
KM
Spudich
J A
JA
D000199:Actins / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000235:genetics*; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D008970:Molecular Weight; D015879:Myosin Subfragments / Q000235:genetics* / Q000302:isolation & purification; D009218:Myosins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic
D000199:Actins; D015879:Myosin Subfragments; D009218:Myosins