Purification of dystrophin from skeletal muscle.
Dystrophin was purified from rabbit skeletal muscle by alkaline dissociation of dystrophin-glycoprotein complex which was first prepared by derivatized lectin chromatography. Dystrophin-glycoprotein complex was isolated from digitonin-solubilized rabbit skeletal muscle membranes by a novel two-step method involving succinylated wheat germ agglutinin (sWGA) chromatography and DEAE-cellulose ion exchange chromatography. Proteins co-purifying with dystrophin were a protein triplet of Mr 59,000 and four glycoproteins of Mr 156,000, 50,000, 43,000, and 35,000, all previously identified as components of the dystrophin-glycoprotein complex. Alkaline treatment of sWGA/DEAE-purified dystrophin-glycoprotein complex resulted in complete dissociation of the dystrophin-glycoprotein complex. In order to separate dystrophin from its associated proteins, alkaline-dissociated dystrophin-glycoprotein complex was sedimented by sucrose gradient centrifugation. The residual glycoproteins which contaminated peak dystrophin-containing gradient fractions were then removed by WGA-Sepharose adsorption. The resulting protein appeared as a single band with an apparent Mr of 400,000 on overloaded Coomassie Blue-stained gels. The absence of WGA-peroxidase staining on nitrocellulose transfers of the pure protein indicated that the pure protein was devoid of contaminating glycoproteins. Antisera raised against the carboxyl terminus of human skeletal muscle dystrophin (which does not cross-react with the carboxyl terminus of the chromosome 6-encoded dystrophin-related protein) recognized the pure protein as did antisera specific for the amino terminus of human dystrophin. These data indicate that the protein isolated is indeed the intact, predominant skeletal muscle isoform product of the Duchenne muscular dystrophy gene.
Ervasti
J M
JM
Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City 52242.
Kahl
S D
SD
Campbell
K P
KP
D000818:Animals; D002498:Centrifugation; D002846:Chromatography, Affinity; D016189:Dystrophin / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000737:chemistry*; D011817:Rabbits; D013386:Succinates; D014909:Wheat Germ Agglutinins / Q000737:chemistry
D016189:Dystrophin; D046911:Macromolecular Substances; D013386:Succinates; D014909:Wheat Germ Agglutinins