Use of a chaotropic anion iodide in the purification of Z-line proteins: isolation of CapZ from fish white muscle.
In the present study, we have described an improved method allowing the isolation of proteins which form tightly associated complexes in organized structures such as Z line in skeletal muscle. This procedure is based on both extraction and chromatography in the presence of a chaotropic agent. KI at medium concentration (0.6 M) was selected, taking into account its dissociating activity and mild effect on the native state of proteins. This procedure was applied to purify and to characterize for the first time a CapZ from fish white muscle, a protein involved in the stabilization of the filaments in Z line. The alpha and beta CapZ subunits were identified using anti-synthetic peptide antibodies directed against conserved sequences derived from chicken CapZ. The protocol can be also used for the isolation of other muscular proteins such as alpha-actinin and actin. Finally this technique may be utilized to obtain a good amount of capping protein which could be employed in experiments of microfilament dynamics.
Papa
I
I
Laboratoire de Recherche sur la Motilité Cellulaire (EPHE), Place E. Bataillon, Montpellier Cedex 05, 34095, France.
Astier
C
C
Kwiatek
O
O
Lebart
M C
MC
Raynaud
F
F
Benyamin
Y
Y
Roustan
C
C
D000096:Acetone; D000199:Actins / Q000378:metabolism; D000818:Animals; D001492:Bass / Q000378:metabolism*; D051347:CapZ Actin Capping Protein; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D066298:In Vitro Techniques; D007454:Iodides; D008840:Microfilament Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009124:Muscle Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry*; D011193:Potassium Iodide; D020836:Protein Structure, Quaternary; D011817:Rabbits
D000199:Actins; D051347:CapZ Actin Capping Protein; D007454:Iodides; D008840:Microfilament Proteins; D009124:Muscle Proteins; D000096:Acetone; D011193:Potassium Iodide