The use of poly(L-proline)-Sepharose in the isolation of profilin and profilactin complexes.
In the purification of proline hydroxylase by affinity chromatography on poly(L-proline)-Sepharose it was found earlier that two other components, profilin and the complex profilin-actin, also bind with high affinity to this matrix. We have exploited this observation to develop a rapid procedure for the isolation of profilin and profilin-actin complexes in high yields directly from high-speed supernatants of crude tissue-extracts. Through an extensive search for elution conditions, avoiding poly(L-proline) as the desorbant, we have found that active proteins can be recovered from the affinity column with a buffer containing 30% dimethyl sulphoxide. Subsequent chromatography on hydroxylapatite separates free profilin and the two isoforms of profilactin, profilin-actin beta and profilin-actin gamma. The profilin-actin complexes produced this way have high specific activities in the DNAase-inhibition assay, give rise to filaments on addition of Mg2+, and can be crystallized. From the isolated profilin-actin complexes the beta- and gamma-actin isoforms of non-muscle cells can easily be prepared in a polymerization competent form. Pure profilin is either obtained from an excess pool present in some extracts or by dissociation of profilin-actin complexes and removal of the actin.
Lindberg
U
U
Department of Zoological Cell Biology, Wenner-Grens Institute, Stockholm University, Sweden.
Schutt
C E
CE
Hellsten
E
E
Tjäder
A C
AC
Hult
T
T
D000199:Actins / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D003285:Contractile Proteins / Q000302:isolation & purification*; D003460:Crystallization; D008840:Microfilament Proteins / Q000302:isolation & purification*; D010455:Peptides; D051304:Profilins; D011506:Proteins / Q000302:isolation & purification*; D013154:Spleen / Q000378:metabolism
D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; D010455:Peptides; D051304:Profilins; D011506:Proteins; C024782:profilactin; C011083:polyproline