Identification of two species of actin depolymerizing factor in cultures of BHK cells.
High-speed supernatant obtained from the lysate of cultured BHK cells has been chromatographed on Sepharose-4B, DEAE-cellulose and hydroxyapatite columns, and a fraction has been identified with characteristics similar to an actin depolymerizing factor (ADF), a small protein previously isolated from embryonic chick brain. Using a rabbit antibody against the chick brain protein, two immunoreactive forms were identified: a 19 kDa form co-migrating in SDS-polyacrylamide gels with embryonic chick brain ADF, and a 20 kDa form. The two species could be separated on a hydroxyapatite or green A dye matrix columns and only the 20 kDa protein was active when assayed for effects on pyrene-G-actin assembly. It enhanced the rate of F-actin assembly, but only after an initial lag phase, and decreased the final proportion of actin in filamentous form. These effects were calcium-independent. Actin depolymerizing factor constituted at least 0.5% of the total protein in the cytoplasmic fraction. A Triton extract of plasma membrane-enriched fraction from BHK cells was fractionated on a Sepharose-4B column and again, a fraction was found which had an ADF-like activity and also contained the two immuno-cross-reactive forms, 19 kDa and 20 kDa. These results suggest a novel regulation of the microfilament system in eukaryotic cells via the control of the ADF activity.
Koffer
A
A
MRC Cell Biophysics Unit, London, U.K.
Edgar
A J
AJ
Bamburg
J R
JR
D051339:Actin Depolymerizing Factors; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002848:Chromatography, DEAE-Cellulose; D006224:Cricetinae; D051345:Destrin; D007118:Immunoassay; D007668:Kidney / Q000032:analysis* / Q000166:cytology; D008840:Microfilament Proteins / Q000032:analysis* / Q000276:immunology / Q000502:physiology
D051339:Actin Depolymerizing Factors; D051345:Destrin; D008840:Microfilament Proteins