Properties of purified actin depolymerizing factor from chick brain.
Actin depolymerizing factor (ADF) from 19-day embryonic chick brains has been purified to greater than 98% homogeneity with a yield of 7.2 mg/100 g of brain. Quantitative immunoblotting with a monospecific antibody to ADF indicated that ADF comprises 0.3% of the total brain protein, resulting in an actual purification yield of about 20%. Brain ADF migrates as a single polypeptide of 19,000 kDa on SDS-containing polyacrylamide gels. The molecular weight of the native protein determined from sedimentation equilibrium in buffers containing from 50 to 200 mM KCl is 20,000. The secondary structure of ADF calculated from the circular dichroic spectrum consists of about 22% alpha-helix, 24% beta-sheet, and 18% beta-turn. ADF contains a blocked N-terminus, a single tryptophan residue located about one-third of the way from one end of the protein, and six cysteine residues (all in reduced form in the native protein). All six cysteine residues could be chemically modified with eosinylmaleimide under nondenaturing conditions; however, ADF activity was lost when more than one cysteine residue was modified. ADF microheterogeneity has been observed upon nonequilibrium pH gradient electrophoresis in polyacrylamide gels containing 9 M urea, the major isoform having a pI of congruent to 7.9-8.0. ADF can interact with either monomeric or filamentous actin to give a complex which can be isolated by gel filtration chromatography. Both major and minor isoforms of the ADF are found in the complex. Assembly-competent actin and active ADF can both be recovered from the complex by chromatography on ATP-saturated DEAE-cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)
Giuliano
K A
KA
Department of Biochemistry, Colorado State University, Fort Collins 80523.
Khatib
F A
FA
Hayden
S M
SM
Daoud
E W
EW
Adams
M E
ME
Amorese
D A
DA
Bernstein
B W
BW
Bamburg
J R
JR
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001923:Brain Chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002642:Chick Embryo; D002845:Chromatography; D002942:Circular Dichroism; D051345:Destrin; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008840:Microfilament Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D013056:Spectrophotometry, Ultraviolet
D051339:Actin Depolymerizing Factors; D000199:Actins; D000596:Amino Acids; D051345:Destrin; D008840:Microfilament Proteins