Purification and partial amino acid sequence of the actin-fragmin kinase from Physarum polycephalum.
The 80-kDa actin-fragmin kinase (AFK) was purified from Physarum polycephalum microplasmodia to apparent homogeneity through a procedure involving six chromatographic steps. Taking the activity at the first purification step as 100%, the kinase was purified more than 1500-fold, with an overall yield of 8%. The specific activity of the purified enzyme was 700 U/mg. The total amount of AFK present could be estimated as 34 ng/mg extracted protein. One of the polyclonal antibodies raised against four tryptic peptides of the purified 80-kDa AFK recognized the 80-kDa band in an immunoblot and could inhibit the AFK activity up to 100%. A minor 78-kDa protein, also displaying AFK activity, appeared to be derived from the 80-kDa AFK. A partial amino acid sequence analysis, covering up to 20% of the protein (150 amino acids), was performed and confirmed the lack of similarity with any of the sequenced kinases. These data are in agreement with the unique physical and enzymatic properties of the AFK.
Laboratory of Physiological Chemistry, University Ghent, Belgium.
J
J
Gettemans
Y
Y
De Ville
J
J
Vandekerckhove
E
E
Waelkens
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006495:Heparin, Low-Molecular-Weight / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D016839:Physarum polycephalum / Q000201:enzymology*; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis
D000199:Actins; D003470:Culture Media; D006495:Heparin, Low-Molecular-Weight; C074377:actin kinase; D017346:Protein Serine-Threonine Kinases