Preparation of alpha s2-casein and its plasminogen activator activity
2004
Igarashi, Y. (Hirosaki Univ., Aomori (Japan). Faculty of Agriculture and Life Science) | Tanifuji, K.
Summary A simple procedure for the preparation of a sub(s2)-casein is presented and the experiments with this fraction and plasminogen suggested that plasminogen activator (PA) activity may be involved in the a sub(s2)-casein fraction. For the preparation of a sub(s2)-casein, acid-precipitated whole casein treated previously with 2-mercaptoethanol was fractionated in 50%ethanol by the addition of ammonium acetate (50 mM at final concentration) at pH 7.2. The precipitate recovered by centrifugation was fractionated again as above and the resultant precipitate was dissolved in deionized water with NaOH, dialyzed against deionized water and freeze dried. For the determination of proteinase activity, dimethylcasein was incubated with casein fractions at 37 deg C for various time intervals in the presence or absence of plasminogen, and the increase in absorbance at 420 nm was measured after the reaction with trinitrobenzensulfonic acid (TNBS) . PA activity was estimated by subtracting proteinase activity in the absence of plasminogen (plasmin activity) from that in its presence. PA activity in a sub(s2)-casein fraction was also observed by urea-polyacrylamide gel electrophoresis (urea-PAGE) . The results of ureaPAGE of this fraction heated at various temperature indicated that the PA activity was relatively heat stable. PA activity in the a sub(s2)-casein fraction was 2.5 times higher than that in unfractionated acid casein.
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