The quaternary structure of carp muscle alkaline protease

Abstract

Carp muscle alkaline protease consists of four kinds of subunits, and its composition was assumed to be ( αβγ 2 δ 2) 4. It dissociated in the presence of 2-mercaptoethanol into an enzyme and α-subunits which upon removal of 2-mercaptoethanol rapidly aggregated to form a precipitate. The composition of the 2-mercaptoethanol-treated enzyme was ( βμ 2 δ 2) 4. The pH of a 2-mercaptoethanol-treated enzyme solution was lowered to 4.5 by the addition of acetic acid in the presence of 0.4 M LiCl and centrifuged to separate the precipitate formed; this exhibited little activity and was mainly composed of β-subunits. The supernatant fluid recovered 53% of activity and contained an enzyme, whose composition was ( γ 4 δ 4) 4. The temperature-activity curve of the native enzyme was the same as that of the 2-mercaptoethanol-treated enzyme and both were unable to hydrolyze casein at all below 55°C. However, the temperature dependence for activity of the LiCl-treated enzyme was ordinary: it hydrolyzed casein at physiological temperatures. When the 2-mercaptoethanol-treated enzyme was incubated with 4.5 M urea at 45°C for 20 min and this was followed by column chromatography, a little activity was recovered and the amount of recovery was parallel with the amount of δ-subunit in the fractions. These findings suggest; (1) the α-subunit does not take any part in activity but is a protein necessary for binding between subunits or between the enzyme and some functional proteins in the cells, (2) the β-subunit is used as inhibitor in the quaternary structure of the enzyme, (3) the δ-subunit is the catalytic one, and (4) binding with the γ-subunit is necessary for the δ-subunit to retain its active comformation.