CHARACTERIZATION OF MULTIPLE FORMS OF POLYPHENOLOXIDASE FROM APPLE FRUIT

Polyphenoloxidase (PPO) (E.C.1.10.3.1) from apple (Pyrus malus L. cv G ranny Smith) pulp thylakoidal membranes presented multiple forms when separated under partially denaturing conditions and revealed by Wester n-blot using apple anti-PPO antibodies: a main active band, a faint ac tive band already described as stable proteolysed form, and a third in active band. The two active forms were studied for their optimum pH wi th or without sodium dodecyl sulfate (SDS). The main active PPO was fo und to have the same activity level at pH 4 (thylakoidal lumen pH) as at pH 6 with SDS, therefore apple PPO does not show any latency. It wa s shown to acquire a broader range of pH conditions by proteolysis or by SDS addition which might signify that a regulatory domain pH-contro ls the active site. The estimated molecular mass of the main active an d proteolysed forms were respectively 64 and 42 kDa. Denaturation by g uanidinium chloride of a native PPO extract led to a single 63-kDa ban d shifted in a 64-kDa band with reduction by dithiothreitol, indicatin g that PPO protein contains internal disulfide bonds. This denaturatio n study led us to suggest that the inactive PPO form present in the na tive extract may be the partially unfolded PPO translocation-competent form entering thylakoidal membranes.