Isolation and Partial Characterization of an Acid Endoprotease Present in Dormant Apple Shoot Bark

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Abstract
A major protease present in dormant bark tissues of the apple (Malus domestica Borkh. cv. “Golden Delicious”) was partially purified by hemoglobin-coupled Sepharose column chromatography. The protease active at pH 4.6 and at temperatures of 30 to 50 C was found to be sulfhydryl-dependent. Phenylmercuric acetate inhibited the enzyme approximately 50% at 2 millimolar, whereas phenylmethylsulfonyl fluoride inhibited less than 30% even at 10 millimolar. Substrate specificity and the separation of the enzyme reaction products indicated that the enzyme is likely to be an endoprotease. We suggest that the storage proteins in apple bark tissue undergo some modification prior to their eventual hydrolysis to amino acids, which requires a multi-enzyme system(s). Evidence is presented that the apple bark protein extracts enzymically release ninhydrin-positive compounds upon storage at 5 C. It is concluded that the activation of the sulfhydryl-dependent acid endoprotease is associated with the rapid metabolism of storage proteins which accompanies bud break upon regrowth.