Spinach Thylakoid Polyphenol Oxidase

Abstract

Polyphenol oxidase activity (EC 1.10.3.2) was identified in 2 enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified > 900-fold by (NH4)2SO4 precipitation, gel filtration and ion exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a MW of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. The higher molecular weight enzyme is the predominant form in freshly isolated preparations but on aging or further purification, the amount of lower molecular weight enzyme increases at the expense of the higher. Sonication releases polyphenol oxidase from the membrane largely in the latent state. C18 fatty acids, especially linolenic acid, are potent activators of the enzymic activity. In the absence of added fatty acids, the isolated enzyme spontaneously but slowly activates with time. Purified polyphenol oxidase utilizes o-diphenols as substrates and shows no detectable levels of monophenol or p-diphenol oxidase activities. The Km values for 3,4-dihydroxyphenylalanine and O2 are 6.5 and 0.065 mM, respectively. Suitable substrates include chlorogenic acid, catechol, caffeic acid, pyrogallol, and dopamine; the enzyme is substrate-inhibited by the last 4 at concentrations near their Km. A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.