The Intracellular Location of Phenol Oxidases, Peroxidase and Phosphatases in the Leaves of Spinach Beet (Beta vulgaris L. Subspecies vulgaris)

Abstract

Phenol oxidase activity was estimated with three substrates: l‐3‐(3,4‐dihydroxyphenyl)ala‐nine, 4‐methylcatechol and p‐coumaric acid (hydroxylase activity). Between 70–85% phenol oxidase activity and 10–20% peroxidase activity was sedimentable in leaf extracts from Beta vulgaris. Some 20–50% of the total phenol oxidase activity in a 6000×g (20 min) fraction could be released by washing with distilled water, incubating at 37°C or freezing and thawing. Acid phosphatase and catalase activities were much more readily released (90%) into solution by these treatments. Disruption of membranes with Triton X‐100 or sonication released the majority of phenol oxidase activity and this paralleled the release of chlorophyll and protein from membranes. Peroxidase was also strongly bound to the membranes. The intracellular location of enzymes was studied exhaustively using a variety of cell fractions and density gradients. Phenol oxidase activity towards all substrates was present in the chlorophyll‐rich chloroplast membranes. Some dihydroxyphenylalanine oxidase and 4‐methylcatechol oxidase was associated with mitochondria. p‐Coumaric acid hydroxylase was restricted to the chloroplasts and supernatant. No phenol oxidase activity sedimented with the peroxisomes. A third membrane fraction containing dihydroxyphenylalanine oxidase (and acid phosphatase) was isolated from a 5 to 20000×g (20 min) fraction, but was apparently not microsomal or mitochondrial in origin. A similar fraction could be isolated when chloroplasts were mechanically disrupted, and it is tentatively suggested this fraction represents the chloroplast envelope. Enzyme distribution among gradient fractions indicated that specific enzyme proteins exist for dihydroxyphenylalanine and catechol/p‐coumaric acid. This does not exclude the existence of isozymes active toward all three substrates. Sedimentable peroxidase was associated with mitochondria and a light membrane fraction. Mixing experiments showed this was not an antifact of homogenization. Chloroplasts were devoid of peroxidase. Different intracellular and intrachloroplastal locations were found for a number of phosphatases. Copyright © 1972, Wiley Blackwell. All rights reserved