Soluble and Bound Apoplastic Activity for Peroxidase, β-d-Glucosidase, Malate Dehydrogenase, and Nonspecific Arylesterase, in Barley (Hordeum vulgare L.) and Oat (Avena sativa L.) Primary Leaves

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Abstract
An intercellular washing solution containing about 1% of the soluble protein, 0.3% or less of the glucose-6-phosphate dehydrogenase activity, but up to 20% of the peroxidase and .beta.-D-glucosidase activity of barley (Hordeum vulgare L.) or oat (.ALPHA.vena sativa L.) primary leaves was obtained by vacuum infiltrating peeled leaves with pH 6.9 buffered 200 millimolar NaCl. After this wash, segments were homogenized in buffer, centrifuged, and the supernatant was assayed for soluble cytoplasmic enzymes. The pellet was washed and resuspended in 1 molar NaCl to sulobilize enzymes strongly ionically bound to the cell wall. The final pellet was assayed for enzyme activity covalently bound in the cell wall. Apoplastic (intercellular washing solution, ionically bound, and covalently bound) fractions contained up to 76% of the .beta.-D-glucosidase activity, 36% of the peroxidase activity, 11% of the nonspecific arylesterase activity, 4% of the malate dehydrogenase activity of peeled leaf segments. The partitioning and salt-solubility of the enzymes between the apoplast and symplast differed considerably between these two species. Intercellular washing fluid prepared by centrifuging unpeeled leaves had higher activity for glucose-6-phosphate dehydrogenase, less soluble protein, and less peroxidase activity per leaf than intercellular washing solution obtained by our peeling-infiltration-washing technique. The results are discussed in relation to the roles of the enzymes in phenolic metabolism in the cell wall.