NAD (P)H-Duroquinone Reductase in the Plant Plasma Membrane

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Abstract
The intracellular distribution of NADPH- and NADH-dependent duroquinone reductase (NAD (P)H-DQR) from etiolated zucchini hypocotyls (Cucurbita pepo L.) was investigated. About 80% of this enzyme is in the supernatant fraction and is probably cytosolic. Particulate NAD (P)H-DQR was largely (42%) found in association with the plasma membrane and was strongly stimulated by TX100. Another 33% of NAD (P)H-DQR was associated with mitochondria, and minor fractions with the endoplasmic reticulum (8%) and other particles. All these fractions were little or not stimulated by TX100. The distribution of detergent-activated NAD (P)H-DQR is thus distinct from microsomal NADH- and NADPH-CCR. The plasma membrane was purified from microsomal fractions by metrizamide plus sucrose density gradient centrifugation or by PEG/dextran phase partitioning. Both types of particle preparations peaked at a density (d) of 1.165 g cm−3 in sucrose gradients and contained substantial TX100-sensitive NADH-DQR, TX100-stimulated NAD (P)H-DQR, together with traces of NADH-CCR and trapped ‘soluble’ enzyme (MDH, NADP-malic enzyme) activities. In isopycnic gradients of unfractionated microsomes, however, trapped enzymes peaked at d 1.155 whereas NAD (P)H-DQR peaked at d 1.165 and GSII at d 1.170, probably revealing plasma membrane heterogeneity. Further evidence of heterogeneity was provided by fractionation of plasma membrane vesicles on dextran step-gradients. Most of the trapped MDH was released to the supernatant by sonication or treatment with 0.0125% TX100. Under these conditions most of the NAD (P)H-DQR sedimented with the membranes. It is concluded that NAD (P)H-DQR is bound to the inside of plasma membrane vesicles, but a fraction (7 to 31%) may be ‘soluble’ and sequestered within the vesicle lumen. Part of the detergent-sensitive NADH-DQR may be externally bound and accessible to non-permeating substrates.