Stereospecificity of NADH‐ferricyanide reductase is a convenient marker for the endoplasmic reticulum of plant cells

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The stereospecificity of NADH‐ferricyanide reductase and NADH‐cytochrome c reductase in the endoplasmic reticulum (ER) for the α‐hydrogen on the nicotinamide ring is presented as a very sensitive and convenient assay to detect ER contamination in preparations of membranes lacking α‐specific NADH‐acceptor reductase, such as the plasma membrane and the tonoplast. The experimental details of the assay are given and the limitations explored (time‐course, amount of protein, possible side reactions, speed, reproducibility, etc.). The NADH‐ferricyanide reductase activity of plasma membranes from spinach and sugarbeet leaf was completely β‐specific and always showed a latency (increase upon addition of Triton X‐100), whereas the α‐specificity in the ER was non‐latent. This is consistent with the presence of mainly right‐side‐out vesicles in preparations of plasma membranes with the binding site for NADH and ferricyanide on the inner, cytoplasmic surface. In contrast, right‐side‐out ER vesicles have the binding site on the outer, cytoplasmic surface. The addition of as little as 1% of the α‐specific ER (on an NADH‐ferricyanide activity basis) to the spinach leaf plasma membrane could be detected with the stereospecificity assay. Wheat root plasma membrane showed some α‐specificity (in addition to β‐specificity) which was probably due to ER contamination since the activity was non‐latent. The stereospecificity assay is also shown to be useful in monitoring the separation of tonoplast vesicles from ER vesicles by countercurrent distribution of a light microsomal fraction. It follows that the NADH‐acceptor reductase activities in preparations of plasma membrane and tonoplast are due to distinct enzymes characteristic for those membranes.