STEREOSPECIFICITY OF NADH-FERRICYANIDE REDUCTASE IS A CONVENIENT MARKER FOR THE ENDOPLASMIC-RETICULUM OF PLANT-CELLS

The stereospecificity of NADH-ferricyanide reductase and NADH-cytochro me c reductase in the endoplasmic reticulum (ER) for the alpha-hydroge n on the nicotinamide ring is presented as a very sensitive and conven ient assay to detect ER contamination in preparations of membranes lac king alpha-specific NADH-acceptor reductase, such as the plasma membra ne 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 beta-specific and always showed a latency (increase up on addition of Triton X-100), whereas the alpha-specificity in the ER was non-latent. This is consistent with the presence of mainly right-s ide-out vesicles in preparations of plasma membranes with the binding site for NADH and ferricyanide on the inner, cytoplasmic surface. In c ontrast, right-side-out ER vesicles have the binding site on the outer , cytoplasmic surface. The addition of as little as 1% of the alpha-sp ecific ER (on an NADH-ferricyanide activity basis) to the spinach leaf plasma membrane could be detected with the stereospecificity assay. W heat root plasma membrane showed some alpha-specificity (in addition t o beta-specificity) which was probably due to ER contamination since t he activity was non-latent. The stereospecificity assay is also shown to be useful in monitoring the separation of tonoplast vesicles from E R vesicles by countercurrent distribution of a light microsomal fracti on. It follows that the NADH-acceptor reductase activities in preparat ions of plasma membrane and tonoplast are due to distinct enzymes char acteristic for those membranes.