Km. Fredlund et al., STEREOSPECIFICITY OF NADH-FERRICYANIDE REDUCTASE IS A CONVENIENT MARKER FOR THE ENDOPLASMIC-RETICULUM OF PLANT-CELLS, Plant journal, 10(5), 1996, pp. 925-933
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.