PLANT PHYSIOLOGY , Vol 104, Issue 3 925-936, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Characterization of the Association of Nitrate Reductase with Barley (Hordeum vulgare L.) Root Membranes
P. A. Meyerhoff, T. C. Fox, R. L. Travis and R. C. Huffaker
Department of Agronomy and Range Science, University of California, Davis, California 95616
The nature of the association between nitrate reductase (NR) and membranes
was examined. Nitrate reductase activity (NRA) associated with the
microsomal fraction of barley (Hordeum vulgare L.) roots amounted to 0.6 to
0.8% of soluble NRA following sonication in the presence of 250 mM KI and
repeated osmotic shock. This treatment removed all contaminating soluble
NRA from microsomes of uninduced barley roots that had been homogenized in
a soluble extract from roots of NO3- -induced plants. On continuous sucrose
gradients, NRA co-migrated specifically with VO4- -sensitive ATPase
activity, a plasma membrane (PM) marker; activity of glucose-6-phosphate
dehydrogenase, assayed as a cytosolic marker, co-migrated with NRA.
Microsomal NRA was absent in barley mutants deficient in soluble NR.
Perturbation and trypsinolysis experiments with PM vesicles isolated by
aqueous two-phase partitioning indicated that NR is associated with the
periphery of the cytoplasmic face of the bilayer. These results demonstrate
that PM and soluble NRs are essentially the same protein but that the
membrane-associated form is tightly bound. Although it is possible that
PM-associated NR exists in vivo, unequivocal evidence for this has yet to
be shown. However, PM NR is definitely present in vitro.
