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Plant and Cell Physiology, 1994, Vol. 35, No. 8 1185-1198
© 1994

Preparation of Monoclonal Antibodies against NADH: Nitrate Reductase from the Red Alga Porphyra yezoensis

Yoshiko Nakamura1, Hikaru Saji2, Noriaki Kondo2 and Tomoyoshi Ikawa1

1Institute of Biological Sciences, University of Tsukuba Tsukuba, Tbaraki, 305 Japan
2Regional Environment Division, National Institute for Environmental Studies Onogawa, Tsukuba, Ibaraki, 305 Japan

Twenty-two monoclonal antibodies were raised against the native form of nitrate reductase (NR) from Porphyra yezoensis, a marine red alga. All the antibodies were able to bind to NR from P. yezoensis, with resultant inhibition of full (NADH:NR) and/or partial (NADH:FR, NADH:CR, FMNH2:NR, and MV:NR) enzymatic activity. Fifteen of the antibodies recognized the denatured form of the enzyme. Size-exclusion gel nitration and immunoblotting of the products of the limited proteolysis of NR from P. yezoensis by trypsin or Staphylococcus aureus V8 protease revealed that 2 out of the 15 subunit-recognizing antibodies bound to the FAD domain, 6 bound to the heme domain, and 7 bound to the Mo-pterin domain. The products of limited proteolysis of NR from P. yezoensis also revealed that the sites of proteolytic cleavage that encompassed the heme domain were inverted as compared to the analogous sites in NRs of higher plants. Some of the monoclonal antibodies cross-reacted with NRs from plants belonged to different phyla. From three to five of the antibodies bound subunits of NR from multicellular red algae, but failed to bind NRs from unicellular red algae. Three or four of the antibodies crossreacted with NRs from higher plants, such as tobacco and spinach. One of the antibodies bound NRs from several types of plant, namely, members of Cryptophyta, Chromophyta, and Chlorophyta. All of the monoclonal antibodies that cross-reacted with NRs from plants other than the red algae were specific for the Mo-pterin domain of NR from P. yezoensis.

(Received May 10, 1994; Accepted September 7, 1994)
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