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Plant Physiology 78:614-618 (1985)
© 1985 American Society of Plant Biologists

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Articles

Nitrate Reductase from Monoraphidium braunii

Immunocytochemical Localization and Immunological Characterization 1

Antonio Lopez-Ruiz, Jose Manuel Roldan, Jean Pierre Verbelen and Jesus Diez

Departamento de Bioquímica, Facultad de Veterinaria, Universidad de Córdoba, Córdoba 14005, Spain, Departamento de Bioquímica, Facultad de Ciencias, Universidad de Córdoba, Córdoba 14005, Spain, Departement Biologie, Universitaire Instelling Antwerpen, Universiteisplein 1, B-2610 Wilrijk, Belgium

Homogeneous nitrate reductase (EC 1.6.6.2) from Monoraphidium braunii was obtained by means of affinity chromatography in blue-Sepharose and gel filtration. After electrophoresis in polyacrylamide, gel slices containing pure nitrate reductase were disrupted and injected into previously unimmunized rabbits. The antiserum produced after several weeks was found to inhibit the different activities of nitrate reductase to a similar degree. Monospecificity of the antiserum was demonstrated by Ouchterlony double diffusion and crossed immunoelectrophoresis. The antibodies were purified by immunoabsorption to Sepharose-bound nitrate reductase.

The intracellular location of nitrate reductase in green algae was examined by applying an immunocytochemical method to M. braunii cells. Ultrathin frozen sections were first treated with immunopurified anti-nitrate reductase monospecific antibodies, followed by incubation with colloidal gold-labeled goat antirabbit immunoglobulin G as a marker. The enzyme was specifically located in the pyrenoid region of the chloroplast.

1 Supported by Comision Asesora de Investigación Científica y Técnica grant 1370/82; a grant of the National Fund for Scientific Research (NFWO-Belgium) to J.-P. Verbelen; and an EMBO short-term fellowship to A. López Ruiz.






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Copyright © 1985 by the American Society of Plant Biologists