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Date of Award

9-1976

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Supervisor

Dr. G.J. Sorger

Abstract

Two nitrite reductase activities were identified in crude extracts of wild type Neurospora exposed to nitrate ions: a first one catalyzes the stoichiometric reduction of nitrate to ammonia (AP); a second causes the disappearance of nitrite to something other than ammonia (ND). The second activity (ND) which is present in all strains under all conditions, was isolated by ultra-filtration through a collodion membrane or by precipitation with 50% acetone. This activity could account for the difference between the nitrite reduction and ammonia production catalyzed by crude extracts of fully induced wild type mycelia. The properties of the ND component were studied by examining the effect of organic solvents, proteases, sulfhydryl reagents, metal-complexing agents and temperature on its activity. The AP activity which is inducible by nitrate or nitrite and is present only in nitrite-utilizing strains, was deduced from a comparison of nitrite non-utilizing mutants and the wild type strain to be the assimilatory enzyme.

Nitrate was found to induce the assimilatory nitrite reductase directly. Four different approaches were employed to show this: a study of the kinetics of induction of nitrate - and nitrate reductase; an examination of the induction of nitrate reductase by nitrate in the presence of tungstate or in the presence of ammonium ions; a comparison of the induction of nitrite reductase by nitrate in nitrate reductaseless mutants and in the Wild type strain. Active nitrate reductase does not appear to be necessary for the induction of nitrite reductase by nitrate as suggested by Cove and Pateman (7). The nitrite reductase apoprotein, on the other hand, seems to be involved in the induction of the assimilatory nitrite reductase.

Ammonium ions repressed the induction of the assimilatory nitrite reductase by nitrate. This repressive effect was an indirect one; ammonium ions had to be metabolized first in order to repress nitrate reductase. This was shown by the lack of repression in strain am-la, a glutamate dehydrogenaseless strain.

A model is proposed to describe the possible mechanism of induction and repression of nitrite reductase in Neurospora crassa.

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