A study of factors influencing in vitro stability of nitrate reductase from wheat leaves

This review and the following chapters are concerned predominantly with the processes occurring in higher and lower plants which regulate the amount of NR present in vitro as controlled by degradation, and the level of activity of the existing enzyme. Those factors regulating the synthesis of NR will not be discussed in any detail but only mentioned where they also affect other mechanisms regulating NR. Nitrate reductase is unstable both in vivo and in vitro (101,193, 252). In vitro instability occurs since the isolation of enzymes and other cell components from plant tissue involves disruption of the plant cell. This results in mixing of substances which in situ were rigidly compartmentalized and is likely to result in the isolation of an enzyme which is modified from its native form. Factors present in plant cells which make plant proteins particularly unstable in vitro have been reviewed by Stahrran (216) and Pirie (157). They include vacuole acids, carbohydrates, hydrolytic and oxidative enzymes and phenolic components and their derivatives. In vivo variation in activity occurs in response to a number of other factors, including tissue age (103,129,166,243,264) and environment (15,72,82,1.03,129,261). Tissue age has been shown to influence the activity or stability of NR extracted from a number of species including corn (194,195,264), wheat (221), oats (194,195), tobacco (195) and barley (48). Nitrate reductase has been demonstrated in nearly all plant parts (16) and its ubiquitous presence suggested in higher plants (16,184). Nevertheless, due to the number of factors involved, detection of activity would only occur given suitable physiological and environmental conditions together with use of the correct extraction and assay procedure. Determining if the level of activity derived is an accurate estimate of the in situ activity is even more difficult. This has been attempted by correlating NR activity and grain or plant nitrogen (28, 36) . In vivo instability is indicated by the decline in NR activity with the onset of darkness, depletion of nitrate supply, and water or heat stress (11,82,121,168,235). Under appropriate conditions these factors could also affect the enzyme in vitro