DETERMINATION OF NITRATE REDUCTASE-ACTIVITY IN ULVA-RIGIDA C AGARDH BY THE INSITU METHOD

Assay conditions for determining in situ nitrate reductase (NR) activity in Ulva rigida C. Agardh were studied. The final composition of the incubation medium was: 0.1 M phosphate buffer, 0.5 mM EDTA, 0.1% 1-propanol, 30 mM KNO3 and 10-mu-M glucose. The optimal pH was 8, which is greater than that usually found in higher plants (7.5) and close to the pH of sea water. Samples were incubated in the dark at 30-degrees-C. A short incubation time (30 min) was more satisfactory than longer ones for determination of the initial rate, and to prevent any limitation of the NO3- reduction rate for the assay conditions themselves. Surfactants such as Tergitol NP-10 and Triton X-100 were less effective than 0.1% 1-propanol. Incubation in anaerobic conditions is a critical point in the in situ NR assay in Ulva rigida. The two methods used; vacuum infiltration for 10 min, and N2-flushing 2 min before and 2 min after introducing the sample of tissue, were almost identically effective. The addition of NADH and glucose as external sources of reducing power was studied and the problems associated with NADH interference in NO2- determination are discussed. The addition of DCMU did not overcome the need for darkness as it does in higher plants, even when the photosynthetic O2-production was completely abolished at the same DCMU concentration.