Comparative Studies on Nitrate Reductase in Agrostemma githago Induced by Nitrate and Benzyladenine

Abstract

NADH-nitrate reductase activity in excised embryos of Agrostemma githago develops in response to nitrate as well as benzyladenine. Induction of nitrate reductase by benzyladenine was much more susceptible to inhibition by a mixture of amino acid analogues and by cordycepin than induction by nitrate. In contrast, only induction of nitrate-nitrate reductase was decreased by chloramphenicol.NADH-cytochrome c reductase and reduced flavin mono-nucleotide-nitrate reductase activities were found to be associated with NADH-nitrate reductase and were induced by both nitrate and benzyladenine. When a partially purified enzyme sample was centrifuged in a linear 5 to 20% sucrose density gradient, a minor and a major band of NADH-cytochrome c reductase activity were observed. NADH-nitrate reductase cosedimented with the major band.The characteristics of nitrate-nitrate reductase and benzyl-adenine-nitrate reductase were compared by four methods but no differences could be detected: (a) Both enzymes sedimented with the same velocity during sucrose density gradient centrifugation. (b) Their distribution among fractions obtained by differential precipitation with (NH(4))(2)SO(4) was identical. (c) The elution profile of nitrate-nitrate reductase and benzyl-adenine-nitrate reductase after chromatography on diethyl-aminoethyl Sephadex A-25 columns showed no significant difference. (d) On polyacrylamide gel, the electrophoretic migration of the two enzymes was also identical.