DIFFERENTIAL RESPONSE OF NITRATE REDUCTASE AND SUCROSE-PHOSPHATE SYNTHASE-ACTIVATION TO INORGANIC AND ORGANIC SALTS, IN-VITRO AND IN-SITU

Studies were conducted to compare the modulation of beta-nicotinamide adenine dinucleotide (NADH):nitrate reductase (NR; EC 1.6.6.1) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) with respect to regulation by the inorganic anions, phosphate (P-i), sulfate and tungstate. Following inactivation of both enzymes in vivo by transferring spinach plants (Spinacia oleracea L. cv. Bloomsdale) to a darkened growth chamber, spontaneous reactivation occurred in vitro when desalted leaf extracts were preincubated at 25 degrees C prior to assay. All three inorganic anions inhibited SPS activation in vitro and also reduced the light activation of SPS in situ when they were fed to excised leaves via the transpiration stream. As expected, feeding tungstate to excised leaves prevented the light-dependent increase in extractable NR activity. However, in contrast to SPS, the light activation of NR in situ was relatively unaffected by P-i and sulfate, and in vitro, both anions stimulated (rather than inhibited) the reactivation of NR. Part of the stimulation by P-i and sulfate was the result of increased ionic strength, and stimulation could also be demonstrated with other inorganic and organic salts. In the presence of high ionic strength (0.1 to 0.2 M KCl), the rate of NR activation in vitro was relatively constant when the pH of the preincubation medium was varied from pH 6.5 to 8.0, whereas in the absence of added salt the rate of activation was nearly zero at pH 6.5 but increased progressively as pH was raised. The stimulation by salts could be reversed, in part, by glycerol and ethylene glycol suggesting that hydrophobic interactions might play some role in the activation of NR.