Intact Tissue AssayforNitrite Reductase

A methodhasbeendevised forthedetection andmeasurementofnitrite reductase activity inintact barley (Hordeum vulgare L.cv.Himalaya) aleurone layers. Thetechnique involves feeding aleurone layers nitrite andmeasuring nitrite disappearance after a giventimeperiod. Themethodalso allows simultaneous determination ofnitrite uptakebythe tissue. Quantitative recovery ofnitrite isobtained byrapid heating oftissue inthepresence ofdimethyl sulfoxide. Usingtheprocedure described, nitrite reductase activity in intact barley aleurone layers wasdetermined. Enzymeactivity wasincreased bypriorincubation ofthetissue withnitrate, butconsiderable activity was present in tissueincubated without nitrate. Nitrate-induced activity was inhibited by cycloheximide butnotbyactinomycin D.Enzymeactivity in induced layers wasinhibited by2,4.dinitrophenol, andpartially byantimycin A and2-n-heptyl4-hydroxyquinoline Noxide. Activity innoninduced tissue appeared tobeless sensitive totheserespiratory inhibitors. Incontrast, bothactivities wereinhibited morethan90% byanaerobiosis; butnitrateinduced andnoninduced aleurone layers wereabletoreduce nitrite anaerobically whentheconcentration ofsubstrate in theassay mediumwasreduced from250pMto25pM.Nitrite uptakewasrelatively insensitive toanaerobiosis andtothe inhibitors tested. Nitrite depletion fromthemediumbyaleurone layers was rapid atpH4.5andnegligible atpH 7.5. Nitrite accumulated atpH 4.5underanaerobic conditions wasrapidly released whenthetissue wastransferred tomediumatpH 7.5. Nitrite release atpH 7.5occurred whether thetissue wasmaintained underanaerobic oraerobic conditions.