Quantitative effects of altering oxidative phosphorylation and respiration on the activity of the enzyme menadione reductase (NAD[P]H2:2-methyl-1,4-naphthoquinone oxidoreductase, E.C. 1 6.5.2), in stabilized polymorphonuclear neutrophil leucocytes of Amphiuma tridactylum, were studied by amplitude-contrast microscopy and microspectrophotometry.
In a general way, the rate of enzymic activity was proportional to ADP concentration and inversely proportional to the concentration of ATP. Terminal respiratory blocking by azide produced selective subtotal inhibition. Uncoupling of phosphorylation by dinitrophenol produced complex results.
Neutrophils of A. tridactylum, irrespective of their stage of maturation in the circulating blood, could be subdivided into 3 metabolic classes: Class I cells, of low enzymic activity (predominantly mitochondrial), greatly activated by ADP, somewhat activated by ATP, and only slightly inhibited by dinitrophenol; Class II cells, twice as active as Class I (in which the endoplasmic reticulum and idiozome were as active as the mitochondria), further activated by ADP used alone or with dinitrophenol, and unaffected by ATP or dinitrophenol; Class III, hyperactive cells (enzymic localization identical with that in Class II), inhibited by ATP and dinitrophenol, and not activated by ADP. Some of the mitochondria of Class III neutrophils retained nearly a third of their reductase activity when the reaction mixture contained 10-1 azide.
There is reason to believe that Class I neutrophils may form a reserve population of vegetative cells; in vitro, they can be transformed into Class II cells when a high concentration of ADP is added. Class II and Class III cells are potentially capable of amoeboid movement and phagocytosis. The metabolic mobilization of neutrophils could be interpreted as being controlled by 2 different feedback mechanisms: activation by ADP in cells of Classes I and II, and inhibition by ATP in cells of Class III.