A STUDY OF THE INDIGOGENIC PRINCIPLE AND IN VITRO MACROPHAGE DIFFERENTIATION
FORT DETRICK FREDERICK MD
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The 5-bromo-4-chloro-substituted indoxyl glycosides were employed in a study of the cytochemistry of macrophages. During short-term 72 hours culture, these cells demonstrated concomitant increases in the number of organelles identified as lysosomes and sites that stained for beta-galactosidase and beta-glucuronidase activities. Moreover, there was a progressive increase in the densities of enzyme reactive centers. Indigo reaction product was not observed over nuclei lipid droplets and cell background were free from spurious precipitations. Both galactosidase and glucuronidase were optimally active at acid pH values. Enzyme activity was completely inhibited by the aldonolactone corresponding to the sugar moiety of the substrate. Unrelated sugars did not influence hydrolysis, whereas substrate analogs caused various degrees of inhibition. Biochemical assays of cell homogenates under the conditions employed in the cytochemical studies revealed the semiquantitative potentialities of the indoxyl methods. Optical density measurements demonstrated that the rate of indoxyl galactoside hydrolysis was dependent on both time and enzyme concentration. Present evidence suggests that the glycosidases reflect and probably contribute to both physiological and pathological processes.