Intracellular distinction between peroxidase and catalase in exocrine cells of rat lacrimal gland: a biochemical and cytochemical study.
The lacrimal gland (Glandula orbitalis externa) of rat contains both peroxidase and catalase and was used as a model for biochemical and cytochemical distinction between peroxidase and catalase. Both enzymes were isolated by ammonium sulfate precipitation from tissue homogenates, and the effects of fixation with glutaraldehyde and various conditions of incubation were investigated colorimetrically using DAB as hydrogen donor. The lacrimal gland peroxidase is strongly inhibited by glutaraldehyde treatment. In contrast, for catalase the fixation with glutaraldehyde is the prerequistie for demonstration of its peroxidatic activity. The maximal peroxidatic activity was obtained after treatment of catalase with 3% glutaraldehyde, higher concentrations being inhibitory. For lacrimal gland peroxidase, the maximal rate of oxidation of DAB is at pH 6.5, whereas for catalase it is at pH 10.5. The optimal concentration of H2O2 for lacrimal gland peroxidase is at 10(-3)M and for peroxidatic activity of catalase at 10(-1)M. These optimal conditions obtained biochemically were applied to tissue sections of rat lacrimal gland. After the fixation of tissue with a low concentration of glutaraldehyde and incubation in the DAB medium at neutral pH containing 10(-3)M H2O2 (Peroxidase medium), the reaction product was localized in the cisternae of the rough endoplasmic reticulum, in elements of the Golgi apparatus, and in secretory granules. After the fixation of tissue with 3% glutaraldehyde and incubation in the DAB-medium containing 10(-1)M H2O2 and at pH 10.5 (catalase medium), the staining in the endoplasmic reticulum, the Golgi-apparatus and in secretory granules was completely inhibited and reaction product was localized exclusively in small (0.2-0.5 mu) particles similar to small peroxisomes described in various other cell-types.
D015100:3,3'-Diaminobenzidine; D000818:Animals; D002374:Catalase / Q000032:analysis*; D003594:Cytoplasmic Granules / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005976:Glutaral; D006056:Golgi Apparatus / Q000201:enzymology; D006651:Histocytochemistry; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D007765:Lacrimal Apparatus / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008830:Microbodies / Q000201:enzymology; D010544:Peroxidases / Q000032:analysis*; D051381:Rats
D015100:3,3'-Diaminobenzidine; D006861:Hydrogen Peroxide; D010544:Peroxidases; D002374:Catalase; D005976:Glutaral