Cytochemical discrimination between catalases and peroxidases using diaminobenzidine

The influence on diaminobenzidine staining of four variables: prefixation in aldehyde, temperature and pH of incubation, and H2O2 concentration, was investigated in catalase-, as well as in peroxydase-containing material. Catalase from five different sources and five types of peroxidase were examined. It is concluded: (a) when cells are incubated without prior fixation, in a DAB medium at room temperature and pH 7.3 with 0.003% H2O2, peroxidases produce a visible cytochemical stain, while catalases do not; (b) the cytochemical reaction elicited by catalases is stimulated by prior aldehyde fixation in specified conditions, and incubation at 45 degrees C and pH 9.7 with 0.06% H2O2; (c) under the latter circumstances several peroxidases also stain. Ultrastructural preservation is satisfactory in tissues incubated prior to fixation. Use of the DAB formula for peroxidases revealed novel localizations: the perinuclear cisterna of smooth muscle, and of capillary endothelium. Other DAB reactivity is seen in the mitochondrial cristae, due most likely to cytochrome c and cytochrome oxidase. Heat-resistant DAB reactivity is found in erythrocytes and liver lipofuscin granules. Pre-oxidized DAB at low pH was shown to stain nucleic acids. 8 light micrographs, 12 electron micrographs, 6 tables

Turnover of peroxisomes during inhibition of catalase syntheesis

During continuous inhibition of catalase synthesis by allylisopropylacetamide, catalase activity in guinea pig liver decreases exponentially, reaching 12% after 15 days. However the number of peroxisomes does not change, when counted in 0.2 µm Epon sections after DAB staining. This refutes the current model that peroxisomes turn over at the rate of their components. During catalase depletion the size of peroxisomes decreases when measured on electron micrographs. Table: Catalase activity; Peroxisome number per volume; Mean peroxisomal volume

Absence of peroxisome turnover during inhibition of catalase synthesis

During its synthesis inhibition by AIA, catalase activity in guinea pig liver declines exponentially, reaching 11% after 15 days. However the number of peroxisomes counted in 0.2 µm Epon sections does not change. The peroxisomal volume in electron micrographs does diminish, to around 50% already after 9 days of synthesis inhibition. These results indicate that peroxisomes are stable structures, in contrast to prior theory. Some of their components turn over at a fast rate, observed also by others. 1 graph, 2 light micrographs, 1 table

Absence of peroxisome turnover during inhibition of catalase synthesis

During continuous inhibition of catalase synthesis by 2-allylisopropylacetamide, catalase activity in guinea pig liver declined exponentially, to 11% after 15 days. The number of peroxisomes however, stained with diaminobenzidine, was unchanged at several time points, when counted in light micrographs of 0.2 µm Epon sections. In electron micrographs peroxisomal volume decreases to approx. 50% after 15 days of inhibition. It is concluded that during turnover of peroxisome content (catalase) the structured organelles themselves are not broken down, in contrast to previous opinions

Cytochemical discrimination between peroxidases and catalases using diaminobenzidine

When cells are incubated before fixation at pH 7.2 and 23° C with 0.003% H2O2, peroxidases give a visible cytochemical stain, while catalases do not. Catalase and peroxisomes staining is strongly promoted by prior fixation in aldehydes, and incubation at pH 9 or 9.7 with 0.03% or 0.07% H2O2. By comparing the results of staining under both conditions, peroxidases can be distinguished from catalase. The effects of fixation on staining differs among peroxidases. 4 electron micrographs

Cytophotometry of extraperoxisomal catalase in the normal and 2-allyl-2-isopropylacetamide treated guinea pig liver

After DAB staining for catalase and preparing 2 µm sections, the absorbance of the reaction product in the cytosol of hepatocytes is measured by a microscope photometer MPV-2 at 450 nm. Synthesis inhibition by daily injections of 2-allyl-2-isopropylacetamide leads to a decrease of absorbance progressing with time: 44% of the untreated animal value after 6 days, 26% after 9 days, 16% after 12 days, 7% (mean value) after 15 days of inhibition. In some animals no cytosolic reaction product can be demonstrated after 15 days of inhibition. Total catalase activity (units Baudhuin) then is 11% of normal. Peroxisomes probably contain the residual catalase