Studies on the catalase inhibitor

In two previous reports, a catalase specific inhibitor which is differentially active in maize scutella during the first few days of germination, has been described. The molecule appears to be proteinaceous in nature, it is not a protease by several different criteria and its molecular weight, estimated by column chromatography is approximately 16,000 daltons. It inhibits catalase from different sources but not other enzymes and not even maize peroxidases, a group of catalytically related hemoproteins (Sorenson and Scandalios, MGCNL 49, 50). Recently evidence has been obtained that a protein in the glyoxysomal membrane inhibits catalase, that suggests that the inhibitor could be compartmentalized in the glyoxysomal membrane (Table 1).

The inhibitor has been purified to homogeneity using conventional chromatographic techniques (DEAE-Sephacell, G-75 Sephadex) and affinity chromatography on immobilized beef liver catalase. This purified molecule appears to have a carbohydrate moiety on it since it positively stained by PAS staining and it is currently being characterized in terms of its MN in SDS denature gel electrophoresis, amino acid composition etc.

Specific antibodies have been raised by injecting rabbits with the purified molecule. Using the antibodies, we are currently trying to develop a double antibody precipitation radioimmunoassay for the inhibitor. The sensitivity of the assay and its independence from the presence of endogenous catalase will help us first to study more accurately its role in post-translation regulation, developmental expression, compartmentation and tissue specificity of catalase, during the first days of seed germination. Secondly, the assay will help us to screen for inhibitor mutants in corn lines and corn seeds recently obtained from chemically mutagenized pollen. The recovery of any mutation will permit the genetic studies of gene(s) coding for the inhibitor protein. Towards that end we have labeled the inhibitor with 125I for use in the radioimmunoassay.

Using different treatments, we have been able to perturb quantitatively catalase gene expression during the first days of germination. Culturing, for instance, isolated scutella in agar media containing 0.1% H202, we have been able to duplicate catalase activity. On the other hand, by adding the drug Allylisopropylacetamide (AIA), catalase levels decrease to almost zero values. These changes in catalase levels were always inversely associated with changes in the activity of the inhibitor. Blocking, for instance, catalase synthesis with AIA during the first days of germination, higher levels of inhibitor activity (assayed against purified catalase) has been obtained, contrary to the controls where on catalase peaks during the 3-4th day of germination the inhibitor drops to almost zero values (Sorenson and Scandalios, Plant Phys., 57:351, 1976).

Table 1.

Athanasios Tsaftaris and John Sorenson


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