Biochemical Characterization of a Catalase Inhibitor from Maize

Abstract

Some biochemical properties of the catalase inhibitor purified from maize scutella are described. The inhibitor is heat-labile and its activity is destroyed by trypsin, indicating that it is a protein. It does not appear to be a lectin nor does the inhibition involve proteolysis. The active inhibitor is a dimer with each subunit having a molecular weight of 5600 as determined by sodium dodecyl sulfate electrophoresis. A kinetic analysis performed in the presence of increasing levels of inhibitor gave unusual Lineweaver-Burk patterns. Possible explanations for these patterns are discussed. The inhibitor is active against all catalases tested from a wide variety of organisms.A substance in maize scutella which inhibits the enzyme catalase (H202:H202 oxidoreductase, EC 1.11.1.6) has been reported; this substance has been purified by affmity chromatography using immobilized catalase (16). The activity of the inhibitor varies inversely with catalase activity in the scutellum of the germinating seed (17) and constitutes one of several mechanisms regulating catalase activity in that tissue (10,12,18). This report describes some of the biochemical properties of this inhibitor. MATERIALS AND METHODSMaterial. Seeds from the highly inbred lines W64A and 229 were used in all studies. Seeds were imbibed in tap water for 12 to 18 h, transferred to plastic trays lined with moist germination papers, and maintained in the dark at 27 C until use. Seedlings were watered daily.Inhibitor Preparation and Assay. The inhibitor was purified by a modification of the affinity chromatography method previously described (16). A scutellar extract was passed through a column containing bovine liver catalase-Sepharose. Following extensive washing with 0.5 M galactose and 0.1 M NaCl, the bound inhibitor was eluted with 1 M NaCl. The galactose wash removed several proteins which apparently adsorbed to the Sepharose matrix. The I M NaCl wash was dialyzed against 10 mM Tris-HCl buffer (pH 7.4) and was stored at 4 C for up to I week. Such preparations lost less than 15% of their original activity during that storage period. The inhibitor was assayed against partially purified maize catalase as previously described (17).Trypsin Digestion. to conduct the digestion. Following incubation, the trypsin was removed by centrifugation at l0,OOOg and the aliquots were assayed for inhibitory activity.After the initial inhibitor action period (approximately 5 min; Fig. 3), no further decrease in catalase activity was observed. This suggests that the centrifugation effectively removed the insolubilized trypsin. Incubation of catalase with insolubilized trypsin under the inhibitor assay times and conditions did not result in a significant decrease in catalase activity.Mol Wt Determination. The mol wt of the native protein was determined by electrophoresis of purified inhibitor under nondenaturing conditions in gels composed of 9.5, 10.0, 10.5, and 11.0% acrylamide as described by Hendrick and Smith (3). The inhibitor does not stain well on gels (see "Dis...

Developmental Genetics of Maize

“…Both isozymes appear to be synthesized on free polysomes, which suggests that they are not directly translocated into this organelle during translation (43). A dimeric protein with a mo lecular weight of approximately 12,000 , isolated fr om maize scutella, is found to bind and inhibit catalase in a reversible but specifi c manner (96,110,111). The galactose associated with this protein is necessary for the inhibitory activity (115).…”