Purification of a catalase inhibitor from maize by affinity chromatography

Sorenson, John C.; Scandalios, John G.

doi:10.1016/s0006-291x(75)80035-0

Cited by 20 publications

(8 citation statements)

References 3 publications

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“…Actual data points were taken to the left of the intersection point, ruling out inaccurate substrate determinations as a possible explanation. The inhibition is reversible under high-salt conditions (16) and under the standard assay conditions presented here as well. The amount of inhibition observed is a function not only of the amount of inhibitor present, but also of the amount of catalase present.…”

Section: Resultsmentioning

confidence: 58%

“…Incubating the inhibitor with trypsin for shorter periods of time results in a partial loss of inhibition. The inhibitor is also heat-labile (16). These data indicate that it is a protein.…”

Section: Resultsmentioning

confidence: 73%

“…The maize inhibitor is not a protease. The activity of immobilized catalase, which is depressed by the addition of inhibitor, is fully restored by the 1 M NaCl wash (16 (Fig. 3) also is not consistent with a typical proteolytic curve.…”

Section: Resultsmentioning

confidence: 78%

“…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.…”

Section: Methodsmentioning

confidence: 99%

“…(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).…”

mentioning

confidence: 99%

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Biochemical Characterization of a Catalase Inhibitor from Maize

Sorenson¹,

Scandalios²

1980

Plant Physiol.

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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...

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Section: Resultsmentioning

confidence: 58%

Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 78%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Biochemical Characterization of a Catalase Inhibitor from Maize

Sorenson¹,

Scandalios²

1980

Plant Physiol.

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Evaluation of a catalase inhibitor in the developmental regulation of maize catalase

Tsaftaris

Sorenson

1979

Dev. Genet.

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A protein which has been shown to inhibit catalase in vitro appears to vary inversely with catalase activity in the maize scutellum during early sporophytic development when assayed using a catalase inhibition assay. This result suggested that the inhibitor protein may play a direct role in regulating catalase activity during this time period. Four experimental approaches were used to evaluate this putative regulatory role, including immunological quantitation of individual catalase isozymes during germination using rocket immunoelectrophoresis, perturbation of normal catalase expression with hydrogen peroxide or allylisopropylacetamide (AIA), examination of a mutant line with an altered catalase developmental program, and direct radioimmunoassay of the inhibitor protein during germination. The results of these experiments indicate that the quantitative changes in catalase activity during development are not mainly due to changes in the expression of the catalase inhibitor. Other possible roles of this protein in catalase regulation are discussed.

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