Turnover of Genetically Defined Catalase Isozymes in Maize

Quail, Peter H.; Scandalios, John G.

doi:10.1073/pnas.68.7.1402

Cited by 59 publications

(34 citation statements)

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“…Thus, no measurable amount of heavy isotopes was incorporated into the ADH molecules within the first 36 hr of germination. Catalase in scutella of the same inbred (W64A) and at the same developmental period showed a density shift as large as 0.019 g/ml when seeds were germinated in the presence of K'NO3 and D20 under similar conditions as described above (8 Figure 2. LDH activity ( 0); ADH activity (Q---0).…”

Section: And Discussionmentioning

confidence: 80%

“…The procedure was essentially as previously described (4,8). Each 4-ml nitrocellulose tube contained 2 ml of saturated CsCl with NAD (3.5 mg/ml), 2 ml of crude enzyme extract, and 20 ,ug of lactate dehydrogenase (E.C.…”

Section: Methodsmentioning

confidence: 99%

“…In order to study the control mechanism(s) regulating the activity of alcohol dehydrogenase during and after germination, we have employed a density labeling technique, which is an effective means for detecting the de novo synthesis of protein molecules (1,4,8) period. We also found that the level of a specific inhibitor for ADH increases during maize seed germination.…”

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Regulation of Alcohol Dehydrogenases in Maize Scutellum during Germination

Ho¹,

Scandalios²

1975

Plant Physiol.

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The pattern of change in the activity of alcohol dehydrogenase in maize (Zea mays L.) scutellum during seed germination is not altered by 10 jug/ml cycloheximide or 50 ,tg/ml actinomycin D. The enzyme does not become density labeled when maize seeds are germinated in the presence of D20 and "NH4Cl, indicating that no new alcohol dehydrogenase molecules are synthesized after the onset of germination. However, the activity of an endogenous inhibitor for alcohol dehydrogenase is increased after germination. The increase of this inhibitor is concomitant with the decline of alcohol dehydrogenase activity, indicating that the activity of alcohol dehydrogenase during seed germination is controlled by the level of the inhibitor.A change in the activity of various enzymes is commonly observed during and after the onset of germination. The activity of those enzymes characteristic of maturing seeds and those required for metabolism in the dry seed will be decreased, while enzymes needed for the germination process will be increased in activity.Alcohol dehydrogenase (alcohol:NAD oxidoreductase, EC 1 . 1 .1 .1; abbreviated as ADH) appears as four distinct zones of activity (ADH-1, ADH-2, ADH-3, and ADH-4) on zymograms of scutellar extracts from mature kernels (10). The genetic basis for some of these isozymes has been determined (11). Physiologically, ADH in plant tissues is probably necessary for anaerobic glycolysis which may be of importance in the metabolism of the resting seed. However, after seed germination the increase in aerobic respiration may obviate the need for ADH.In order to study the control mechanism(s) regulating the activity of alcohol dehydrogenase during and after germination, we have employed a density labeling technique, which is an effective means for detecting the de novo synthesis of protein molecules (1,4,8), and have found that there is no de novo synthesis of ADH molecules during this developmental period. We also found that the level of a specific inhibitor for ADH increases during maize seed germination. These facts lead us to suggest that the structural genes for ADH are probably repressed in the scutellum by some mechanism before or during the onset of the germination process and that the decline of ADH activity is due to the increase of the ADH specific inhibitor.MATERIALS AND METHODS Preparation of Seeds. Maize (Zea mays L.) seeds, inbred line W64A, were surface-sterilized with 5% sodium hypochlorite for 10 min, washed with sterilized deionized H20, and germinated between moistened germination paper (Kimpak) in the dark at 24 C. In the density labeling experiments, the germination paper was replaced by two pieces of Whatman No. 1 filter paper moistened either with 10 mm '4NH4Cl in H20 as control, or with 10 mm 'NH4C1 in 70% D,O. For the drug inhibitor experiments, the scutella were excised under sterile conditions after 24 hr of germination and placed on filter paper moistened with Hoagland's solution. Extracts were prepared by homogenizing the scuteila with sand in a mortar and p...

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Section: And Discussionmentioning

confidence: 80%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Regulation of Alcohol Dehydrogenases in Maize Scutellum during Germination

Ho¹,

Scandalios²

1975

Plant Physiol.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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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“…Assuming that barley a-amylase has a half-life greater than 18h, these conditions were met. The third prerequisite is that turnover of the enzyme should not be faster than the rate of equilibration of the available amino acid pool with density marker (Quail & Scandalios, 1971). Otherwise labelling will be strictly a function of the state ofthe amino acid pool and will give no indication ofthe dynamics of the enzyme population.…”

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Phytochrome-induced synthesis of ribonuclease de novo in lupin hypocotyl sections

Acton

Schöpfer

1974

Biochemical Journal

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1. Density-labelling with 99 atoms % of 2H20 distinguished pre-existing from newly synthesized ribonuclease molecules in sections of developing hypocotyl tissue. 2. Activity profiles ofenzyme extracted from the fraction pelletable at 1000OOg showed heterogeneity after isopycnic centrifugation in CsCl gradients. 3. Measurement of density shifts of the entire heterogeneous band shows that ribonuclease protein is synthesized de novo in both continuous far-red light and darkness. 4. A twofold increase in enzyme activity after irradiation was accompanied by band-broadening and a significantly faster rate of labelling than in darkness. 5. The conclusion is drawn from the experimental evidence and theoretical arguments presented that phytochrome regulates the synthesis of new enzyme molecules against a background of continuous (dark-rate) synthesis and degradation. 6. Further information has been deposited as Supplementary Publication SUP 50033

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Turnover of Genetically Defined Catalase Isozymes in Maize

Cited by 59 publications

References 12 publications

Regulation of Alcohol Dehydrogenases in Maize Scutellum during Germination

Regulation of Alcohol Dehydrogenases in Maize Scutellum during Germination

Biochemical Characterization of a Catalase Inhibitor from Maize

Phytochrome-induced synthesis of ribonuclease de novo in lupin hypocotyl sections

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