Detection of protease activities using specific aminoacyl or peptidyl
            <i>p</i>
            ‐nitroanilides after sodium dodecyl sulfate — polyacrylamide gel electrophoresis and its applications

Hou, Wen Chi; Chen, Hsien-Jung; Chen, Tzeng E.; Lin, Yaw Huei

doi:10.1002/(sici)1522-2683(19990301)20:3<486::aid-elps486>3.0.co;2-#

Cited by 10 publications

(1 citation statement)

References 26 publications

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“…In plant tissues, specific proteases are involved in the mobilization of reserve proteins, developmental processes and senescence (Hou et al 1999). In this study, we found that increasing Cd concentrations had a negative effect on protease activity.…”

Section: Discussionmentioning

confidence: 54%

Cadmium stress affects seed germination and seedling growth in Sorghum bicolor (L.) Moench by changing the activities of hydrolyzing enzymes

2007

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Seed germination, one of the most important phases in the life cycle of a plant, is highly responsive to existing environment. Hydrolyzing enzymes play a major role in the mobilization of food reserves by hydrolyzing carbohydrates, proteins and fats. This paper reports on the effect of Cd toxicity on seed germination and the activities of hydrolyzing enzymes, like acid phosphatases (ACPs), proteases and a-amylases in Sorghum bicolor (L.) Moench. The metal uptake by embryonic axes and seeds was quantified. We found that sorghum could tolerate up to 0.5 mM Cd. At concentrations above 3.0 mM, seed germination was adversely affected with a complete cessation of seedling growth. All investigated hydrolyzing enzymes exhibited a significant decrease in activity with increasing Cd concentrations. The isozyme profiles indicated the loss of one or two isozymes of ACP, induction of a new isozyme for total protease (at 3.0 mM Cd) and a decline in the intensity of aamylase isozymes. SEM studies revealed that Cd affected a change in root hair density. SEM investigations also confirmed the assay results of the inhibition of starch mobilization from endosperm. This suggested an inhibition of the hydrolysis of reserve carbohydrates and translocation of hydrolyzed sugars, ultimately resulting in decreased germination and disruption of seedling growth. Because sorghum is an important dryland crop, its response to the presence of Cd in agro-ecosystems and Cd-induced phytotoxicity during seed germination and seedling growth needs critical investigation.

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Section: Discussionmentioning

confidence: 54%

Cadmium stress affects seed germination and seedling growth in Sorghum bicolor (L.) Moench by changing the activities of hydrolyzing enzymes

2007

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Activity staining of isocitrate lyase after electrophoresis on either native or sodium dodecyl sulfate polyacrylamide gels

et al. 2001

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Isocitrate was cleaved into succinate and glyoxylate by isocitrate lyase (ICL) in the glyoxylate cycle. We used lactate dehydrogenase as an ancillary enzyme to further metabolize the glyoxylate to glycolate in the presence of NADH. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2,6-dichlorophenol-indolphenol (DCPIP) were used in the coupling reactions for detecting ICL activity after electrophoresis on either native or sodium dodecyl sulfate (SDS) polyacrylamide gels. This fast and sensitive method can be used in the process of ICL enzyme purification and characterization.

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Activity staining of glutathione peroxidase after electrophoresis on native and sodium dodecyl sulfate polyacrylamide gels

2002

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Glutathione peroxidase (GSH-Px), from commercial bovine erythrocytes or ammonium sulfate fractionations (30-45%, 45-60%, 60-75% and 75-90% saturations) of ginger rhizome, was detected on polyacrylamide gels after native polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate (SDS)-PAGE. The gel was submerged in a 50 mM Tris-HCl buffer (pH 7.9) containing 13 mM glutathione and 0.004% hydrogen peroxide with gentle shaking for 10-20 min. The GSH-Px activity was stained with a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 10 min. The clear zone of GSH-Px activity on a purple background was found in both native and SDS-PAGE gels. This fast and sensitive method can be used in the process of enzyme purification and characterization of mammalian or plant cells.

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Detection of protease activities using specific aminoacyl or peptidyl p ‐nitroanilides after sodium dodecyl sulfate — polyacrylamide gel electrophoresis and its applications

Cited by 10 publications

References 26 publications

Cadmium stress affects seed germination and seedling growth in Sorghum bicolor (L.) Moench by changing the activities of hydrolyzing enzymes

Cadmium stress affects seed germination and seedling growth in Sorghum bicolor (L.) Moench by changing the activities of hydrolyzing enzymes

Activity staining of isocitrate lyase after electrophoresis on either native or sodium dodecyl sulfate polyacrylamide gels

Activity staining of glutathione peroxidase after electrophoresis on native and sodium dodecyl sulfate polyacrylamide gels

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