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High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)
Published on: June 16, 2018
Limited proteolysis of maize NADP-malic enzyme
S Pinto1, S R Rao, A S Bhagwat
1Molecular Biology and Agricultural Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
Indian Journal of Biochemistry & Biophysics
|August 22, 2012
Summary
Maize malic enzyme activity was rapidly inactivated by trypsin, which cleaved the enzyme into smaller fragments. Enzyme substrates like NADP protected against this inactivation, suggesting a specific interaction site.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Maize malic enzyme is a crucial enzyme in plant metabolism.
- Understanding its regulation by proteolysis is important for metabolic studies.
Purpose of the Study:
- To investigate the effect of limited proteolysis on maize malic enzyme activity and structure.
- To identify proteases that can inactivate the enzyme and characterize the modified enzyme's properties.
Main Methods:
- Incubation of maize malic enzyme with trypsin and other endoproteases.
- Analysis of enzyme activity and fragmentation using SDS-PAGE.
- Kinetic analysis (Km values) and fluorescence spectroscopy of the modified enzyme.
Main Results:
- Trypsin rapidly inactivated maize malic enzyme by cleaving it into 40 kDa and 20 kDa fragments.
- Substrates, particularly NADP, protected the enzyme from inactivation.
- Endoprotease Lys-C was also effective in inactivating the enzyme.
- Limited proteolysis did not significantly alter malate Km but slightly increased NADP Km, indicating reduced affinity for NADP.
- Fluorescence properties and NADPH binding remained unaffected.
Conclusions:
- Limited proteolysis, especially by trypsin and Lys-C, significantly impacts maize malic enzyme activity.
- The NADP binding site appears to be involved in the protective effect against proteolysis.
- The enzyme's catalytic and coenzyme binding properties are altered, but not completely abolished, after proteolysis.
