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Cottonseed malate synthase : purification and immunochemical characterization
R N Trelease1, C A Hermerath, R B Turley
1Department of Botany and Microbiology, Arizona State University, Tempe, Arizona 85287.
Plant Physiology
|August 1, 1987
Summary
Malate synthase, an enzyme in the glyoxylate cycle, was purified from cotton seedlings. This study characterized its structure and presence in different cellular compartments and seed developmental stages.
Area of Science:
- Plant Biochemistry
- Enzymology
- Molecular Biology
Background:
- The glyoxylate cycle is crucial for plant metabolism, particularly in oilseed germination.
- Malate synthase (EC 4.1.3.2) is a key enzyme in this cycle, but its purification and characterization have been challenging.
- Understanding malate synthase is vital for comprehending carbon flux during seed development.
Purpose of the Study:
- To purify and characterize malate synthase from cotton (Gossypium hirsutum L.) seedlings.
- To investigate the enzyme's quaternary structure and subcellular localization.
- To examine the enzyme's presence and potential modifications during seed development.
Main Methods:
- Enzyme purification using a six-step protocol without large-scale glyoxysome isolation.
- Assessment of enzyme homogeneity via SDS-PAGE and silver staining.
- Determination of native and subunit molecular weights using sucrose gradient sedimentation and SDS-PAGE.
- Antibody production and characterization (immunotitration, double immunodiffusion, Western blotting) for immunological analysis.
- Cross-reactivity studies with other oilseed extracts.
Main Results:
- Malate synthase was purified to homogeneity from 72-hour dark-grown cotton seedlings.
- The purified enzyme exists as a dodecamer (750,000 MW) in glyoxysomes, with identical subunits (~63,000 MW).
- A monomeric form (5S) was identified in the cytosol, showing partial immunological identity with the glyoxysomal form.
- Antibodies raised against malate synthase were monospecific and cross-reacted with other oilseed extracts.
- Malate synthase protein was detected in immature, desiccated, and germinated seeds, with consistent subunit molecular weight.
Conclusions:
- A robust purification method for malate synthase was developed, bypassing glyoxysome isolation.
- Cotton malate synthase exhibits distinct cytosolic and glyoxysomal forms with different quaternary structures.
- The enzyme is immunologically conserved across several oilseed species.
- Malate synthase protein is present throughout cotton seed development, correlating with enzyme activity.