Purification, properties and subunit structure of arginase from Iris bulbs

Area of Science:

• Biochemistry
• Enzymology
• Plant Science

Background:

• Arginase (L-arginine amidinohydrolase, EC 3.5.3.1) plays a crucial role in the urea cycle and amino acid metabolism.
• Understanding plant arginases is important for insights into nitrogen metabolism and physiological processes in plants.

Purpose of the Study:

• To purify and characterize arginase from Iris hollandica bulbs.
• To investigate the enzyme's kinetic properties, cofactor requirements, and quaternary structure.
• To compare the structural characteristics of plant arginase with known arginases from other sources.

Main Methods:

• Purification using DEAE-Sephacel chromatography, aminohexyl-Sepharose 4B chromatography, and gel filtration.
• Enzyme activity assays under varying conditions (pH, Mn2+, dithiothreitol).
• Molecular weight determination using gel filtration and SDS-PAGE; structural analysis via glutaraldehyde cross-linking.

Main Results:

• Iris hollandica arginase was purified ~20,000-fold, showing high specificity for L-arginine.
• The enzyme requires Mn2+ and dithiothreitol for activity and stability, with optimal conditions at pH 9.0.
• The native enzyme is a hexamer (Mr ~191,000) that dissociates into dimers (Mr ~36,500) upon Mn2+ removal, a novel structural characteristic.

Conclusions:

• Iris hollandica arginase possesses a unique hexameric structure that is sensitive to Mn2+ concentration.
• The dissociation into dimers upon Mn2+ loss is a previously unreported feature for arginases.
• This study provides new insights into the structural diversity and regulation of arginase enzymes in plants.