Arginine decarboxylase from Lathyrus sativus seedlings. Purification and properites.
Arginine decarboxylase which makes its appearance in Lathyrus sativus seedlings after 24 h of seed germination reaches its highest level around 5-7 days, the cotyledons containing about 60% of the total activity in the seedlings at day 5. The cytosol enzyme was purified 977-fold from whole seedlings by steps involving manganese chloride treatment, ammonium sulphate and acetone fractionations, positive adsorption on alumina C-gamma gel, DEAE-Sephadex chromatography followed by preparative disc gel electrophoresis. The enzyme was shown to be homogeneous by electrophoretic and immunological criteria, had a molecular weight of 220,000 and appears to be a hexamer with identical subunits. The optimal pH and temperature for the enzyme activity were 8.5 and 45 degrees C respectively. The enzyme follows typical Michaelis-Menten kinetics with a Km value of 1.73 mM for arginine. Though Mn2+ at lower concentrations stimulated the enzyme activity, there was no dependence of the enzyme on any metal for the activity. The arginine decarboxylase of L. sativus is a sulfhydryl enzyme. The data on co-factor requirement, inhibition by carbonyl reagents, reducing agents and pyridoxal phosphate inhibitors, and a partial reversal by pyridoxal phosphate of inhibition by pyridoxal-HCl suggests that pyridoxal 5'-phosphate is involved as a co-factor for the enzyme. The enzyme activity was inhibited competitively by various amines including the product agmatine. Highest inhibition was obtained with spermine and arcain. The substrate analogue, L-canavanine, homologue L-homoarginine and other basic amino acids like L-lysine and L-ornithine inhibited the enzyme activity competitively, homoarginine being the most effective in this respect.
D000596:Amino Acids; D002262:Carboxy-Lyases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007700:Kinetics; D046911:Macromolecular Substances; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D063245:Plant Development; D010944:Plants / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature
D000596:Amino Acids; D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D008345:Manganese; D011732:Pyridoxal Phosphate; D002262:Carboxy-Lyases