Arginine decarboxylase is a component activity of the multifunctional.

A homogenous preparation of putrescine synthase, the versatile multifunctional enzyme involved in agmatine→putrescine conversion in Cucumis sativus was found to catalyze enzymatic decarboxylation of arginine also. Similarly, the purified arginine decarboxylase mediated the component as well as the complete set of coupled reactions harboured by putrescine synthase. Both the enzyme preparations exhibited identical electrophoretic and chromatographic behaviour and were immunologically indistinguishable. All the enzymic activities are stabilized concurrently by feeding arginine to the intact seedlings. Therefore, it is concluded that the multifunctional putrescine synthase in Cucumis sativus seedlings also harbours arginine decarboxylase activity unlike its counterpart in Lathyrus sativus.

Purification and characterization of putrescine synthase from cucumber seedlings. A multifunctional enzyme involved in putrescine biosynthesis.

The multifunctional enzyme, putrescine synthase has been purified from Cucumis sativus and characterized. This enzyme harbours agmatine iminohydrolase, ornithine transcarbamylase, putrescine transcarbamylase and carbamate kinase activities, whose concerted action results in agmatine → putrescine conversion. The enzyme resolved into two aggregation forms, enzyme aggregated and enzyme monomer upon electrophoresis at pH 8·3. Evidence has been provided by two-dimensional gel electrophoresis that both enzyme aggregated and enzyme monomer comprise of identical polypeptide chains. Under non-reducing conditions on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the protein moves as a single 150 KDa polypeptide; however, in the presence of 2-mercaptoethanol on sodium dodecyl sulphate-polyacrylamide gel elec trophoresis, it migrates as 3 polypeptides of molecular weight 48,000, 44,000 and 15,000. The enzyme undergoes age-dependent in vivo proteolytic degradation from a 66 KDa polypeptide (primary translational product), through 48 KDa polypeptide to 44 KDa species and finally to small molecular weight peptides.

Decarboxylation of arginine and ornithine by arginine decarboxylase purified from cucumber (Cucumis sativus) seedlings.

A purified preparation of arginine decarboxylase from Cucumis sativus seedlings displayed ornithine decarboxylase activity as well. The two decarboxylase activities associated with the single protein responded differentially to agmatine, putrescine and Pi. While agmatine was inhibitory (50 %) to arginine decarboxylase activity, ornithine decarboxylase activity was stimulated by about 3-fold by the guanido arnine. Agmatine-stimulation of ornithine decarboxylase activity was only observed at higher concentrations of the amine. Inorganic phosphate enhanced arginine decarboxylase activity (2-fold) but ornithine decarboxylase activity was largely uninfluenced. Although both arginine and ornithine decarboxylase activities were inhibited by putrescine, ornithine decarboxylase activity was profoundly curtailed even at 1 mM concentration of the diamine. The enzyme-activated irreversible inhibitor for mammalian ornithine decarboxylase, viz. α-difluoromethyl ornithine, dramatically enhanced arginine decarboxylase activity (3-4 fold), whereas ornithine decarboxylase activity was partially (50%) inhibited by this inhibitor. At substrate level concentrations, the decarboxylation of arginine was not influenced by ornithine and vice-versa. Preliminary evidence for the existence of a specific inhibitor of ornithine decarboxylase activity in the crude extracts of the plant is presented. The above results suggest that these two amino acids could be decarboxylated at two different catalytic sites on a single protein.

Purification and characterization of arginine decarboxylase from cucumber (Cucumis sativus) seedlings.

A simple, reproducible and rapid protocol for the purification of arginine decarboxylase from Cucumis sativus seedlings has been standardised. The purification steps involved ion-exchange chromatography on diethylaminoethyl-cellulose followed by gel filtration on Sephadex G-l 50. The purified enzyme preparation migrated as a single stainable band on Polyacrylamide gels at both basic and acidic pH, but under denaturing and reducing conditions on sodium dodecyl sulphate-polyacrylamide gels resolved into polypeptides of molecular weight 48,000,44,000 and 15,000. However, in the absence of 2-mercaptoethanol on electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, the enzyme moved as single band with a molecular weight of 150,000. Evidence was obtained to indicate that these three polypeptides were probably derived from a single larger molecular weight enzyme. On storage of the purified protein, the 48,000 species was preferentially degraded to smaller polypeptides. The preliminary data suggested that the 48,000 and 44,000 species shared many common tryptic peptides as revealed by finger printing of the [125I ]-labelled protein. The purified enzyme was a glycoprotein and had a Km of 0·5 mM for arginine. Its activity was stimulated by dithiothrietol and pyridoxal phosphate. EDTA did not inhibit the enzyme activity. Mn2+ at 1 mM stimulated arginine decarboxylase activity but was inhibitory at higher concentration.