Graduate Thesis Or Dissertation

 

Partial purification and characterization of a zeatin Cis-trans isomerase from Phaseolus vulgaris L. Public Deposited

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  • Conversion of exogenous cis-zeatin to trans-zeatin in immature seeds of Phaseolus vulgaris L. led to the isolation of a cis-trans isomerase from the endosperm. The enzyme was purified over 2000-fold by chromatography on a series of FPLC (anion exchange, gel filtration, and hydrophobic interaction) and Concanavalin A columns. Non-enzymatic isomerization occurred under standard assay conditions but the presence of the enzyme enhanced conversion significantly. The enzymatic reaction favors conversion from the cis to the trans form and requires flavin, blue light, and DTT. Optimal conditions for isomerization were identified using a Phenyl Superosepurified enzyme extract. Concentrations of 0.1-0.316 mM FAD and 0.5 mM DTT resulted in high conversion. The pH optimum for the reaction was 7.5. Although the isomerase was heat stable, a temperature of 35°C was chosen for the assay reaction in order to minimize non-enzymatic thermal isomerization. Retention on the Concanavalin A column as well as shifting in mobility of all visible protein bands on SDS-PAGE following N-glycosidase-F treatment indicated that the enzyme is a glycoprotein. The enzyme was stable for at least 8 weeks when stored at -80°C. The isomerase is capable of converting cis-zeatin riboside and trans-zeatin riboside. Changing the isoprenoid side chain location from N⁶ in zeatin to the 9-position of the adenine ring in 9-(4-hydroxy-3- methylbut-2-enyl)adenine also resulted in isomerization. The occurrence of cis-trans isomerization suggests that cis-zeatin and cis-zeatin riboside formed by tRNA degradation could be precursors of biologically active cytokinins.
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