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| Abstract or Summary |
- 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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| Digitization Specifications |
- File scanned at 300 ppi (Monochrome, 256 Grayscale) using Scamax Scan+ V.1.0.32.10766 on a Scanmax 412CD by InoTec in PDF format. LuraDocument PDF Compressor V.5.8.71.50
used for pdf compression and textual OCR.
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Previous issue date: 1994-07-25
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