Paper and thin-layer chromatography

Zweig, Gunter; Sherma, Joseph

doi:10.1021/ac50028a006

Cited by 12 publications

(1 citation statement)

References 617 publications

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“…The plate was developed in a sealed tank containing 1-propano1:ethyl acetate:water in the ratio of 7:1:2 (v/v/v) for 4 h to overnight, air dried, and redeveloped in the same tank for another 4 h. An analytic imaging scanner (System 200, Bioscan, Inc., Washington, DC) was used to identify and quantify labeled galactinol. The identity of galactinol was established by running authentic galactinol (courtesy of Dr. D. Pharr) on TLC and staining for sugar with the silver nitrate-alkaline method (Zweig and Sherma, 1972).…”

Section: Enzyme Assaymentioning

confidence: 99%

Galactinol Synthase from Kidney Bean Cotyledon and Zucchini Leaf (Purification and N-Terminal Sequences)

1995

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Calactinol synthase (CS) was purified 1591-fold with a 3.9% recovery from the cotyledon of kidney bean (Phaseolus vulgaris) by a nove1 scheme consisting of ammonium sulfate fractionation followed by diethylaminoethyl, Affi-Cel Blue, and UDP-hexanolamine affinity chromatography. The purified enzyme had a specific activity of 8.75 pmol mg-' min-', a p H optimum of 7.0, and requirements for manganese ion and DTT. The enzyme exhibited a K,,, = 0.4 mM for UDP-galactose and a K,,, = 4.5 mM for myo-inositol. It was identified as a 38-kD peptide that co-purified with a 41 -and a 43-kD peptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PACE). Purification t o homogeneity was achieved by isolating the 38-kD peptide from the SDS-PACE gel. To clarify conflicting reports in the literature about the relative molecular mass of purified CS from zucchini leaf (Cucurbita pepo), a similar scheme with modified eluting conditions was used to purify CS from this source. Zucchini leaf CS was purified t o homogeneity and identified as a 36-kD peptide on SDS-PACE. Partial N-terminal sequences of the 38-kD peptide from kidney bean cotyledon and the 36-kD peptide from zucchini leaf were obtained. To facilitate identification of CS during the purification, an assay utilizing thin-layer chromatography and an isotopic analytic imaging scanner was developed.

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Section: Enzyme Assaymentioning

confidence: 99%