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Purification of UDP-galactose: diacylglycerol galactosyltransferase from chloroplast envelopes of spinach (Spinacia oleracea L.)

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Abstract

Uridine 5′-diphosphate(UDP)-galactose: 1,2-diacylglycerol 3-O-β-d-galactopyranosyltransferase (EC 2.4.1.46) is an integral protein of chloroplast envelope membranes from which it has been partially purified (Covès et al., 1986, FEBS Lett. 208, 401–406). We have worked out a purification procedure which after removal of peripheral membrane proteins, solubilization and two chromotographic steps allowed us to identify a 22-kDa protein as the galactosyltransferase. Enrichment of enzymatic activity was paralleled by an enrichment of this protein and its radioactive derivative obtained by photoaffinity labelling with [α-−32P]UDP which is a potent inhibitor of the enzyme. The purification factor of about 350 is substantially higher than achieved previously and indicates that the enzyme represents less than 0.3% of the envelope proteins. The purified enzyme has a Km of 87 μM for UDP-galactose with dioleoylglycerol as acceptor and could not be activated by addition of other lipids.

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Abbreviations

CHAPS:

3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate

DTE:

dithioerythritol

MGD:

monogalactosyl diacylglycerol

PMSF:

phenylmethanesulfonyl fluoride

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Authors and Affiliations

  1. Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, W-2000, Hamburg 52, Germany

    T. Teucher & E. Heinz

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  1. T. Teucher
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  2. E. Heinz
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This work was supported by the Deutsche Forschungsgemeinschaft.

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Teucher, T., Heinz, E. Purification of UDP-galactose: diacylglycerol galactosyltransferase from chloroplast envelopes of spinach (Spinacia oleracea L.). Planta 184, 319–326 (1991). https://doi.org/10.1007/BF00195332

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  • DOI: https://doi.org/10.1007/BF00195332

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