Solubilization of UDPglucose-ceramide glucosyltransferase from the Golgi apparatus.
The choice of a suitable detergent for solubilization of UDPglucose-ceramide glucosyltransferase from Golgi membranes has been investigated. Among the various classes of detergent, CHAPS, a zwitterionic detergent, was used as it produced a substantial activation of the enzyme activity. 30% of the enzyme activity and 50% of proteins were solubilized in the first attempts. Further experiments were conducted with addition of a second detergent, Zwittergent 3-14 which increased enzyme recovery to 45%. Lastly, reducing the concentrations of buffer and divalent cations Mn2+, Mg2+ and introducing glycerol (20%, v/v) allowed 80% of proteins to be solubilized together with 68% of the ceramide glucosyltransferase activity.
Durieux
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Laboratoire de Biochimie des Membranes (LBTM-CNRS UM 380024), Université Claude Bernard Lyon I, Villeurbanne, France.
Martel
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MB
Got
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D000818:Animals; D002793:Cholic Acids; D004795:Enzyme Stability; D005724:Ganglia / Q000201:enzymology*; D005964:Glucosyltransferases / Q000302:isolation & purification*; D006056:Golgi Apparatus / Q000201:enzymology*; D012995:Solubility; D013552:Swine
D002793:Cholic Acids; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate