Solubilization and partial purification of yeast chitin synthetase. Confirmation of the zymogenic nature of the enzyme.
Chitin synthetase was solubilized with digitonin from a particulate yeast fraction. The solubilized enzyme, which did not sediment at 200,000 X g and emerged after the void volume in a Sepharose 6B column, was active only after treatment with a protease. This confirms that chitin synthetase exists in the plasma membrane as a zymogen and that initiation of the chitin septum occurs by localized activation of the enzyme. By differential extraction with sodium cholate and digitonin, followed by chromatography on Sepharose 6B, a 20-fold purification of the enzyme was achieved with respect to the crude particles. The purified enzyme showed a requirement for a phospholipid; phosphatidylserine and lysophosphatidylserine were the best activators. Unsaturated fatty acids strongly inhibited synthetase activity, whereas their saturated counterparts were inert. The solubilized enzyme catalyzed the formation of insoluble chitin in the absence of added primer. The synthetic polysaccharide was examined by electron microscopy and found to consist of lozenge-shaped particles about 60 nm long and 10 nm wide.
D002687:Chitin Synthase / Q000302:isolation & purification* / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000302:isolation & purification* / Q000378:metabolism; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology; D006602:Hexosyltransferases / Q000302:isolation & purification*; D007700:Kinetics; D010743:Phospholipids / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014357:Trypsin / Q000494:pharmacology
D004792:Enzyme Precursors; D005230:Fatty Acids, Nonesterified; D010743:Phospholipids; D006602:Hexosyltransferases; D002687:Chitin Synthase; D014357:Trypsin; D004072:Digitonin