Chitin synthesis in Candida albicans
1) Membrane fractions synthesising chitin were isolated from homogenates of yeast and mycelial tissue of Candida albicans. The plasma membrane was the major chitin-synthesising membrane fraction. 2) Chitin synthase exists mainly as a zymogen, being proteolytically activated. 3) Chitin synthase was solubilised, preferentially as zymogen, from membrane preparations of both yeast and mycelial tissue, by homogenisation in 1% (w/v) cold digitonin buffer. 4) Kinetic studies on yeast and mycelial solubilised preparations yielded no significant differences. 5) Primer was not required for chitin synthesis. Divalent cations were required e.g, Mg2+, Mn2+.6) Optimum pH using Tris-Chloride buffer was 8.5. Optimum temperature using HEPES-NaOH buffer was 35°C.7) Chitin synthase exhibited allosterio enzyme kinetics. Both UDPNAG and NAG were allosteric activators. 8) Addition of 20 mU NAG to assays at 0.1 mM substrate concentration stimulated activity up to 200 times; less so in the presence of higher substrate concentrations, 9) Addition of 20 mM NAG to the assay nearly abolished co-operative enzyme kinetics. 10) Chitin synthase was competitively inhibited by UDP (K~, 0.2 mm) and polyoxin D (0.75ju M). 11) UDPase appeared to be present in the digitonin solubilised preparations. 12) No chitinase and very little protease activity was detected in the solubilised preparations. 13) Chitin synthase aggregated extensively in the absence of 0.1% (w/v) digitonin during continuous sucrose gradient centrifugation and column chronatography. 14) Both Ultrogel AcA34 and Sophacryl 8300 gel filtration media afforded useful purification steps. 15) Polyacrylamide gel electrophoresis of solubilised preparations yielded 20-E5 separate protein fractions under non-dissociating conditions and 30 plus fractions under dissociating (SDS) conditions. 16) Three fractions upon a non-dissociating polyacrylamide gel (R 0.38, 0.66, 0.88) yielded chitin c-nthase activity. 17) It would appear that chitosomes exist in C. albicans yeast and mycelial tissue.