DISCRIMINATION BETWEEN CHITINASE AND CHITOBIASE ACTIVITIES IN CANDIDA-ALBICANS

Measurement of chitinase activity in crude homogenates of Candida albi cans with the artificial substrates 4-methylumbelliferyl-beta-D-N,N',N '' triacetylchitotrioside and 4-methylumbelliferyl-beta-D-N, N'-diace tylchitobioside (MU-[GlcNAc](3,2)) showed that both chitobiase and chi tinase were involved in the liberation of the fluorophore methylumbell iferone (MU). The substrate MU-[GlcNAc](3) appeared to be digested by means of two separate mechanisms, resulting in a lag-phase in the time curve of the MU-release, and thus making this release non-linear with time. The first mechanism is the action of an endochitinase that rele ased MU in one step; this reaction could be inhibited by allosamidin. The second probable mechanism involves the digestion of the substrate by the stepwise release of the outer two GlcNAc residues by a chitobia se, followed either by a third chitobiase-catalyzed release of the fin al GlcNAc or by the action of a beta-N-acetylhexosaminidase, that is a lso present in C. albicans, Hydrolysis of the substrate MU-[GlcNAc](2) was not inhibited by allosamidin, suggesting that the two GlcNAc resi dues in this substrate are probably released in two steps, either by t he action of chitobiase alone, or by the successive action of chitobia se (step 1) and beta-N-acetylhexosaminidase (step 2). Our results show that the activities of the individual enzymes involved in chitin degr adation cannot be determined on the basis of MU release in unfractiona ted homogenates of C. albicans.