Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 16 Dec 2018 19:29:07 423 The Release of Acid Phosphatase and Polysaccharide- and Pro tein-containing Components from the Surface of the Dimorphic Forms of Candida albicans by Treatment with Dithiothreitol By F. W. CHATTAWAY AND S. SHENOLIKAR Department oj Bioclienii,stry, University of Leech, Leeds LS2 9LS AND A. J. E. BARLOW Depar ti I I tw t of Derrna t o logy, The Roy a1 InJirn iary , HuddersJield, Yo rksh ir e (Receii7ed 8 April I 974) INTRODUCTION It is well established that in the preparation of protoplasts from yeasts by enzymic treat- ment, a sulphydryl reagent accelerates the process by reducing disulphide bonds in proteins on the cell surface (Davies & Elvin, 1964; Kidby & Davies, 1970). Acid phosphatase is released from Saccharoniq'ces rnellis by exposure to 2-mercaptoethanol (Weimberg, I 97 I), and invertase from S. carlsbergensis and S. cereiisiae by the action of dithiothreitol (Som- mer & Lewis, 1971). In the course of work on the nature of components released from the surface of both blastospore and mycelial forms of Candida albicans during protoplast for- mation by enzyme digestion it was found that a number of products containing saccharide and peptide material, and including 'acid phosphatase, were released by exposure of the cells to dithiothreitol. MET 13 0 D S The organism used was Candida albicans, ca I. It was grown on medium C, and blasto- spores or mycelium were harvested as described by Chattaway, Holmes & Barlow (1968). Cells (10 mg dry wt for 4 h cells and 30 mg dry wt for 18 h cells) were suspended in mannitol (800 mM)-tris-HCI (50 mM) buffer, pH 7.5; when required, dithiothreitol (12 mM, BDH) was added. The suspension was incubated under nitrogen for 24 h at 37 "C and then centrifuged at 8oog. The supernatant was freeze-dried and the residue (I g/ro ml) redissolved in tris-HC1 buffer (50 mM), p H 7.5 and then fractionated on a Sephadex G-75 column with elution by the same buffer. Fractions were monitored for protein content by measurement of for carbohydrate by the phenol-sulphuric acid method of Dubois et a/. (1956)~ and for acid phosphatase according to Linhardt & Walter (1963) (specific activity as pmol p-nitrophenollhlmg protein). lsoelectric focusing was carried out at 4 "C with an LKB 440 ml I.E.F. column using standard sucrose density gradient with I ampholine (pH range 3 to 10) at 300 V. Eluates were monitored as above. Protein was determined from extinction measurements at 260 and 280 nm. RESULTS AND DISCUSSION Blastospores and mycelium grown for 4 and 18 h were treated with dithiothreitol and the qualitative pattern of material released was the same in all cases. Figure I shows its frac- tionation on Sephadex G-75. There was a carbohydrate-rich fraction (Fig. I: I and 3)