Abstract
A particle-bound enzyme preparation from the fungus imperfectus Cryptococcus laurentii var. flavescens (NRRL Y-1401) catalyzes transfer of mannosyl units from GDP-mannose to mono- and oligosaccharide acceptors. Four separate enzymes can be differentiated by some or all of the following criteria: pH optimum, Km values for GDP-mannose, acceptor specificity, heat lability, metal ion requirement, and cross inhibition. These four enzymes catalyze the formation of four distinct mannosyl linkages, namely mannosyl-α-1,2-mannosyl (Enzyme I); mannosyl-α-1, 6-mannosyl (Enzyme II); mannosyl-α-1,3-mannosyl (Enzyme III); mannosyl-xylosyl (Enzyme IV). The occurrence of at least two of these linkages in cell wall heteropolysaccharides of C. laurentii suggests involvement of at least two, and possibly all four of these enzymes in cell wall biosynthesis of this organism.
Highlights
Analytical Methods-Protein was determined by the procedure of Lowry et al [11]
Radioactivity was measured in a gas flow counter (Nuclear-Chicago) or in a liquid scintillation counter (Packard) using Bray’s solution or toluene scintillation fluid (Spectrafluor, Amersham/Searle)
Paper chromatograms and electrophoretograms we1.e analyzed for radioactivity with a Nuclear-Chicago Actigraph III or by autoradiography
Summary
Protein was determined by the procedure of Lowry et al [11]. Radioactivity was measured in a gas flow counter (Nuclear-Chicago) or in a liquid scintillation counter (Packard) using Bray’s solution or toluene scintillation fluid (Spectrafluor, Amersham/Searle).Paper chromatograms and electrophoretograms we1.e analyzed for radioactivity with a Nuclear-Chicago Actigraph III or by autoradiography.Chromatography and Electrophoresis-I’apel, chsomatography was carried out on Whatman No I paper, either ascending or descending, The solvents used were [1] I-prolranol-ethyl acetate-water, 7 : 1: 2; [2] et,hyl ~Lcct,:Lte-pyritlinc-~~,at,cr, 8: 2 : I ;(3) ethyl acetate-acetic acid-water, 3: 1: 1. Elect’rophorcsis was carried out on Whattnan No I paper at 30 volt,s lrcr cm for 8 hours in 0.04 M sodium borate, pII 9.2. (:arbohydrates were detected with p-anisidine pht,h:tlatc or by silver nitrateacetone followed by alcoholic sodium hydroxide slimy.Enzyme Assay-Reaction mixtures contained GDP-mannoser4C (0.05 MCI; 151 m
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