Purification and properties of a glycoprotein acid phosphatase from Candida albicans.
An acid phosphomonoesterase was purified 87-fold with a 4% recovery from disintegrated cells of Candida albicans by four stages of column chromatography. The purified enzyme was homogeneous by ultracentrifugal, electrophoretic, and immunological analyses. The fully corrected sedimentation coefficient, s(20,w), was calculated to be 5.51s. Molecular weight estimated from ultracentrifugal data was 124.3 x 10(3), from gel chromatography was 115 x 10(3), and from acrylamide gel electrophoretic data was 131 x 10(3). Buoyant density in sucrose was 1.15 g/cm(3). The enzyme was a mannoprotein with a hexose to protein ratio of 7: 1. The Michaelis constant of the enzyme was 3.3 x 10(-4) M for p-nitrophenyl phosphate as substrate, and the pH optimum was 4.5. The enzyme was competitively inhibited by inorganic phosphate (K(i) = 10(-4) M) and by arsenate (K(i) = 0.5 x 10(-4) M). A wide range of inorganic cations and anions did not affect enzyme activity, but Hg(2+), Cd(2+), and Cu(2+) were inhibitory. F(-) was also inhibitory at low concentrations, but the effect was reversed at higher concentrations. Phosphatase activity was completely destroyed by exposure of the enzyme to 70 C for 12 min, but was destroyed only slowly by proteolytic hydrolysis. The purified glycoprotein enzyme gave a line of identity with the "b" antigen of crude C. albicans homogenates in immunodiffusion and immunoelectrophoresis tests with sera from rabbits inoculated with intact C. albicans cells and from humans with proven candidiasis. Preliminary evidence suggests that the mannan and not the protein portion of the enzyme molecule is responsible for this antigenicity.
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D002176:Candida albicans / Q000201:enzymology* / Q000254:growth & development / Q000276:immunology; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D006023:Glycoproteins; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D008722:Methods; D008970:Molecular Weight; D010710:Phosphates / Q000494:pharmacology; D011817:Rabbits / Q000276:immunology; D012964:Sodium / Q000494:pharmacology; D013696:Temperature; D014461:Ultracentrifugation
D006023:Glycoproteins; D007106:Immune Sera; D010710:Phosphates; D012964:Sodium; D000135:Acid Phosphatase