scholarly journals Purification and properties of cytochrome P-450-dependent 14 α-sterol demethylase from Candida albicans

1989 ◽  
Vol 263 (2) ◽  
pp. 573-579 ◽  
Author(s):  
C A Hitchcock ◽  
K Dickinson ◽  
S B Brown ◽  
E G V Evans ◽  
D J Adams
Keyword(s):  
Candida Albicans ◽  
Polyacrylamide Gel Electrophoresis ◽  
Membrane System ◽  
Sodium Cholate ◽  
Blue Staining ◽  
Purification And Properties ◽  
Opportunistic Fungal Pathogen ◽  
High Degree ◽  
Cytochrome P 450 ◽  
Coomassie Brilliant

The purification of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) from the important opportunistic fungal pathogen, Candida albicans, is described. Optimal purification (875-fold) was achieved by extracting the cytochrome from microsomes with sodium cholate followed by hydroxyapatite, octyl-Sepharose and CM-Sepharose chromatographies, giving a cytochrome preparation of 17.5 nmol/mg of protein. By the use of SDS/polyacrylamide-gel electrophoresis the cytochrome was judged to be highly purified on the basis of Coomassie Brilliant Blue staining of protein. The Mr of P-450DM was estimated to be 51,000. The absorption spectrum of oxidized P-450DM was characteristic of a low-spin cytochrome, and its reduced CO complex had a Soret absorption peak at 447 nm. When reconstituted in a model membrane system of dilauroylphosphatidylcholine with NADPH and O2, P-450DM catalysed the complete 14 alpha-demethylation of lanosterol, which was inhibited by CO. The cytochrome appeared to have a high degree of substrate specificity; it was unable to oxidize a number of xenobiotic compounds in the reconstituted assay.

scholarly journals Cytochrome P-450-dependent 14 α-demethylation of lanosterol in Candida albicans

1989 ◽  
Vol 260 (2) ◽  
pp. 549-556 ◽  
Author(s):  
C A Hitchcock ◽  
S B Brown ◽  
E G V Evans ◽  
D J Adams
Keyword(s):  
Enzyme Activity ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Reciprocal Relationship ◽  
Sterol Biosynthesis ◽  
Assay System ◽  
Opportunistic Fungal Pathogen ◽  
Cytochrome P 450

A novel assay for cytochrome P-450-dependent 14 alpha-sterol demethylase of the important opportunistic fungal pathogen, Candida albicans, is described. The enzyme was assayed in microsomal preparations (microsomes) by measuring the incorporation of [14C]lanosterol into (4,14)-desmethylated sterols. The efficacy of different cell-breakage methods was compared; desmethylated-sterol biosynthesis was maximal when cells were broken with a Braun disintegrator. The solubilization of [14C]lanosterol with detergent in the assay system was essential for enzyme activity, which was enhanced considerably when microsomes were gassed with O2. Under these conditions, there was a reciprocal relationship between the amount of radioactivity incorporated into desmethylated sterols and that lost from lanosterol. The major radiolabelled desmethylated sterol was ergosterol. The enzyme had an apparent Km of 52.73 +/- 2.80 microM and an apparent Vmax of 0.84 +/- 0.14 nmol/min per mg of protein (n = 3). Enzyme activity was decreased greatly when microsomes were treated with CO or the triazole antifungal ICI 153066.

scholarly journals The purification and properties of yeast proteinase B from Candida albicans

1986 ◽  
Vol 236 (1) ◽  
pp. 177-184 ◽  
Author(s):  
P C Farley ◽  
M G Shepherd ◽  
P A Sullivan
Keyword(s):  
Candida Albicans ◽  
Polyacrylamide Gel Electrophoresis ◽  
Serine Proteinase ◽  
Aromatic Amino Acids ◽  
Peptide Bond ◽  
Amino Acid Ester ◽  
Heat Stable ◽  
Purification And Properties ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.

scholarly journals Identification of vitronectin as a major plasma protein adsorbed on polymer surfaces of different copolymer composition

Blood ◽  
1989 ◽  
Vol 74 (8) ◽  
pp. 2698-2706 ◽  
Author(s):  
MD Bale ◽  
LA Wohlfahrt ◽  
DF Mosher ◽  
B Tomasini ◽  
RC Sutton
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Polymer Surfaces ◽  
Blue Staining ◽  
Enzyme Linked Immunosorbent Assay ◽  
Copolymer Composition ◽  
Major Protein ◽  
Protein Layer ◽  
Adsorbed Protein ◽  
Coomassie Brilliant Blue Staining ◽  
Coomassie Brilliant

The arrays of proteins adsorbed from plasma onto a series of polystyrene copolymeric latexes were analyzed by enzyme-linked immunosorbent assay (ELISA) of washed beads and immunoblotting of proteins desorbed from the beads and separated by polyacrylamide gel electrophoresis (PAGE). Beads were prepared by continuous emulsion polymerization in the absence of surfactant. Coomassie brilliant blue staining of gel electropherograms of desorbed proteins indicated that the presence of small amounts of comonomers (1 to 10 mole %) significantly influenced the composition of the adsorbed protein layer. Immunoblotting revealed that fibrinogen, fibronectin, and vitronectin were adsorbed by all surfaces investigated. C3 and Clq adsorption varied significantly with copolymer composition. The ELISAs revealed that although the concentrations of vitronectin and fibronectin in plasma are similar, the extent of vitronectin adsorption from 70% to 85% plasma was greater by two orders of magnitude than fibronectin adsorption. Vitronectin adsorbed on carboxylic acid-containing copolymers reacted more strongly with a conformationally sensitive antivitronectin monoclonal antibody (MoAb) than vitronectin adsorbed to polystyrene and was more susceptible to cleavage by plasma proteases(s). The results show that vitronectin is a major protein adsorbed from concentrated plasma and that small changes in the chemical composition of a copolymer profoundly affects the extent and nature of protein adsorption from complex mixtures such as plasma.

scholarly journals Identification of vitronectin as a major plasma protein adsorbed on polymer surfaces of different copolymer composition

Blood ◽  
1989 ◽  
Vol 74 (8) ◽  
pp. 2698-2706 ◽  
Author(s):  
MD Bale ◽  
LA Wohlfahrt ◽  
DF Mosher ◽  
B Tomasini ◽  
RC Sutton
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Polymer Surfaces ◽  
Blue Staining ◽  
Enzyme Linked Immunosorbent Assay ◽  
Copolymer Composition ◽  
Major Protein ◽  
Protein Layer ◽  
Adsorbed Protein ◽  
Coomassie Brilliant Blue Staining ◽  
Coomassie Brilliant

Abstract The arrays of proteins adsorbed from plasma onto a series of polystyrene copolymeric latexes were analyzed by enzyme-linked immunosorbent assay (ELISA) of washed beads and immunoblotting of proteins desorbed from the beads and separated by polyacrylamide gel electrophoresis (PAGE). Beads were prepared by continuous emulsion polymerization in the absence of surfactant. Coomassie brilliant blue staining of gel electropherograms of desorbed proteins indicated that the presence of small amounts of comonomers (1 to 10 mole %) significantly influenced the composition of the adsorbed protein layer. Immunoblotting revealed that fibrinogen, fibronectin, and vitronectin were adsorbed by all surfaces investigated. C3 and Clq adsorption varied significantly with copolymer composition. The ELISAs revealed that although the concentrations of vitronectin and fibronectin in plasma are similar, the extent of vitronectin adsorption from 70% to 85% plasma was greater by two orders of magnitude than fibronectin adsorption. Vitronectin adsorbed on carboxylic acid-containing copolymers reacted more strongly with a conformationally sensitive antivitronectin monoclonal antibody (MoAb) than vitronectin adsorbed to polystyrene and was more susceptible to cleavage by plasma proteases(s). The results show that vitronectin is a major protein adsorbed from concentrated plasma and that small changes in the chemical composition of a copolymer profoundly affects the extent and nature of protein adsorption from complex mixtures such as plasma.

scholarly journals Copper Availability Influences the Transcriptomic Response of Candida albicans to Fluconazole Stress

2021 ◽  
Vol 11 (4) ◽  
Author(s):  
Elizabeth W Hunsaker ◽  
Chen-Hsin Albert Yu ◽  
Katherine J Franz
Keyword(s):  
Candida Albicans ◽  
Time Course ◽  
Transcriptional Response ◽  
Metal Homeostasis ◽  
Metal Availability ◽  
Drug Induced ◽  
Transcriptomic Response ◽  
Opportunistic Fungal Pathogen ◽  
Whole Transcriptome Analysis ◽  
Whole Transcriptome

Abstract The ability of pathogens to maintain homeostatic levels of essential biometals is known to be important for survival and virulence in a host, which itself regulates metal availability as part of its response to infection. Given this importance of metal homeostasis, we sought to address how the availability of copper in particular impacts the response of the opportunistic fungal pathogen Candida albicans to treatment with the antifungal drug fluconazole. The present study reports whole transcriptome analysis via time-course RNA-seq of C. albicans cells exposed to fluconazole with and without 10 µM supplemental CuSO4 added to the growth medium. The results show widespread impacts of small changes in Cu availability on the transcriptional response of C. albicans to fluconazole. Of the 2359 genes that were differentially expressed under conditions of cotreatment, 50% were found to be driven uniquely by exposure to both Cu and fluconazole. The breadth of metabolic processes that were affected by cotreatment illuminates a fundamental intersectionality between Cu metabolism and fungal response to drug stress. More generally, these results show that seemingly minor fluctuations in Cu availability are sufficient to shift cells’ transcriptional response to drug stress. Ultimately, the findings may inform the development of new strategies that capitalize on drug-induced vulnerabilities in metal homeostasis pathways.

scholarly journals CAP1, an Adenylate Cyclase-Associated Protein Gene, Regulates Bud-Hypha Transitions, Filamentous Growth, and Cyclic AMP Levels and Is Required for Virulence of Candida albicans

2001 ◽  
Vol 183 (10) ◽  
pp. 3211-3223 ◽  
Author(s):  
Yong-Sun Bahn ◽  
Paula Sundstrom
Keyword(s):  
Candida Albicans ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Germ Tube ◽  
Filamentous Growth ◽  
Tube Formation ◽  
Camp Signaling ◽  
Solid Media ◽  
Opportunistic Fungal Pathogen ◽  
Camp Levels

ABSTRACT In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains withCAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in thecap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles ofCAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficientcap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.

scholarly journals Decrease in hepatic cytochrome P-450 by cobalt. Evidence for a role of cobalt protoporphyrin

1982 ◽  
Vol 204 (1) ◽  
pp. 103-109 ◽  
Author(s):  
J F Sinclair ◽  
P R Sinclair ◽  
J F Healey ◽  
E L Smith ◽  
H L Bonkowsky
Keyword(s):  
Chick Embryo ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Cobalt Protoporphyrin ◽  
Cytochrome P 450 ◽  
Hepatic Cytochrome

Exposure of cultured chick-embryo hepatocytes to increasing concentrations of CoCl2 in the presence of allylisopropylacetamide results in formation of cobalt protoporphyrin, with a reciprocal decrease in haem and cytochrome P-450. Treatment of rats with CoCl2 (84 mumol/kg) and 5-aminolaevulinate (0.2 mmol/kg) also results in formation of cobalt protoporphyrin and a decrease in cytochrome P-450 in the liver. Hepatic microsomal fractions from rats treated with phenobarbital, CoCl2 and 5-aminolaevulinate were analysed by polyacrylamide gel electrophoresis. Cobalt protoporphyrin was associated mainly with proteins of 50000-53000 mol.wt. The results suggest that the formation of cobalt protoporphyrin occurred at the expense of the synthesis of haem, leading to a decrease in cytochrome P-450. Furthermore, the cobalt protoporphyrin that was formed may itself have been incorporated into apocytochrome P-450.

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