Comparative production and rapid purification of Candida acid proteinase from protein-supplemented cultures
| Autor: | C D Payne, T L Ray |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Immunology
Serum albumin Biology Monospecific antibody Microbiology Mice chemistry.chemical_compound Casein Endopeptidases Animals Aspartic Acid Endopeptidases Pathogen Antibodies Fungal Serum Albumin Candida chemistry.chemical_classification Mice Inbred BALB C Caseins Molecular biology Yeast Corpus albicans Culture Media Molecular Weight Infectious Diseases Enzyme chemistry biology.protein Keratins Parasitology Pepstatin Research Article |
| Zdroj: | Infection and Immunity. 58:508-514 |
| ISSN: | 1098-5522 0019-9567 |
| DOI: | 10.1128/iai.58.2.508-514.1990 |
| Popis: | Six Candida spp. that were previously characterized for cutaneous pathogenicity were assessed for Candida acid proteinase (CAP) production in albumin-supplemented, nitrogen-restricted media. C. albicans CAP production was compared in media supplemented with albumin, casein, collagen, hemoglobin, or keratin and in TC medium 199. C. albicans, C. stellatoidea, and C. tropicalis, which are cutaneous pathogens in murine infections, produced 3.3 to 4.7 times more CAP than did the nonpathogens C. parapsilosis and C. guilliermondii. C. krusei, a nonpathogen, produced negligible amounts of enzyme. C. albicans CAP production was similar in each protein-supplemented medium, and only a single acid proteinase was recovered from each one. Rapid CAP purification from culture supernatants was achieved by hollow fiber and stirred cell ultrafiltration followed by Affi-Gel blue and Sephacryl column chromatographies. The highest yield, purity, and specific activity of CAP were obtained from keratin-supplemented medium supernatants, producing 2.86 mg of purified CAP from a 7-liter culture. CAP was characterized as a 41,500-dalton glycoprotein, with a pI of 4.5; a pH range of 2.5 to 5.5; and broad substrate specificity, including that for keratin, denatured collagen, hemoglobin, casein, and albumin. Isolation of CAP also isolated the keratinolytic proteinase of Candida spp. CAP was inhibited by pepstatin A, but not by EDTA or phenylmethylsulfonyl fluoride. Monospecific antibody to CAP was produced in mice and reacted only to the 41,500-dalton protein, as determined by immunoblot analysis. High CAP production by cutaneous pathogenic Candida spp. supports the fact that CAP is a potential virulence factor that may facilitate Candida colonization and invasion of skin. CAP production from keratin-supplemented medium was superior to that from the other media that were tested and yielded sufficient and suitable enzyme for use in immunoassays of CAP antigen and antibody. |
| Databáze: | OpenAIRE |
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