K5-type yeast killer protein in the culture supernatant of Pichia anomala NCYC 434 cells was concentrated by ultrafiltration and purified to homogeneity by ion-exchange chromatography with a POROS HQ/M column followed by gel filtration with a TSK G2000SW column. The protein migrated as a single band on discontinuous gradient SDS-PAGE and had a molecular mass of 49,000 Da. The pI value of the K5-type killer protein was measured at pH 3.7 by high voltage vertical gel electrofocusing. The result of an enzyme immuno assay revealed that it was a glycosylated protein. Its internal amino acid sequencing yielded the sequences LNDFWQQGYHNL, IPIGYWAFQLLDNDPY, and YGGSDYGDVVIGIELL, which are 100% identical to exo-beta-1,3-glucanase (accession no. AJ222862) of Pichia anomala (strain K). The purified protein was highly stable at pH values between 3 and 5.5 and temperatures up to 37 degrees C.
Killer proteins that are produced and secreted into the environment by certain yeast strains are considered as promising antifungal agents. In this study, in vitro activity of Pichia anomala NCYC 434 (K5) killer protein, panomycocin, which is a 49 kDa glycoprotein with an exo-beta-1,3-glucanase activity was tested against 41 isolates of dermatophytes. Minimum inhibitory concentrations (MICs) were determined by a broth microdilution method based on the reference document M38-A of Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS). For panomycocin MIC determinations two end point criteria MIC-2 (prominent growth inhibition) and MIC-0 (complete growth inhibition) were recorded. All the tested isolates (Microsporum spp. and Trichophyton spp.) were found susceptible to panomycocin. The MIC-2 values ranged from 0.25 to 2 microg ml(-1) and MIC-0 values ranged from 1 to 8 microg ml(-1). These results showed that panomycocin is active in vitro against fungal strains that cause superficial infections and highlighted its probable use as a topical antifungal agent.
K5-type yeast killer toxin secreted by P. anomala NCYC 434 cells has a broad killing spectrum. Competitive inhibiton of killer activity showed that glucans, mainly the -1,3 glucan, represent the primary toxin binding site within the cell wall of sensitive cells. Its hydrolytic activity on laminarin in an exo-like fashion revealed that the toxin exerts its killing effect by exo--1,3-glucanase activity. Its specific activity on laminarin was 120 U/mg, and the Michaelis constants K m and V max for laminarin hydrolysis were 0.25 mg/ml and 370 mol/min/mg. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Production of the toxin by the cells was induced only when they were grown in culture media rich in -glucan sources, and the addition of glucose increased the specific production rate. The enzymic activity of the toxin was fully inhibited by Hg þ2 , but increased with some other metal ions, most of all by Pb þ2 .
Allosuppressor (sal) mutations enhance the efficiency of the yeast ochre suppressor SUQ5 and define five unlinked loci, SAL1-SAL5. A number of sal4 mutants were isolated and found to have pleiotropic, allele;specific phenotypes, including hypersensitivity in vivo to paromomycin and other antibiotics that stimulate translational errors in yeast. To examine further the nature of the SAL4 gene product, the wild type SAL4 gene was isolated by complementation of a conditional lethal allele sal4-2, and demonstrated to be a single copy gene encoding a single 1.6 kb transcript. Restriction mapping and DNA hybridisation analysis were used to demonstrate that the SAL4 gene is identical to the previously identified omnipotent suppressor gene SUP45 (SUP1). Our results implicate the SAL4 gene product as playing a major role in maintaining translational accuracy in yeast.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.