RESUMO
1. Mitochondrial DNAs from Dactylium dendroides, Hypomyces rosellus, Fusarium graminearum, Gibberella fujikuroi, Fusarium tricinctum strains and a galactose oxidase (GAO)-producing mold (original strain) presented distinctive restriction enzyme fragment patterns with the endonucleases Hind III and EcoRI. 2. A small number of comigrating bands was found when the GAO-producing mold was compared with the others. The molecular size of mtDNA from the GAO-producing mold, as judged by summation of fragment sizes produced by digestion with EcoRI, Hind III and Bgl II, is 61.3 +/- 2.16 kb. 3. The results suggest that the mtDNA from the GAO-producing mold strain is distinct from that of D. dendroides and all other ascomycetes analyzed.
Assuntos
DNA Fúngico/isolamento & purificação , DNA Mitocondrial/isolamento & purificação , Galactose Oxidase/biossíntese , Fungos Mitospóricos/classificação , Polimorfismo de Fragmento de Restrição , Basidiomycota , Galactose Oxidase/genética , Fungos Mitospóricos/enzimologiaRESUMO
1. Mitochondrial DNAs from Dactylium dendroides, Hypomyces rosellus, Fusarium graminearum, Gibberella fujikuroi, Fusarium tricinctum strains and a galactose oxidase (GAO)-producing mold (original strain) presented distinctive restriction enzyme fragment patterns with the endonucleases Hind III and EcoRI. 2. A small number of comigrating bands was found when the GAO-producing mold was compared with the others. The molecular size of mtDNA from the GAO-producing mold, as judged by summation of fragment sizes produced by digestion with EcoRI, Hind III and Bgl II, is 61.3 +/- 2.16 kb. 3. The results suggest that the mtDNA from the GAO-producing mold strain is distinct from that of D. dendroides and all other ascomycetes analyzed
Assuntos
DNA Fúngico/isolamento & purificação , DNA Mitocondrial/isolamento & purificação , Galactose Oxidase/biossíntese , Fungos Mitospóricos/classificação , Polimorfismo de Fragmento de Restrição , Basidiomycota , Galactose Oxidase/genética , Fungos Mitospóricos/enzimologiaRESUMO
The effect of ethanol and tunicamycin on synthesis and secretion of galactose oxidase was studied in resting cells of Dactylium dendroides. Ethanol promoted an overall decrease in both intra- and extracellular enzyme levels to the same extent that it inhibited [14C]glucosamine incorporation into total protein. The carbohydrate content of the intracellular enzyme was also depressed (44%) with a simultaneous decrease in O-Ser linked oligosaccharides. The intracellular galactose oxidase obtained after exposure of mycelia to ethanol plus tunicamycin lost 86% of its carbohydrate moieties, whereas the extracellular form lost only 35%. In both cases, residual sugar moieties were not eliminated by mild alkaline treatment. These data suggest that ethanol affects O-glycosylation of galactose oxidase. O-Underglycosylation did not affect the S0.5 values for galactose but diminished the molar catalytic activity. The absence of O-Ser/Thr-linked saccharides turned the intracellular enzyme into a form more susceptible to proteolysis than that devoid of N-linked sugars (tunicamycin-treated). O-Underglycosylation had a significant effect on the renaturation-reactivation of the enzyme after denaturation with 2.4 M Gdn-HCl.
Assuntos
Basidiomycota/enzimologia , Etanol/farmacologia , Galactose Oxidase/biossíntese , Glucosamina/metabolismo , Polyporaceae/enzimologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Galactose Oxidase/genética , Glicosilação , Cinética , Leucina/metabolismo , Polyporaceae/efeitos dos fármacos , Polyporaceae/crescimento & desenvolvimento , Tunicamicina/farmacologiaRESUMO
The stability of intracellular, extracellular, and deglycosylated forms of galactose oxidase was compared with respect to the denaturing effects of heat, pH, and guanidine hydrochloride. The highly glycosylated forms were found to be more stable to pH and thermal inactivation. All forms were reversibly denaturated by guanidine hydrochoride, but the extent was dependent on the carbohydrate content. Deglycosylation did not affect the affinity of the enzyme for dihydroxyacetone and galactose. Exposure of different forms of galactose oxidase to proteases like pronase and trypsin resulted in a rapid degradation of the glycoenzymes with the formation of stable products. After pronase digestion of intra- and extracellular forms of galactose oxidase catalytic species were isolated by gel filtration. The species (61 and 42 kDa) isolated from pronase-digested extracellular enzyme lost their ability to oxidize primary alcohols. Species (67 and 46 kDa) obtained from the intracellular enzyme kept the specificity of the original enzyme. Active pronase-derived peptides (42 and 46 kDa, respectively) had a higher carbohydrate content than the inactive ones.
Assuntos
Carboidratos/isolamento & purificação , Galactose Oxidase/isolamento & purificação , Fungos Mitospóricos/enzimologia , Carboidratos/fisiologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Peptídeos/isolamento & purificação , Desnaturação Proteica , Especificidade por SubstratoRESUMO
1. A morphological mutant of the mold Dactylium dendroides was isolated and the phenotype characterized as D-Gal- and L-Ara-. 2. The transport system for D-galactose seemed to be inducible in wild type and mutant and was altered in the mutant. 3. Galactose-1-P-uridylyl transferase activity was absent in the mutant. 4. The levels of intracellular galactose oxidase activity were similar in the wild type and in the mutant, thereby excluding a possible participation of this enzyme in galactose catabolism in the mold. 5. The low level of galactose oxidase activity found in the extracellular medium indicates a defect in galactose oxidase secretion by the mutant.
Assuntos
Galactose Oxidase/metabolismo , Galactose/metabolismo , Fungos Mitospóricos/metabolismo , Mutação , Arabinose/metabolismo , Galactose Oxidase/deficiência , Fungos Mitospóricos/enzimologia , Fungos Mitospóricos/genética , Fungos Mitospóricos/crescimento & desenvolvimentoRESUMO
1. A morphological mutant of the mold Dactylium dendroides was and the phenotype characterized as D-Gal-and L-Ara-. 2. The transport system for D-galactose seemed to be inducible in wild type and mutant and was altered in the mutant. 3. Galactose-1-P- uridylyl transferase activity was absent in the mutant. 4. The levels of intracellular galactose oxidase activity were similar in the wild type and in the mutant, theraby excluding a possible participation of this enzymes in glactose catabolism inthe mold. 5. The low level of galactose oxidase activity found in the extracellular medium indicates a defect in galactose oxidase secretion by the mutant