RESUMO
Pyruvate kinase was purified from the filamentous fungus Aspergillus nidulans with a 45-55% yield. The procedure involved dye-affinity chromatography and fast protein liquid chromatography, resulting in highly active and pure enzyme in milligram quantities within 2 days. The purified enzyme, a tetramer with a subunit molecular weight of 65,000 and an isoelectric point of 4.7, was used to determine the amino acid composition.
Assuntos
Aspergillus nidulans/enzimologia , Piruvato Quinase/isolamento & purificação , Aminoácidos/análise , Fenômenos Químicos , Físico-Química , Cromatografia de Afinidade/métodos , CorantesRESUMO
A selective method for the isolation of Aspergillus nidulans mutants defective in the pyruvate dehydrogenase complex was devised. The essential steps in the procedure were a mutagenic treatment of conidia with X-rays to about 50% survival, followed by filtration enrichment in minimal medium with D-galacturonate as sole carbon source, and rescue on complete medium with acetate. The mutants thus isolated were phenotypically characterized on the basis of growth tests, and different genotypes were assigned on the basis of complementation tests. The majority of the mutants that were unable to utilize galacturonate were defective in one of the components of the pyruvate dehydrogenase complex. In addition, mutants defective in pyruvate carboxylase, mutants defective in glycerol catabolism and some novel mutants which were only unable to use D-galacturonate as carbon source were found. At least two genes were shown to be involved in D-galacturonate metabolism.
Assuntos
Aspergillus nidulans/genética , Ácidos Hexurônicos/metabolismo , Ácidos Urônicos/metabolismo , Aspergillus nidulans/metabolismo , Genes Fúngicos , Glicerol/metabolismo , Ácidos Hexurônicos/genética , Mutação , Fenótipo , Piruvato Carboxilase/metabolismo , Complexo Piruvato Desidrogenase/metabolismoRESUMO
Several mutants aspergillus nidulans defective in carbohydrate metabolism were tested for growth on different carbon sources. D-Galacturonate was found to be a substrate, useful to discriminate between mutants in pyruvate kinase, pyruvate dehydrogenase complex or pyruvate carboxylase. The results of these tests indicate how particular classes of mutants can be obtained and which substrates can be used preferentially for a rapid phenotypical screening of unknown mutants.
Assuntos
Aspergillus nidulans/metabolismo , Metabolismo dos Carboidratos , Aspergillus nidulans/genética , Aspergillus nidulans/crescimento & desenvolvimento , Glicerol/metabolismo , Mutação , Pentosefosfatos/metabolismo , Piruvato Carboxilase/genética , Complexo Piruvato Desidrogenase/genética , Piruvato Quinase/genéticaRESUMO
In order to investigate the special function of tryptophan in peptide hormones, six tryptophan analogues have been synthesized, in which structural resemblance has been grossly retained and potentially essential properties have only partially been varied. The L-enantiomers have been isolated after enzymatic digestion of racemic dipeptide derivatives, and charge-transfer properties of the compounds have been studied.