RESUMO
The present study was designed to identify alkaline phosphatases in non-permeabilized hyphal cells of the fungus Neurospora crassa by staining these enzymatic activities with a modified azo dye coupling method. Our strategy allowed the identification of three non-specific alkaline phosphatase activities, one of them possibly being a novel putative enzyme, which is not responsive to either Mg2+ or EDTA. Another alkaline phosphatase activity, whose location in the hyphal cell is regulated by phosphate, is stimulated by Mg2+, inhibited by EDTA, and somehow dependent on the expression of the pho-2+-encoded Pi-repressible alkaline phosphatase.
Assuntos
Fosfatase Alcalina/análise , Hifas/enzimologia , Neurospora crassa/enzimologia , Fosfatase Alcalina/genética , Membrana Celular , Ácido Edético , Histocitoquímica , Neurospora crassa/citologia , Neurospora crassa/genética , Coloração e Rotulagem , Fatores de TempoRESUMO
A cobalt-resistant wall-less mutant of N. crassa (Cor-sl) characterized previously was also found to be 3-fold more resistant to nickel when compared to the parent wall-less mutant (W-sl). The Cor-sl strain accumulates relatively lower amounts of nickel when compared to W-sl. Sub-cellular fractionation showed significant quantities of nickel to be associated with nuclear and mitochondrial fractions in both the wall-less mutants. However significant differences were observed in vacuolar fractions of W-sl and Cor-sl strains. Fractionation of cell-free extracts on Sephadex G-10 column resolved nickel into two peaks, of which the peak II in Cor-sl constituted 70% of nickel, while the same in W-sl was about 30%. A 3-fold increase in histidine content was observed in case of Cor-sl as compared to W-sl strain, suggesting its role in Ni-resistance.