RESUMO
1. The mycelial Pi-repressible acid phosphatase presented p-nitrophenylphosphatase activity with negative cooperativity and Michaelian behavior when synthesized by the wild-type and pho-2A mutant strains of Neurospora crassa, respectively. 2. The major acid phosphatase present in cell extracts of the pho-2A mutant of N. crassa grown in low Pi medium is more thermolabile (t1/2 = 4 min at 54 degrees C, pH 5.4) than that of the wild strain (stable for at least 80 min at 54 degrees C, pH 5.4). 3. The pho-2A mutant of N. crassa secreted a more thermolabile acid phosphatase (t1/2 = 30 min at 50 degrees C, pH 5.4) than the wild strain (t1/2 of at least 80 min at 50 degrees C, pH 5.4). 4. The pho-2A mutant of N. crassa synthesized a more thermolabile acid phosphatase (t1/2 = 37 min at 54 degrees C, pH 5.4) than the wild strain in high Pi medium (t1/2 = 14 min at 54 degrees C, pH 5.4). 5. The pleiotropic nature of the pho-2 locus and its possible involvement in the mechanism of phosphatase secretion by N. crassa are proposed.
Assuntos
Fosfatase Ácida/biossíntese , Fosfatase Alcalina/biossíntese , Mutação/fisiologia , Neurospora/enzimologia , 4-Nitrofenilfosfatase/metabolismo , Fosfatase Ácida/deficiência , Fosfatase Ácida/genética , Fosfatase Alcalina/deficiência , Fosfatase Alcalina/genética , Repressão Enzimática , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Neurospora/genética , TemperaturaRESUMO
The production of exoenzymes which degrade cellulose, polygalacturonic acid and xylan was studied in mycelial and wall-less phenotypic derivatives of Neurospora crassa obtained by vegetative selection applied to a single fz;sg;os-1 ('slime'-like) segregant (strain RCP-3) of a cross 'slime' x wild type. The unrelated stable 'slime' strain FGSC 1118 was also studied. The synthesis of polysaccharide-degrading enzymes was normally induced by polysaccharidic substrates and was sensitive to carbon-catabolite repression for both mycelium-forming phenotypes (mycelial intermediate and spheroplast-hyphal intermediate) of strain RCP-3. The stable 'slime' from RCP-3 produced cellulose-degrading activity and xylan-degrading activity constitutively but was fully sensitive to glucose repression. The stable 'slime' RCP-3 did not synthesize polygalacturonic-acid-degrading activity, even in the presence of inducers. For the stable 'slime' FGSC 1118, all of the polysaccharide-degrading activities were produced constitutively and were markedly resistant to glucose repression. The possible epigenetic origin of the different properties of stable 'slimes' RCP-3 and FGSC 1118 is considered. These results may relate to the role of the cell surface in the processing of regulatory signals which control the adaptation of the fungal cell to the nutritional environment.
Assuntos
Parede Celular/fisiologia , Glicosídeo Hidrolases/biossíntese , Glicosídeos/metabolismo , Neurospora/enzimologia , Carboximetilcelulose Sódica/metabolismo , Glicosídeo Hidrolases/genética , Mutação , Neurospora/genética , FenótipoRESUMO
Adenylate cyclase catalytic subunits from Neurospora crassa membranes may interact with regulatory factors from membranes of bovine retinal rod outer segments (pretreated with N-ethylmaleimide), reconstituting a heterologous system which, in the presence of light, is catalytically active in assay mixtures containing MgATP. Maximal activation was observed at 550 nm. Transducin-depleted retinal membranes were not capable of reconstituting the heterologous light-stimulated adenylate cyclase system. Addition of a transducin preparation to depleted membranes restored the reconstitution capacity of these membranes. A similar heterologous adenylate cyclase system was reconstituted with Neurospora and mouse retinal whole membranes (pretreated with N-ethylmaleimide). Membranes from mice suffering photoreceptor degeneration (rd homozygotes) did not reconstitute an heterologous adenylate cyclase system.
Assuntos
Adenilil Ciclases/análise , Luz , Neurospora crassa/enzimologia , Neurospora/enzimologia , Retina/enzimologia , Adenosina Difosfato Ribose/metabolismo , Animais , Bovinos , GMP Cíclico/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Monoclonal antibodies to Neurospora crassa cyclic nucleotide phosphodiesterase (PDE I) were selected by their capacity to inhibit the enzyme activity. The monoclonal immunoglobulin, coupled to Sepharose 4B, was used for the affinity purification of PDE I activity. After SDS-polyacrylamide gel electrophoresis the affinity purified PDE I fractions showed a single polypeptide band of about 41 kDa. This band reacted in Western blots with the above mentioned monoclonal immunoglobulin.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/isolamento & purificação , Neurospora crassa/enzimologia , Neurospora/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/imunologia , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Anticorpos Monoclonais , Cromatografia de Afinidade/métodos , Cadeias Pesadas de Imunoglobulinas , Cadeias Leves de Imunoglobulina , Cinética , Peso MolecularRESUMO
An endogenous thermostable activator of Protein kinase III (PKIII) was purified from 100,000 X g supernatants of Neurospora crassa mycelial extracts. This 38,000 dalton polypeptide, clearly separable from calmodulin on P-60 gel filtration, specifically stimulated N. crassa PKIII activity on casein or phosvitin "in vitro" phosphorylation. The factor was only present in the initial growth phase of the fungus. The mechanism of PKIII activation and its possible regulatory role are discussed.
Assuntos
Neurospora crassa/enzimologia , Neurospora/enzimologia , Proteínas Quinases/metabolismo , Cromatografia em Gel , Ativação Enzimática , Proteínas Fúngicas/metabolismo , Cinética , Peso Molecular , Neurospora crassa/crescimento & desenvolvimento , FosforilaçãoRESUMO
We show that N. crassa represses the production of acid phosphatase at pH higher than 8.0, irrespective of the carbon source used, whereas production was stimulated by sucrose at slightly acidic pH. The same profile of acid phosphatase production was observed in the pho-2A, pho-3A, nuc-1A, nuc-2A and pregc mutant strains. We also show that acid phosphatase synthesized by the pregc mutant strain grown on high phosphate medium has pronounced differences when compared to the enzyme synthesized by the wild-type strain grown on low phosphate medium in terms of heat stability, steady-state kinetic properties and DEAE-cellulose chromatography. In addition, the synthesis and/or secretion of only phosphate-repressible alkaline phosphatase is affected by mutations in acu-1, and acu-5 and acu-7 genes. These results, which indicate distinct pathways for the synthesis and secretion of acid and alkaline phosphatases in N. crassa, contradict the dosage titration model proposed by Metzenberg et al. (1974) whereby the synthesis of these enzymes should occur through a single hierarchical regulatory circuit as a response to phosphate starvation.
Assuntos
Fosfatase Ácida/biossíntese , Fosfatase Alcalina/biossíntese , Regulação da Expressão Gênica , Genes Fúngicos , Genes , Neurospora crassa/enzimologia , Neurospora/enzimologia , Fosfatase Ácida/genética , Fosfatase Ácida/isolamento & purificação , Fosfatase Alcalina/genética , Fosfatase Alcalina/isolamento & purificação , Cinética , Neurospora crassa/genética , Especificidade da EspécieRESUMO
The adenyl cyclase deficient cr-1 mutant of Neurospora crassa grew poorly in bovine serum albumin as an alternative and only source of either sulfur, nitrogen or carbon. The low growth of the cr-1 mutant in protein was correlated with limited secretion of extracellular alkaline protease. The defect was specific for the cr-1 mutant and was suppressed by exogenous cyclic AMP. Cyclic AMP relieved protease deficiency under carbon, nitrogen or sulfur limiting conditions to unequal extents. Protease stimulation was greatest under carbon-limited conditions, but the resulting growth was least. Most of the cyclic AMP-mediated increase of alkaline protease was extracellular.
Assuntos
Adenilil Ciclases/deficiência , AMP Cíclico/farmacologia , Neurospora crassa/enzimologia , Neurospora/enzimologia , Peptídeo Hidrolases/metabolismo , Neurospora crassa/genética , FenótipoRESUMO
1. N-Acetyl galactosaminoglycan deacetylase was purified from Neurospora mycelium 215-fold in 25% yield to electrophoretic homogeneity. A single band corresponding to a molecular weight of 76,000 was obtained by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. 2. The enzyme activity had pH optima at pH 5.0 and 9.0. Sodium molybdate, 2 mM, stimulated enzyme activity 4-fold at pH 5.0 but had no effect at pH 9.0. Cupric ion, 1 mM, inhibited activity by more than 85% at pH 5.0 and 9.0. The Km of the enzymatic reactions was 16 microM on the basis of the concentration of N-acetylgalactosamine. 3. This enzyme may be involved in determining the properties of the hyphal apex of the colonial form of Neurospora crassa and thus could play a role in morphogenetic regulation.
Assuntos
Amidoidrolases/isolamento & purificação , Hidrolases de Éster Carboxílico , Neurospora crassa/enzimologia , Neurospora/enzimologia , Amidoidrolases/metabolismo , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Peso Molecular , Morfogênese , Neurospora crassa/crescimento & desenvolvimentoRESUMO
Previous findings in the literature that rhein inhibits DPNH-linked mitochondrial oxidations by acting in the DPNH dehydrogenase region of the respiratory chain have been confirmed and extended. In the micromolar range rhein inhibits DPNH oxidase and DPNH-ferricyanide activities and the energy-linked reduction of DPNH by succinate in membrane preparations from heart, as wellas the DPNH dehydrogenase and transhydrogenase activities of the soluble, purified enzyme. The inhibition of the activities of the soluble enzyme are purely competitive with respect to substrate. These facts localize the primary inhibition site of rhein between substrate and FMN. In heart ETP a second noncompetitive inhibition is also present but is detectable only at very low (<10æM) rhein concentrations. Rhein also inhibits DPNH dehydrogenase in Candida utilis mitochondria and the purified enzyme from liver. On conversion of the heart enzyme to the low molecule weight DPNH-cytochrome reductase the typical effect of rhein disappears and is replaced by a slight stimulation or inhibition, depending on the electron acceptor used, showing that the substrate binding site is modified in this form of the enzyme. In beef liver mitochondria DPNH oxidation may appear insensitive to rhein, probably because of the strong binding of rhein to other proteins. To a lesser extent unspecific binding of rhein and resultant interference with the inhibition of DPNH dehydrogenase is also shown by BSA and by proteins in heart ETP. Rhein also inhibits transhydrogenations in mitochondria and at higher concentrations lactate and malate dehydrogenases but has no effect on sccinate, alcohol (liver nad yeast), and glucose-6-p dehydrogenases or on Neuospora DPN-ase, glucose-6-phosphatase, and amine oxidase. (SUMMARY)