Die Phenoloxydasen des Ascomyceten Podospora anserina

Summary The ascomycete Podospora anserina forms two different phenoloxidases. According to their substrate specifity they can be classified as laccase and tyrosinase, respectively. While laccase is found in freshly prepared extracts in a highly active form, the tyrosinase needs to be activated by cold- or heat-treatment. Further characteristics of the two enzymes are: their different solubilities in ammoniumsulfate solution and their different heat inactivation rates. The half life times of partially purified extracts at 60° C are for laccase 2.5 min and for tyrosinase 23 min.     

Die Phenoloxydasen des AscomycetenPodospora anserina

Zusammenfassung Die Tyrosinase der Mutantezonata (z) vonPodospora anserina wurde untersucht. Die Mutante erzeugt im Vergleich zum Wildstamm große Mengen Tyrosinase.z ist eine morphologische Mutante mit rhythmischen Wuchs. Das Genz liegt auf dem rechten Arm der Koppelungsgruppe II und hat einen Abstand von 46 Kartierungseinheiten vom Centromer.Die Reinigung des Enzyms erfolgte durch Präzipitation mit Protaminsulfat, Ammoniumsulfat und anschließende Säulenchromatographie an DEAE-Sephadex und Hydroxylapatit. Die Einheitlichkeit des gereinigten Enzyms wurde in der Ultrazentrifuge und der Elektrophorese festgestellt. Das gereinigte Enzym ist in verdünnter Lösung instabil.Im Konzentrationsbereich von 2–15 mg/ml hat die Tyrosinase eine Sedimentationskonstante vons _20,w ⁰ =6,04. Die Molekulargewichtsbestimmung aus Diffusion und Sedimentation und aus dem Sedimentationsgleichgewicht nachYphantis ergab in demselben Konzentrationsbereich ein Molekulargewicht um 100.000. Bei einer Enzymkonzentration von 0,04 mg/ml fanden wir mit der Gelfiltrations-methode nur ein Molekulargewicht von 42.000. Aus diesen Resultaten läßt sich in Übereinstimmung mit den Ergebnissen an anderen Tyrosinasen aus Pilzen eine konzentrationsabhängige Assoziation und Dissoziation des Enzyms folgern.Der Kupfergehalt beträgt 0,214±0,008%. Das gereinigte Enzym hat ein Absorptionsmaximum bei 280 nm und eine Schulter bei 290 nm und von 320 bis 380 nm.Das Verhältnis von Dehydrogenierungen an Brenzcatechin zu Hydroxylierungen an Hydrochinon konnte polarographisch bestimmt werden. Es läuft im Mittel auf zwei Dehydrogenierungen nur eine Hydroxylierung ab. Natriumazid wirkt als reversibler und kompetitiver Inhibitor der Dehydrogenierungsreaktion.Im Rohextrakt liegt das Enzym in einer latenten Form vor. Es sind nur 10–20% der späteren Aktivität nachzuweisen. Das Enzym wird im Verlauf der Reinigungsprozedur oder durch Temperaturbehandlung (10 min bei 60°C) aktiviert. Der Aktivierungsvorgang ist von einer Veränderung der elektrophoretischen Beweglichkeit des Enzyms begleitet. Latentes und aktives Enzym unterscheiden sich nicht in ihrem Sedimentationsverhalten.

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The phenoloxidases of the ascomycetePodospora anserina IV. Purification and properties of tyrosinase

Summary Tyrosinase of the mutantzonata (z) ofPodospora anserina was investigated. In comparison to the wild strain the mutant (z) produces large quantities of tyrosinase.z is a morphological mutant with rhythmic growth. The genez is localized on the right arm of linkage group II at a distance of 46 map-units from the centromere.The enzyme was purified by precipitation with protamine-sulphate and ammonium sulphate and by subsequent column-chromatography on DEAE-Sephadex and hydroxylapatite. The homogeneity of the purified enzyme was checked by ultracentrifugation and disc-electrophoresis. The purified enzyme is unstable in dilute solution.At a concentration of 2–15 mg/ml the tyrosinase has a sedimentation constant ofs _20,w ⁰ =6.04. The determination of molecular weight from sedimentation and diffusion constants and from sedimentation-equilibrium according toYphantis showed a molecular weight of approximately 100,000 in the same concentration range. At an enzyme concentration of 0.04 mg/ml we found a molecular weight of only 42,000 using the gel-filtration technique. From these results, in accordance with the results obtained from other tyrosinases in fungi, association and dissociation of the enzyme dependent on concentration may be concluded.The copper content reaches a level of 0.214±0.008%. The purified enzyme has an absorption maximum at 280 nm and a shoulder at 290 nm and from 320 to 380 nm.The quotient of catechol-dehydrogenations to hydroquinone-hydroxylation was determined polarographically. The mean ratio is two dehydrogenations to only one hydroxylation. Sodium azide acts as a reversible and competitive inhibitor of the dehydrogenation-reaction.In the crude extract the enzyme is found in a latent form. Only 10–20% of its later activity can be demonstrated. The enzyme is activated in the course of the purification procedure or by temperature treatment (10 min at 60°C). The activation process is accompanied by a change in the electrophoretic mobility of the enzyme. Latent enzyme and active enzyme do not differ in their sedimentation behaviour.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft     

Die Phenoloxydasen des AscomycetenPodospora anserina

Ohne ZusammenfassungProfessorG. Melchers zum 60. Geburtstag.     

The phenoloxidases of the Ascomycete Podospora anserina

Summary In order to learn the internal conditions for the production of the various phenoloxidases produced by the Ascomycete Podospora anserina the wild strain has been grown under controlled conditions in a fermenter for a period of 34 days. Samples were withdrawn at regular intervals and assayed for mycelial yield and intra- and extracellular phenoloxidase production.Maximal yield was obtained at the following age of the culture: Mycelial production 9 d, tyrosinase 4 d, the high molecular weight laccase I between 9 and 19 d. The low molecular weight laccases II and III, initially present in medium concentrations, dropped to an early minimum after 4 days, followed by an increase with a maximum in the late autolytic phase.The changes in the phenoloxidase spectrum and the antiparallel production curve for the high molecular weight against the low molecular weight laccases are discussed in relation to the earlier observed genetical and physiological control of phenoloxidase synthesis and in relation to the possibility of laccase I being composed of active subunits of low molecular weight laccases.With support of the Deutsche Forschungsgemeinschaft, Bad Godesberg (Germany).     

The phenol oxidases of the ascomycete Podospora anserina

For the low molecular weight laccases II and III of Podospora anserina the kinetic parameters Michaelis constant (K _M) and maximum reaction velocity (V) were determined polarographically under pH optimum conditions for representative substrates of different substitution patterns.Laccase II showed two peaks in its pH optimum curve, each with a different substrate specificity, indicating structural differences to laccase III which exhibits only one broad peak.Under optimum conditions the affinities of various substrates are determined by their substitution patterns: high affinity for simple o-and p-diphenols, low affinity for m-phenols. The maximal velocity remains largely uninfluenced.This study of the effect of substitution on substrate utilization leads to the assumption that there is no specific reactive site for m-phenols in either laccase. Oxidation of m-phenols, however, takes only place at high pH values.     

Infectious Fold and Amyloid Propagation in Podospora anserina

Amyloid protein aggregation is involved in serious neurodegenerative disorders such as Alzheimer''s disease and transmissible encephalopathies. The concept of an infectious protein (prion) being the scrapie agent was successfully validated for several yeast and fungi proteins. Ure2, Sup35 and Rnq1 in Saccharomyces cerevisiae and HET-s in Podospora anserina have been genetically and biochemically identified as prion proteins. Studies on these proteins have revealed critical information on the mechanisms of prions appearance and propagation. The prion phenotype correlates with the aggregation state of these particular proteins. In vitro, the recombinant prion proteins form amyloid fibers characterized by rich β sheet content. In a previous work on the HET-s prion protein Podospora, we demonstrated the infectivity of HET-s recombinant amyloid aggregates. More recently, the structural analysis of the HET-s prion domain associated with in vivo mutagenesis allowed us to propose a model for the infectious fold of the HET-s prion domain. Further investigations to complete this model are discussed in this review, as are relevant questions about the [Het-s] system of Podospora anserina.Key Words: prion, HET-s, Podospora, amyloid, infectious, β sheet, mutagenesis, fold, propagation