Dora M. Rast

Metabolic products of microorganisms

SummaryBoromycin, at a concentration of 0.05 μg/ml inhibits the synthesis of protein, RNA and DNA in whole cells of Bacillus subtilis. It is being antagonised by surface active compounds and is being bound to lipoprotein. Binding of the boromycin within the cell especially takes place at the cytoplasmic membrane.The inhibitory effect to Bacillus subtilis is being reversed by high concentration of potassium salts (e.g. 0.2 m KCl). The reversion is specific of potassium salts. After the adding of boromycin a discharge of potassium ions from the cells can be observed. The K+-Na+-activated ATP-ase of the cytoplasmic membrane is not influenced by boromycin. On an artificial membrane of carbon tetrachloride boromycin shows a low selectivity for potassium ions compared with sodium and lithium ions.The degradation of boromycin through alkaline and acid hydrolysis leads to a loss of antibiotic activity, due to the splitting off the boric acid from the molecule.

Cell wall-associated enzymes in fungi

This review compiles and discusses previous reports on the identity of wall-associated enzymes (WAEs) in fungi and addresses critically the widely different terminologies used in the literature to specify the type of bonding of WAEs to other entities of the cell wall compartment, the extracellular matrix (ECM). A facile and rapid fractionation protocol for catalytically active WAEs is presented, which uses crude cell walls as the experimental material, a variety of test enzymes (including representatives of polysaccharide synthases and hydrolases, phosphatases, gamma-glutamyltransferases, pyridine-nucleotide dehydrogenases and phenol-oxidising enzymes) and a combination of simple hydrophilic and hydrophobic extractants. The protocol provides four fully operationally defined classes of WAEs, with constituent members of each class displaying the same basic type of physicochemical interaction with binding partners in situ. The routine application of the protocol to different species and cell types could yield easily accessible data useful for building-up a general objective information retrieval system of WAEs, suitable as an heuristic basis both for the unravelling of the role and for the biotechnological potentialities of WAEs. A detailed account is given of the function played in the ECM by WAEs in the metabolism of chitin (chitin synthase, chitinase and beta-N-acetylhexosaminidase) and of phenols (tyrosinase).

The biosynthesis and possible function of γ-glutaminyl-4-hydroxybenzene in Agaricus bisporus

Abstract The occurrence of γ- L -glutaminyl-4-hydroxybenzene (GHB) and γ- L -glutaminyl-3,4-dihydroxybenzene (GDHB) has been studied in the different de

Zur stoffwechselphysiologischen bedeutung von mannit und trehalose in Agaricus bisporus (Eine gaschromatographische studie)

ZusammenfassungEs wird eine gaschromatographische Methode zur simultanen Bestimmung von Mannit und Trehalose in Pilzextrakten beschrieben und damit die Zusammensetzung der Neutralfraktionen einiger Basidiomyceten-species untersucht.Die Anwendung des Verfahrens zur Analyse der einzelnen Teile von Sporokarpen verschiedener Entwicklungsstadien von Agaricus bisporus zeigt, daß dieser entgegen früheren Literaturangaben wie die meisten Basidiomyceten ebenfalls Trehalose enthält (0,03–0,3% des Frischgewichtes).Es besteht ein negativer Gradient von Mannit in Richtung der Pilzlängsachse, welcher während der ganzen Lebensdauer des Fruchtkörpers aufrechterhalten wird und sich mit zunehmendem Alter akzentuiert. Für offene, sporulierende Fruchtkörper wurden Mannitgehalte von 4% (des FG) im Stiel, 3,3% im Hutplectenchym und 1% im Hymenium festgestellt. Hingegen waren keine dem Mannit entsprechende oder zu ihm inverse Konzentrationsgradienten von Trehalose nachzuweisen. Das Verhältnis von Mannit zu Trehalose weist zeitliche Variationen und örtliche Unterschiede innerhalb des Basidiokarps auf; der Quotient M/T erreicht in den “Pinheads” und in den Sporen den kleinsten Wert (4).Die experimentellen Ergebnisse werden in bezug auf die stoffwechselphysiologische Bedeutung von Mannit und Trehalose in höhern Pilzen diskutiert.SummaryA gaschromatographic method for the simultaneous determination of mannitol and trehalose in fungal extracts is described. It is used to determine the composition of the neutral fractions of some basidiomycetous species.The application of the procedure to the analysis of the individual parts of sporocarps from Agaricus bisporus in different developmental stages shows that this species contains trehalose (0.03–0.3% of the fresh weight) like most basidiomycetes, contrary to the citations in the literature.There exists a negative gradient of mannitol in the direction of the longitudinal axis of the mushroom which is maintained during the whole life of the fruit body and accentuated with increasing age. The mannitol contents of open, sporulating fruit bodies were 4% (of the fresh weight) in the stipe, 3.3% in the pileus and 1% in the hymenium. However, no gradient of trehalose corresponding to or inverse to that of mannitol was found. The ratio of mannitol to trehalose shows temporal and topographic differences within the basidiocarp; in the “pinheads” and the spores the ratio of M/T is lowest (4).The experimental results are discussed in relation to the physiological rôle of mannito and trehalose in higher fungi.

The physiological role of malic enzyme in grape ripening

The high specificity of malic enzyme (ME; EC 1.1.1.40) from grape berries (Vitis vinifera L.) for the naturally occurring l-enantiomer of malic acid, its very selective C4-decarboxylation, and certain allosteric properties, reported previously, favour the conjecture of a regulatory function of ME in fruit malic acid degradation. On the other hand, high ME activity was detected even during the acid-accumulating phase of berry development. Also, the in vitro reversibility of the reaction supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate, notably high CO2/HCO3-and NADPH/NADP ratios. However, a very limited incorporation of 14C into malate and the uniform labeling pattern of the dicarboxylic acid after administration of [U-14C] alanine to grape berries before and after the onset of ripening, indicate that the ‘reverse” reaction does not contribute essentially to grape malate synthesis. A regulatory mechanism mediating malic acid remetabolization on the basis of cosubstrate availability, comparable to the control of the hexose monophosphate shunt, is discussed.

Isolation and properties of chitin synthetase from Agaricus bisporus mycelium

Abstract Using the standard procedure for the isolation of chitosomes ( S. Bartnicki-Garcia, C. E. Bracker, E. Reyes, and J. Ruiz-Herrera, 1978 , Exp. Mycol. 2, 173) a 50-fold purification has been achieved of chitin synthetase (ChS) from Agaricus bisporus hyphae grown in stirred, pellet-free liquid culture. Some properties of the enzyme were determined and compared with those of chitosomal ChS of other organisms. Effects of some antifungal compounds upon enzyme activity were also investigated. Inclusion of digitonin into the extraction medium afforded a second species of chitosomes, which have a higher sedimentation coefficient than the “standard” chitosomes. As regards ultrastructure, behavior in sucrose density gradients, level of activities, zymogenicity, cofactor requirements, kinetics, apparent K m values for UDPGlcNAc and GlcNAc, inhibitory constants for nikkomycin X, nikkomycin Z, and amphotericin B methyl ester (AME), as well as modulation by digitonin, the mushroom chitosomal ChS displayed basically the same features as the corresponding enzyme from Mucor rouxii yeast cells. Some differences were, however, observed: Rennilase was almost ineffectual in overcoming latency of the enzyme, and trypsin performed satisfactorily only in a narrow concentration range, whereas pronase was very good. Furthermore, stimulation of ChS by digitonin in low concentrations was considerably higher (attaining more than 300%) with the mushroom than with the Mucor enzyme. The strong stimulatory effect of digitonin seems to be specific for the spirostanol glycoside since it could be produced by neither the closely related saponin α-tomatine, nor by AME—both compounds, like digitonin, sterol-complexing agents. The suggestion is made that stimulation of ChS by digitonin may be causally related to its antimycotic activity. Several possibilities are discussed as to its mode of action in stimulating ChS.

The polyol pattern of some fungi not hitherto investigated for sugar alcohols

Abstract Polyols in the mycelia (or vegetative cells) of 27 species, including representatives from each of the major classes of fungi, were analyzed using thin-layer chromatography, paper electrophoresis, and gas chromatography. The polyol contents (as determined by gas chromatography) were always considerably less than 1% of the fungal dry matter in the Phycomycetes and usually well above this figure in the higher fungi, reaching maximum values in some Ascomycetes and Deuteromycetes (16, 15, and 13% in Endothia parasitica, Penicillium oxalicum , and Alternaria tenuissima , respectively). Polyols were absent in Achlya radiosa and Phytophthora cinnamomi , a result confirming earlier reports that Oomycetes lack sugar alcohols. In the Zygomycetes and Chytridiomycetes, generally considered to be also devoid of polyols, glycerol was present. Zygorhynchus moelleri and Mucor miehei contained ribitol. The polyol fraction of Blastocladiella emersonii and Allomyces arbusculus consisted almost exclusively of mannitol. This also dominated the polyol pattern of the higher fungi. In addition to mannitol, these usually contained glycerol. Erythritol was present in Monascus ruber, Penicillium brefeldianum, Penicillium italicum, P. oxalicum , and Verticillium fungicola , arabitol in A. tenuissima, P. italicum, P. oxalicum , and Rhizoctonia solani . In the discussion, special consideration is given to the phylogenetic implications of the results.

Purification and properties of mannitol dehydrogenase from Agaricus bisporus sporocarps

Abstract Mannitol dehydrogenase (mannitol: NADP + 2-oxidoreductase: EC 1.1.1.138) was isolated from Agaricus bisporus by fractionation with protamine sulphate and (NH 4 ) 2 SO 4 , followed by chromatography on DEAE-Sephadex, then by affinity and gel chromatography. The products of enzyme reaction were identified by GLC and TLC. K m , optimum pH, MW and pI of the enzyme as well as the influence of temperature, ions and inhibitors on enzymic activity were determined. In the sugar reducing reaction, the enzyme was specific for fructose but, in the reverse direction, some structurally related polyols could substitute for mannitol. The enzyme was very sensitive to alterations in the NADP + /NADPH ratio. The results are discussed in relation to the possible role of mannitol dehydrogenase in fungal metabolism.

Biochemische beziehung zwischen mannitbildung und hexosemonophosphatzyklus in agaricus bisporus

Zusammenfassung Mannitol accumulation occurs in almost all higher fungi, but its physiological significance is unknown. The polyol is neither a carbon source for growth nor a respiratory substrate, and polymeric compounds in which it might be incorporated have not so far been found. Experiments have been designed to investigate whether the biosynthesis of mannitol, rather than its mere presence, could be of prime importance for organisms synthesizing this sugar alcohol. The labelling patterns of mannitol isolated after application of glucose-1-3H, −6-3H or −3-3H to slices of Agaricus bisporus fruit bodies were determined. The results show that the biogenesis of mannitol involves the reoxidation of NADPH formed in the hexose monophosphate shunt. These observations are discussed in relation to the known role of this pathway, and the fact that the overall rate of glucose catabolism via the shunt is normally limited by the availability of NADP. On this basis, it is suggested that mannitol formation has a growth-regulating function in this fungus.

Partitioning of photosynthate in leaves of Vitis vinifera infected with Uncinula necator or Plasmopora viticola

Carbohydrate concentrations, 14 CO 2 -incorporationand partitioning of photosynthates in grapevine leaves as affected by association with Uncinula necator or Plasmopara vilicola were determined. Homogeneity of replicate samples was checked using arabitol as a marker of relative fungal biomass for the former and trehalose for the latter. U. necator showed typical biotrophic behaviour even at the time of sporulation. Increased concentrations of sugar in infected leaves were attributed to import from uninfected tissue. Photosynthetic carbon flux to arabitol was slow compared with that to mannitol. Sporulating P. viticola , on the other hand, induced depletion of host sucrose and an extensive diversion of glucose to trehalose. These results indicate that the metabolic host/parasite interface is likely to include sucrose cleavage and transformation of the resultant hexoses to hexitol and/or trehalose. Invertase of plant or fungal origin in crude extracts from diseased material was shown to hydrolyse sucrose at a higher rate than that in extracts from healthy plant tissue.