A procedure for the isolation of highly purified labeled and unlabeled DSqAs from fungi representing three major groups has been described. The yield of DI~A per g weight of freeze-dried mycelia is always higher in Neurospora crassa than Coprlnus lagopus and Mucor azygos79ora. Thermal melting profiles show that the N. crassa and C. lagopus DNAs have one low G-}-C (32 mole percent) and another high Gq-C (52 mole percent) component, whereas the M. azygospora DNA has only one low G-t-C (38 mole percent) component. Based on DNA:DNA reassociation kinetics studies in this and other laboratories the genome size of M. bacilli/ormis, N. crassa and C. lagopus was found to be 2× 101°, 2.2× 101° and 2.5× 10 l° daltons respectively. N. crassa asp DNA when hybridized with Coprinus and Mucor DNAs gave an average of 14 and 11% homologies respectively but when hybridized with DNAs of other species of the same genus gave a homology of 83 % on an average. When asp Coprinus DNA was hybridized with N. crassa and M. azygospora DNAs an average of 10 and 8 % hybridization, respectively, was obtained. The DNA:DNA homology and thermal stability data indicate that the two Neurospora species, N. intermedia and N. sitophila, are equally distant from N. crazsa and probably originated from the same ancestor in the evolutionary scale. On the basis of genome size comparison between the three fungi, the Coprinus (basidiomyeetes) seems to be highly evolved whereas Mucor (phyeomyeetes) is a relatively primitive one, Neurospora (aseomycetes) being somewhere in between these two groups in the evolutionary scale.
Immunogold labeling of calcium-dependent neutral protease II (CDPII) with specific antibodies in near median longitudinal ultrathin sections of Allomyces arbuscula showed that the enzyme is predominantly localized in the growing hyphal and rhizoidal apices. The tips in both cell type had more enzyme than the distal regions and showed a gradient distribution. Labeling of the ultrathin sections and western blot analysis of purified subcellular fractions showed that CDPII is mainly cytosolic. Catalytic activity of the enzyme measured with synthetic substrate (Bz-Arg-pNA) showed that 90% of its activity is present in the soluble fraction, although a small amount is associated with the nuclei (0.2%), plasma membranes (0.7%) and microsomes (3.9%). This association is discussed in the context of the functional role of the enzyme and its possible localized activation. Western blot analysis of the crude extract and indirect immunofluorescence of the fixed permeabilized hypahe after treatment with CDPII showed that theα-tubulin is a specific target of the enzyme.
A Ca2"-activated neutral protease was purified to homogeneity from an aquatic Phycomycete fungus, Allomyces arbuscula. It requires millimolar concentrations of Ca24 for activation (1.8 to 2 mM for 50% activation). Sr2' can replace Ca2' but at higher concentrations (4 mM for 50% activation). The enzyme is a dimer of 40-kilodalton subunits and contains six cysteine residues, three of which are revealed only after the addition of micromolar concentrations of Ca24; the other three are free. Enzyme activity is strongly inhibited by SH-group inhibitors and some trypsin inhibitors (leupeptin and a-N-tosyl-L-lysine chloromethyl ketone). The enzyme lacks general trypsinlike specificity, since substrates containing tryptic cleavage sites are not cleaved nor is enzyme activity inhibited by other trypsin inhibitors. The enzyme has many functional similarities to the extensively characterized mammalian and avian Ca24-activated neutral proteases but differs in its substrate specificity, inhibition by a-N-tosyl-L-phenylalanine chloromethyl ketone, and subunit structure.It is, nevertheless, presumed that this enzyme has a similar high order of specificity and is involved in the regulation of a specific growth function.We have reported that Allomyces arbuscula, an aquatic fungus, contains two major neutral proteases (17). Both enzymes contain functionally important cysteines, since their activities are potently inhibited by SH-binding reagents. The enzyme from the exponentially growing vegetative mycelia has an absolute requirement for Ca2" for its activity and is, therefore, strongly inhibited by metal chelators like EDTA and EGTA [ethylene glycol-bis(,B-aminoethyl ether)-N-N-N'-N'-tetraacetic acid]. It is inhibited by phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor, but only at relatively high concentrations. The preparation from differentiated mycelia, which does not require Ca2" for its activity, is very sensitive to PMSF. In this report we present details of the purification and some properties of the vegetative enzyme. MATERIALS AND METHODSOrganism and cultural conditions. The experimental strain of A. arbuscula Butl. was maintained on YpSs agar medium (6) as previously described (16). Zoospores liberated from a young sporophytic culture were inoculated on filter pads placed on the solidified YpSs medium in petri plates 14 cm in diameter. After 4 to 5 days of incubation at 32°C, the pads were removed, placed in sterile plastic petri plates, and flooded with approximately 60 ml of sterile distilled water.After 30 min at room temperature the water was replaced, and the incubation was continued up to 4 h for the induction and liberation of zoospores. The dense zoospore suspension was pipetted off and inoculated into GCY medium (21). A single pad gave approximately 6 x 106 spores. The suspension obtained from two pads was used to inoculate a 10-liter carboy containing 6 liters of medium. The cultures were incubated with forced aeration at 32°C for 24 h, and the mycelia were harvested by filtration, squeeze-dr...
The Ca'+-dependent protease antisera and the purified specific antibodies from Allomyces arbuscula have shown very specific recognition when blotted against the total protein extract or the purified 43340 kDa Ca"-dependent protease from this aquatic fungus. By immunoblotting and immunofluorescence techniques using specific antibodies, we have shown that the enzyme activity is developmentally regulated and is related to the presence of antigen and not to any specific inhibitor. The immunofluorescence was absent in zoospores but appeared in polarized forms in germinating spores. In elongating hyphae the protease was mainly localized along the cytoplasmic membrane and in the cytoplasm, with predominance at the apex.
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