Intracellular adenylate pools and protein degradation during zoosporangial differentiation in Allomyces arbuscula

In Allomyces arbuscula Butl., strain Bali, the ratio of protein to dry weight remained constant in exponentially growing but decreased in differentiating cultures. The adenylate pools (ATP, ADP, AMP) and energy charge which integrates them, increased during zoospore germination and stabilized around 0.9 during differentiation. The level of ATP increased early during the induction of zoosporangia for up to 1 h and then declined. The ADP and AMP remained low for most of the time except for a transient increase in ADP (first 30 min induction). The energy charge was low in spores. The rate of turnover of proteins during growth and differentiation was more or less similar for up to 1.5 h after transfer. Subsequently very little turnover of proteins occurred in the growing plants. In differentiating plants, the rate of degradation was maintained and by the end of the 4 h experimental period 30% of the vegetative proteins were degraded. The intracellular ammonium showed a peak between 30 to 60 min of induction and was higher in the differentiating mycelia than in actively growing plants, while the glutamate pool remained around 1 μmol (mg protein)⁻¹ in both types of plants. The physiological role of these protein degradation products is discussed.     

Evolution of tubulin isoforms during the sporophytic development of Allomyces arbuscula

Abstract Tubulins extracted from the sporophytic developmental stages of Allomyces arbuscula have been characterised by one- and two-dimensional SDS-PAGE gels immunoblotted with monoclonal antibodies as α-, acetylated α- ( M _r 57 kDa both) and β- ( M _r 55 kDa) isoforms. The zoosporangial isoforms could be characterised only when precautions were taken to inhibit the strong tubulin proteolytic activity at this stage. The zoospores and zoosporangia contained greater amounts of the acetylated α- and β-isoforms than the mycelium, while the non acetylated α-isoform was present in greater quantity in the mycelium than in the zoospores or zoosporangia.     

Polyribosomes in different stages of the life cycle of the water mold Allomyces arbuscula.

Synchronous gametogenesis in the water mold Allomyces arbuscula is blocked by actinomycin D added at the onset of the process. Formation of the male gametangium can be selectively inhibited by administering actinomycin one hr after the induction of gametogenesis. The polyribosome pattern obtained after density gradient centrifugation remains virtually unchanged throughout gametogenesis until a stage immediately preceding maturation of the gametes. When ribosome from gametes and swarming zygotes are analyzed on gradients, some RNase-sensitive materials is found to band in the heavier portion of the gradient. Its presence suggests that some messenger RNA associated with ribosomes is conserved in the swarming cells. During gametogenesis RNA is de novo synthesized and becomes associated with the polyribosomes.     

Cell wall formation in zoospores of Allomyces arbuscula

Carbohydrate composition was determined in isolated cell walls of meiospores of Allomyces arbuscula after incubation for 15 min (encysted meiospores: cysts), 150 min (germlings: cysts + rhizoids) and 24 h (cysts + rhizoids + hyphae). The principal constituent in all cell wall samples is chitin, accounting for about 75% of the recovered carbohydrates. In addition, cell walls of all stages examined contain polysaccharides which release galactose, glucose, mannose, arabinose, xylose, fucose, and rhamnose on acid hydrolysis. While different developmental stages show minor quantitative changes in chitin, the ratio of galactose to glucose decreases sharply during differentiation of ungerminated cysts into germlings with rhizoids and hyphae. The increase in glucose is accompanied by a decrease in the amount of xylose and/or fucose and of galactose.List of Abbreviation TFA trifluoroacetic acid     

Chemotaxis and transport of amino acids in Allomyces arbuscula

Among a number of amino acids tested, l-lysine and l-arginine are the principal attractants in the chemotaxis of the zygotes of Allomyces arbuscula. The reaction can be stimulated to a greater or lesser extent by a number of compounds chemically related to l-leucine. No relationship between transport of attracting amino acids and their effect on chemotaxis has been found.     

Immunocharacterization of actin and its immunofluorescent localization during the developmental gametophytic stages of Allomyces arbuscula

Highly specific polyclonal antibodies against actin from Allomyces arbuscula were produced in rabbits, immunopurified by immunoblotting and specified with actin isolated from Neurospora crassa and mouse skeletal muscle. Used as immunofluorescence probes, they allowed localization of actin in the sequential gametophytic stages of the mould.     

Protein and Ribonucleic Acid Synthesis During the Diploid Life Cycle of Allomyces arbuscula

The diploid life cycle of Allomyces arbuscula may be divided into four parts: spore induction, germination, vegetative growth, and mitosporangium formation. Spore induction, germination, and mitosporangium formation are insensitive to inhibition of actinomycin D, probably indicating that stable, pre-existing messenger ribonucleic acid (RNA) is responsible for these developmental events. Protein synthesis is necessary during the entire life cycle except for cyst formation. A system for obtaining synchronous germination of mitospores is described. During germination there is a characteristic increase in the rate of synthesis of RNA and protein although none of the other morphogenetic changes occurring during the life cycle are necessarily accompanied by an appreciable change in the rate of macromolecular synthesis.     

Novel Ca2+-activated neutral protease from an aquatic fungus, Allomyces arbuscula.

A Ca2+-activated neutral protease was purified to homogeneity from an aquatic Phycomycete fungus, Allomyces arbuscula. It requires millimolar concentrations of Ca2+ 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 Ca2+; the other three are free. Enzyme activity is strongly inhibited by SH-group inhibitors and some trypsin inhibitors (leupeptin and alpha-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 Ca2+-activated neutral proteases but differs in its substrate specificity, inhibition by alpha-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.     

Comparative studies of Ca2+-dependent proteases (CDP I and CDP II) from Allomyces arbuscula.

Allomyces arbuscula, an aquatic fungus, contains two Ca2+-dependent neutral cysteine proteases (CDP I and CDP II), eluting respectively, at 0.07 and 0.2 M NaCl from DEAE cellulose columns. The purified CDP I has a Mr of 39 kDa whereas CDP II appears as a doublet of 43 and 40 kDa. Both enzymes require free thiol, the same concentration of Ca2+ for half maximal activation, and are inactivated by thiol protease inhibitors. Our results show that despite these similarities the two enzymes are different because affinity-purified CDP II antibodies do not cross-react with CDP I antigen in Western blots. In contrast, there is a strong cross-reaction between the two 43 and 40 kDa CDP II peptides and their respective antibodies. Both enzymes cleave preferentially the carboxy terminus of Arg and to a limited extent Lys on the cleavage site. This primary specificity is governed by the nature of the amino acids in the P2 and P3 positions. In general either Pro or Gly in P2 is required, with preference for Pro and in P3 position, Gly over Val. CDP II has higher catalytic activity than CDP I. The sulfhydryl reagent NEM is a more potent inhibitor of CDP I than CDP II. Although the function of the phosphorylable site(s) is not clear, both CDP I and CDP II contain phosphorylable serine residue(s).     

Comparative inhibitory effects of cytochalasins on the gametangial differentiation inAllomyces arbuscula

In contrast to cytochalasin D which selectively prevents differentiation of female gametangia, cytochalasins A, B and E also masculinizeAllomyces arbuscula by preventing septation between female and male gametangia, thereby allowing the dominant expression of the male characteristics.     

Changes in the basic nuclear proteins of the cellular slime mould Dictyostelium discoideum during growth and differentiation.

Nuclei isolated from myxamoebae and differentiating cells (slug stage) of Dictyostelium discoideum contain similar ratios of DNA, RNA and protein (1:8:29) and acid soluble proteins present in amounts equal in weight to the nuclear DNA can be extracted therefrom. On urea polyacrylamide gels these basic proteins were shown to be very similar with the exception of one band, present in the myxamoebae, which was virtually absent from the differentiating cells.