Cyclic AMP binding proteins and cyclic AMP-dependent protein kinase from Blastocladiella emersonii.

The stoichiometry of cyclic AMP binding protein to cyclic AMP in sporulating cells of Blastocladiella emersonii and the resistance of protein-bound cyclic AMP to enzyme-catalyzed hydrolysis suggest that the distribution of cyclic AMP between free and protein-bound pools is an important factor in cyclic AMP metabolism. Most but not all of the cyclic AMP binding protein in sporulating cells is associated with a cyclic AMP-dependent protein kinase.     

Chitosome-like vesicles from gamma particles of Blastocladiella emersonii synthesize chitin

When gamma particles isolated from the aquatic fungus, Blastocladiella emersonii, were incubated in a supernatant derived from a homogenate of zoospores previously triggered to encyst with 50 mM KCl, they exhibited a three-fold increase in chitin synthetase activity and produced chitosome-like vesicles. Passage of such vesicles through a linear sucrose gradient resulted in a symmetrical distribution of chitin synthetase activity over a broad portion of the gradient, and the specific activity of the peak fraction was seven times greater than that of the gamma particles. After isopycnic sucrose gradient centrifugation, chitin synthetase activity occurred in a band of particles with a peak buoyant density of 1.18 g/cm³. Ultrastructural examination of negatively stained samples from the peak fraction revealed spheroidal, chitosome-like particles 70–120 nm in diameter. Suspension of these particles produced chitin microfibrils when incubated with uridine diphosphate N-acetylglucosamine, the substrate for chitin synthetase.Non Standard Abbreviations Used GlcNAc N-acetylglucosamine - UDP-GlcNAc uridine diphosphate N-acetylglucosamine - PYG agar 1.25 g of peptone, 1.25 g of yeast extract, 3 g of glucose, and 20 of agar per 1000 ml of water, the pH being adjusted to 6.8 with KOH after autoclaving - EGTA ethyleneglycol-bis (-aminoethylether)-N,N-tetraacetic acid     

Isolation and characterization of lipid globules from the zoospores of Blastocladiella emersonii

Lipid globules were isolated and characterized both chemically and morphologically. They were composed mainly of triglyceride and free sterol, which accounted for over 90% of the total globule content. Smaller amounts of diglyceride, carotenoid, free fatty acid, phospholipid and protein were found. No sterol esters or monoglycerides were detected. Morphologically, the isolated lipid globules resembled the lipid globules in situ. They were spherical, 0.4–1.5 m in diameter and lacked a trilaminar membrane.Non-Standard Abbreviations PL phospholipids - TG triglycerides - FS Tree sterols - DG diglycerides - SE sterol esters - MG monoglycerides     

The induction of sporulation in the aquatic fungus Blastocladiella emersonii is dependent on extracellular calcium

Induction of sporulation in Blastocladiella emersonii is absolutely dependent on extracellular calcium. Vegetative cells grown in media with or without calcium do not sporulate in media devoid of calcium or in CaCl₂ with EGTA. Calcium channel blockers, CoCl₂ and nifedipine, and ionophore A23187 inhibited the induction of sporulation. The calmodulin antagonists trifluoperazine and chlorpromazine inhibited the sporulation when present in the cultures at least 60 min after induction. So, calcium that is accumulated during growth is not sufficient or is not mobilized to initiate sporulation, and a calcium influx is likely to occur by type II calcium channel functions, essential for the response to nutritional starvation. A calmodulin-like protein has been suggested to mediate calcium events in sporulation.     

Autophosphorylation and rapid dephosphorylation of the cAMP-dependent protein kinase from Blastocladiella emersonii zoospores

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate and affinity chromatography on N6-(2-aminoethyl)-cAMP-Sepharose were used to analyze the cAMP-binding proteins present in cell-free extracts of Blastocladiella emersonii zoospores. In the presence of a mixture of protease inhibitors, 8-azido[32P]cAMP was specifically and quantitatively incorporated into a major protein band of Mr = 58,000, and three minor protein bands of Mr = 50,000, Mr = 43,000, and Mr = 36,000 respectively, after autoradiography following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. In the absence of the protease inhibitors, the Mr = 58,000 protein band was converted into the lower molecular weight cAMP-binding proteins, indicating a high sensitivity of the intact Mr = 58,000 protein band to endogenous proteases. The Mr = 58,000 protein corresponded to the regulatory subunit (R), of the cAMP-dependent protein kinase of zoospores, as shown by their identical behavior on DEAE-cellulose chromatography. The partially purified protein kinase incorporated 32P from [gamma-32P] ATP . Mg2+ into R as demonstrated by the specific adsorption of the 32P-labeled protein with N6-(2-aminoethyl)-cAMP-Sepharose. The incorporated 32P group was rapidly removed by endogenous phosphoprotein phosphatases in the presence of cAMP, as shown by pulse-chase experiments with [gamma-32P]ATP. Dephosphorylation of R-cAMP and rapid proteolysis may indicate two other mechanisms, in addition to cAMP, for the control of this protein kinase in vivo.     

Changes in cyclic AMP binding and protein kinase activities during growth and differentiation of Blastocladiella emersonii.

Multiple protein kinases in the water mould Blastocladiella emersonii are described. A cyclic AMP-independent protein kinase which prefentially phosphorylates casein remains unchanged during vegetative growth of the cells and in the two phases of differentiation: germination and sporulation. In contrast, cyclic AMP-dependent protein kinase activity and cyclic AMP binding components are induced during the sporulation.     

Partial purification and properties of D-glucosamine 6'-phosphate N-acetyltransferase from zoospores of Blastocladiella emersonii

A d-glucosamine 6-phosphate N-acetyltransferase from Blastocladiella emersonii zoospores was partially purified to a specific activity of 2.41 IU per mg of protein. Its pH optimum was 8.05 and its K(m) values were 2.4 x 10(-4) M d-glucosamine 6-phosphate and 0.38 x 10(-4) M Na(3)S-acetyl coenzyme A.     

Properties of the nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from Blastocladiella emersonii.

The nicotinamide adenine dinucleotide phosphate (NADP)-specific isocitrate dehydrogenase from Blastocladiella emersonii was purified. The enzyme was very unstable. Satisfactory stability was obtained in the presence of 0.2% ovalbumin. The enzyme had a molecular weight of about 100,000. It did not exhibit homotropic cooperativity for any of it substrates and was not affected by the allosteric modifiers citrate and adenosine monophosphate, diphosphate, and tri-phosphate. The substrate saturation studies showed both intercept and slope effects in Lineweaver-Burk plots. The Km values for isocitrate and NADP were found to be 20 and 10 muM, respectively. The product inhibition pattern was compatible with a random sequential reaction mechanism. The enzyme catalyzed the oxidative decarboxylation of isocitrate about six times better than the reductive carboxylation of alpha-ketoglutarate. The enzyme was inhibited by glyoxylate plus oxalacetate. Assays conducted in the presence of low Mg2+ concentrations exhibited a lag. This lag could be abolished by the addition of reduced NADP to the assay mixture.     

Structural and thermodynamic studies of KM+, a d-mannose binding lectin from Artocarpus integrifolia seeds

The KM+ lectin exhibits a novel and unusual circular dichroism (CD) spectrum that could be explained by a high proline content that would be inducing deformation of the beta-structure and/or unusual turns. KM+ was shown to be a very rigid lectin, which was very stable under a broad variety of conditions (urea, guanidine, hydrolysis, pH, etc.). Only incubation for 60 min at 333-338 K and extreme basic pH were able to induce conformational changes which could be observed by CD and fluorescence measurements. Data from CD are typical for protein denaturing associated with changes in the overall secondary structure. Data from high-performance size exclusion chromatography (SEC) showed that the denatured forms produced at pH 12.0 are eluted in clusters that co-elute with the native forms. A significant contribution from the tyrosines to the fluorescence emission upon denaturation was observed above 328 K. In fact at 328 K some broadening of the emission spectrum takes place followed by the appearance of a shoulder (approx. 305 nm) at 333 K and above. The sensitivity of tryptophan fluorescence to the addition of sugar suggests a close proximity of the tryptophan residues to the sugar binding site, K(a)=(2.9+/-0.6)x10(3) M(-1). The fraction of chromophore accessible to the quencher obtained is f(a)=0.43+/-0.08, suggesting that approximately 50% of the tryptophan residues are not accessible to quenching by d-mannose. KM+ thermal denaturation was found to be irreversible and was analyzed using a two-state model (N-->D). The results obtained for the activation energy and transition temperature from the equilibrium CD studies were: activation energy, E(a)=134+/-11 kJ/mol and transition temperature, T(m)=339+/-1 K, and from the fluorescence data: E(a)=179+/-18 kJ/mol and T(m)=337+/-1 K. Kinetic studies gave the following values: E(a)=108+/-18 kJ/mol and E(a)=167+/-12 kJ/mol for CD and fluorescence data, respectively.     

Alkaline phosphatase of Blastocladiella emersonii: partial purification and characterization.

Alkaline phosphomonoesterase (EC 3.1.3.1) activity from Blastocladiella emersonii, while displaying typically broad substrate specificity for phosphorylated organic compounds, exhibited nearly complete substrate preference for N-acetylglucosamine-6-phosphate over N-acetylglucosamine-1-phosphate. Enzyme in zoospore extracts was purified 43-fold by differential centrifugation followed by gel filtration (Sephadex G-200) and then by ion-exchange chromatography (diethylaminoethyl-cellulose). The partially purified enzyme displayed an apparent molecular weight (Sephadex G-200) of approximately 170,000. The activity of partially purified enzyme exhibited a pH optimum of pH 8.5, did not require a metal divalent cation, but was inhibitable by ethylenediaminetetraacetic acid. During the life cycle of the organism, the specific activity of the phosphatase decreased slightly during germination and early exponential growth but then increased about 4.5-fold during sporulation. B. emersonii alkaline phosphatase does not appear to be a repressible enzyme.