Pyruvate carboxylase from Aspergillus nidulans. Effects of regulatory modifiers on the structure of the enzyme

Pigment-binding protein of the facultatively phototrophic bacterium Rhodospeudomonas capsulata could be selectively synthesized in toluene-treated cells as well as in homologous and heterologous cell-free translation systems by isolated polysomes. It is shown that the pigment-binding polypeptides of the light-harvesting complexes are encoded by messenger RNA of extreme longevity. The dependence of their synthesis on the concomitant synthesis of tetrapyrroles was demonstrated in the toluene-treated cells. The large Mr-28 000 polypeptide of the reaction center and the Mr-10 000 pigment-binding polypeptide of the light-harvesting complex II were found to be synthesized by free (water-soluble) polysomes without a cleavable 'leader' or 'signal' peptide [reviewed by W. Wickner (1979) Annu. Rev. Biochem. 48, 23-45]. The Mr-10 000 polypeptide, as synthesized in vitro, was studied in more detail. Unlike the membrane-assembled polypeptide in vivo it was insoluble in an organic solvent mixture (chloroform/methanol 1:1, v/v). After detergent denaturation in the presence of membrane isolated from the organism it became organic-solvent-soluble. Obviously the polypeptide could be induced to assume alternative conformations in which its apolar residues were either exposed to the solvent or buried within. These findings, in agreement with Wickner's hypothesis, indicate that the Mr-10 000 polypeptide may enter the lipid bilayer by a 'membrane-triggered' conformational change.     

Protoplasts from Aspergillus nidulans.

A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.     

Pyruvate carboxylase from rainbow trout liver

Summary A method is described for the partial purification of pyruvate carboxylase from rainbow trout liver. The enzyme has a pH optimum of about 8.0, possesses an absolute requirement for activation by acetylCoA, and prefers MgATP over other nucleoside triphosphates. K⁺ causes a decrease in the apparentK _m for HCO ₃ ^– . AcetylCoA activation shows positive cooperativity withK _a=0.072 mM andn _H=1.78 at pH 7.7, 2.5 mM free Mg²⁺, 100 mM K⁺, and saturating concentrations of substrates. A high acetylCoA concentration causes a decrease in the apparentK _m values for MgATP and HCO ₃ ^– and a biphasic double reciprocal plot with pyruvate as the varied substrate. MgADP and AMP are competitive inhibitors with respect to MgATP. The enzyme shows a U-type response to the adenylate energy charge and retains considerable activity throughout a wide range of energy charge values. It is proposed that intramitochondrial acetylCoA concentration and the adenylate energy charge control the rate of pyruvate carboxylation in vivo.Abbreviations DTT dithiothreitol - PMSF phenylmethylsulfonylfluoride     

Purification and properties of aldehyde dehydrogenase from Aspergillus nidulans

• 1.1. Aspergillus nidulans produces aldehyde dehydrogenase (ALD-DH) only when grown in the presence of ethanol, threonine or acetoacetic acid as inducer. Enzyme formation is inhibited by glucose in the growth medium.
• 2.2. ALD-DH is purified by a rapid procedure using Cibacron Blue Affinity Chromatography with specific inhibitoe elution by NAD plus 2:2′ dithiodipyridine or 2:4 disulfiram.
• 3.3. The pure native enzyme has a M_r=265,000 and a subunit M_r of 540,000. Its optimum pH is 8.5; its preferred substrate is acetaldehyde and it can use either NAD or NADP.

     

Purification, properties and primary structure of thioredoxin from Aspergillus nidulans.

This paper reports the purification and the properties of a thioredoxin from the fungus Aspergillus nidulans. This thioredoxin is an acidic protein which exhibits an unusual fluorescence emission spectrum, characterized by a high contribution of tyrosine residues. Thioredoxin from A. nidulans cannot serve as a substrate for Escherichia coli thioredoxin reductase. Corn NADP-malate dehydrogenase is activated by this thioredoxin in the presence of dithiothreitol, while fructose-1,6-bisphosphatase is not. The amino acid sequence of Aspergillus thioredoxin was determined by automated Edman degradation after cleavage with trypsin, SV8 protease, chymotrypsin and cyanogen bromide. The masses of tryptic peptides were verified by plasma-desorption mass spectrometry. The mass of the protein was determined by electrospray mass spectrometry and shown to be in agreement with the calculated mass derived from the sequence (M(r) = 11,564). Compared to thioredoxins from other sources, the protein from A. nidulans displays a maximal sequence similarity with that from yeast (45%).     

Isolation of DNA from Aspergillus nidulans.

A procedure for isolation of DNA from Aspergillus nidulans on a preparative scale is described. Mechanical disruption of lyophilized material in high-salt medium and treatment with proteinase K, followed by sedimentation of the lysate into saturated CsC1 solution yielded pure, highly polymerized DNA.     

Purification and properties of purine hydroxylase II from Aspergillus nidulans

Purine hydroxylase II from Aspergillus nidulans has been purified to near homogeneity. The enzyme has a pI of 5.7, a molecular weight of 300,000, and two subunits with molecular weight of 153,000 each. The enzyme contains 2 FAD, 2 molybdenum atoms, and 4 (2 Fe-2S) iron-sulfur centers per molecule and exhibits broad specificity for reducing and oxidizing substrates. Among the more notable characteristics are the ability to oxidize hypoxanthine and nicotinic acid but not xanthine and virtually complete inactivity with oxygen. Moreover, while the enzyme is inactivated by borate and methanol, it is very resistant to cyanide and arsenite and it not inactivated by allopurinol. At infinite concentrations of reducing and oxidizing substrates, the Km for hypoxanthine was 119 microM, for nicotinic acid was 136 microM, and for NAD+ was 525 microM.     

Regulatory properties of a fructose 1,6-bisphosphatase from the cyanobacterium Anacystis nidulans.

A fructose 1,6-bisphosphatase (EC 3.1.3.11) (FBPase) was purified over 100-fold from Anacystis nidulans. At variance with a previous report (R. H. Bishop, Arch. Biochem. Biophys. 196:295-300, 1979), the regulatory properties of the enzyme were found to be like those of chloroplast enzymes rather than intermediate between chloroplast (photosynthetic) and heterotrophic FBPases. The pH optimum of Anacystis FBPase was between 8.0 and 8.5 and shifted to lower values with increasing Mg2+ concentration. Under the experimental conditions used by Bishop, we found the saturation curve of the enzyme to be sigmoidal for Mg2+ ions and hyperbolic for fructose 1,6-bisphosphate. The half-maximal velocity of the Anacystis FBPase was reached at concentrations of 5 mM MgCl2 and 0.06 mM fructose 1,6-bisphosphate. AMP did not inhibit the enzyme. The activity of the FBPase was found to be under a delicate control of oxidizing and reducing conditions. Oxidants like O2, H2O2, oxidized glutathione, and dehydroascorbic acid decreased the enzyme activity, whereas reductants like dithiothreitol and reduced glutathione increased it. The oxido-reductive modulation of FBPase proved to be reversible. Reduced glutathione stimulated the enzyme activity at physiological concentrations (1 to 10 mM).l The reduced glutathione-induced activation was higher at pH 8.0 than at pH 7.0.     

Pyruvate carboxylase from a thermophilic Bacillus: some molecular characteristics.

Analysis of the native enzyme and of the subunits produced upon its denaturation shows that pyruvate carboxylase from a thermophilic Bacillus is a tetramer with a molecular weight (mean value) of 558,000 and that the four polypeptide subunits are probably identical. The three functions (carboxyl carrier, carboxylation, and carboxyl transfer) in the pyruvate carboxylation reaction must therefore reside in this quarter-molecular polypeptide. The enzyme molecule contains four atoms of zinc and four molecules of D-biotin, and in the electron microscope the disposition of its four subunits presents a rhombic appearance. Reaction of the denatured enzyme with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reveals 10 sulfhydryl groups/subunit. In the native enzyme less than one of these groups reacts with DTNB. By contrast, all of these groups (11/subunit) of the native chicken liver pyruvate carboxylase are accessible to DTNB. The thermophile enzyme is also more resistant to other sulfhydryl reagents and to denaturation under certain conditions than the avian enzyme.     

Supersuppressors in Aspergillus nidulans.

Summary Simultaneous reversion of mutations in two different Aspergillus nidulans loci adA and metG was found to be due monogenic suppressor mutations. Prelimirary evidence for the existance of supersuppressors in A. nidulans is presented.     

Electrofusion of protoplasts from Aspergillus nidulans

Abstract Electrical parameters were determined and quantified for the stimulation of the optimum alignment and fusion of Aspergillus nidulans protoplasts. In a non-homogeneous alternating electrical field A. nidulans protoplasts aligned to form pearl chains associated with the electrodes of the fusion chamber. Most protoplasts were in pearl chains in an alignment field frequency of 3.0 MHz but maximum pair formation occurred at 1.0 MHz. At a field strength between 100 and 1000 V · cm⁻¹ pearl chain formation occurred with minimal protoplast rotation or lysis. The application of DC pulses resulted in protoplast fusion. Most fusion events were observed after two 500 V · cm⁻¹ DC pulses with a 0.5 s interpulse period. Using 1 × 10³ protoplasts · cm⁻³ in a 7 μm fusion chamber a maximum of 17.2 ± 2.0% fusion events were achieved.     

A New Metabolite from Aspergillus nidulans

Among the bacteria isolated from polluted water and viscid sludges in the factories manufacturing sweet potato starch, a group of strains was ascertained to be capable of producing slimy materials keeping fairly stable viscosity through the alterations in pH. Representative strain A-1 of the group was assigned to Agrobacterium radiobacter. The polysaccharide produced by culturing the strain in the medium containing glucose, yeast extracts and CaCO₃ was estimated to be Gal: Glc: succinic acid: pyruvic acid = 1: 7.2~7.3: 1: 0.85 in a molar ratio. The IR spectra, basicity and other determinations indicated that the one of the moieties showing acidic function was succinic acid linking in ester bond, and another one was pyruvic acid linking to glucose in ketal.