Metabolism of D-glucose in a wall-less mutant of Neurospora crassa examined by 13C and 31P nuclear magnetic resonances: effects of insulin

13C NMR and 31P NMR have been used to investigate the metabolism of glucose by a wall-less strain of Neurospora crassa (slime), grown in a supplemented nutritionally defined medium and harvested in the early stationary stage of growth. With D-[1-13C]- or D-[6-13C]glucose as substrates, the major metabolic products identified from 13C NMR spectra were [2-13C]ethanol, [3-13C]alanine, and C1- and C6-labeled trehalose. Several observations suggested the existence of a substantial hexose monophosphate (HMP) shunt: (i) a 70% greater yield of ethanol from C6- than from C1-labeled glucose; (ii) C1-labeled glucose yielded 19% C6-labeled trehalose, while C6-labeled glucose yielded only 4% C1-labeled trehalose; (iii) a substantial transfer of 13C from C2-labeled glucose to the C2-position of ethanol. 31P NMR spectra showed millimolar levels of intracellular inorganic phosphate (Pi), phosphodiesters, and diphosphates including sugar diphosphates and polyphosphate. Addition of glucose resulted in a decrease in cytoplasmic Pi and an increase in sugar monophosphates, which continued for at least 30 min. Phosphate resonances corresponding to metabolic intermediates of both the glycolytic and HMP pathways were identified in cell extracts. Addition of insulin (100 nM) with the glucose had the following effects relative to glucose alone: (i) a 24% increase (P less than 0.01) in the rate of ethanol production; (ii) a 38% increase (P less than 0.05) in the rate of alanine production; (iii) a 27% increase (P less than 0.05) in the rate of glucose disappearance. Insulin thus increases the rates of production of ethanol and alanine in these cells, in addition to increasing production of CO2 and glycogen, as previously shown.     

Intracellular Localization of Nitrate Reductase in Neurospora crassa

Nitrate reductase was localized in mycelial cells of Neurospora crassa by immunohistochemical labeling with ferritin. The enzyme is found in the cell wall-plasmalemma region and in the tonoplast membranes.     

Intracellular localization of Neurospora crassa endo-exonuclease and its putative precursor.

Endo-exonuclease of rapidly growing mycelia of Neurospora crassa was found to be distributed in a ratio of about 1.6:1 in vacuoles and in mitochondria where it is associated with the inner membrane. Although the activity in vacuoles was readily released by osmotic shock, very little of that in mitochondria was released by this method. The mitochondrial activity was partially (60 to 70%) released by sonication, and the remaining activity was solubilized in the presence of Triton X-100. An inactive form of endo-exonuclease, activated in vitro by treatment with trypsin, is present in mycelia at a level over four times that of active enzyme. It was found to be distributed in a ratio of about 2.5:1 in the cytosol and in the inner membrane of mitochondria. The mitochondrial protein was more tightly bound than the active enzyme. Very little of the inactive enzyme was released by sonication, but it was solubilized in the presence of Triton X-100. The intracellular distribution of active and inactive forms of endo-exonuclease differs in a mutagen-sensitive mutant of Neurospora crassa (uvs-3) which shows many pleiotropic effects. The most striking difference in distribution is in the mitochondria where endo-exonuclease is present almost entirely in the inactive form at a level 30% higher than in wild-type mitochondria.     

Cobalt resistance in Neurospora crassa: overproduction of a cobaltoprotein in a resistant strain

A cobalt-resistant strain of Neurospora crassa (cor) is 20-fold more resistant to Co when compared with the wild type. DEAE-cellulose and metal-chelate affinity chromatography of cell-free extracts separated cobalt into protein-bound and free ionic fractions. In N. crassa cor about 80% cobalt of cell-free extracts was protein bound while the same in wild type was only 25%. Cobalt content of the protein-bound fraction increased with time and cobalt concentration in the growth medium, and was not influenced by related metal ions. A cobaltoprotein (CBP) which is overproduced in N. crassa cor and constitutes up to 12% of total protein of extracts was purified. CPB is a brown coloured (absorption peaks at 275, 350 and 440nm), small molecular weight glycoprotein (Mr 8200 daltons) with 28 - 30% carbohydrate (mannose). CBP has 70 g cobalt mg-1 protein. Cysteine, glycine, glutamic acid and aspartic acid are the major amino acid constituents. The role of CBP in cobalt resistance is discussed in relation to other known metalloproteins involved in resistance. © Rapid Science 1998.     

Cobalt-resistance in wall-less mutant (fz; sg; os-1) of Neurospora crassa

A cobalt-resistant wall-less mutant (slime) of Neurospora crassa was obtained by repeated sub-culturing of the sensitive wall-less mutant (W-sl) on agar medium containing toxic concentrations of cobalt. Resistance was stable on culturing Cor-sl on cobalt-free medium up to 15 weekly subcultures. Cor-sl is 10-fold more resistant to cobalt when compared to W-sl. It is also cross-resistant to Cu (10-fold) and Ni (3-fold). Cobalt accumulated by Cor-sl during growth and in short-term uptake experiments was lower when compared to W-sl. Cells previously loaded with cobalt was released into medium in both mutants, while in case of Cor-sl most of cobalt taken up (>80%), was released back into the medium when compared to W-sl. Metabolic inhibitor (Sodium azide) and magnesium ions inhibited cobalt uptake in both the mutants. Fractionation of cell-free extracts showed that most of the cobalt (70%) taken up by Cor-sl was bound to an inducible protein fraction which bound to DEAE-Cellulose, while in W-sl only 20% of cobalt was associated with this fraction. Subcellular localization of cobalt in W-sl indicated most of it to be cytoplasmic (70%) while nuclei and mitochondria had 10% and 5% respectively. In case of Cor-sl, mitochondrial cobalt accounted for only 2% while no significant differences were noted for other fractions. Our data implicate both transport block and intracellular sequestration of cobalt to play a major role in resistance.     

Intracellular localization and properties of glycerokinase and glycerophosphate dehydrogenase in Neurospora crassa.

Glycerokinase and glycerol-3-phosphate dehydrogenase activities have been examined in cell extracts obtained from Neurospora crassa after growth in media containing glycerol. The glycerokinase is located in the cytosol and has been partially purified by ion exchange and gel-filtration chromatography. The molecular weight of the enzyme has been estimated by sucrose density centrifugation to be approximately 120,000. No effect of either fructose-1,6-bisphosphate or other sugar phosphates on enzyme activity was observed. The G3P dehydrogenase activity in cell extracts is apparently catalyzed by a flavin-linked enzyme as no dependence for either NAD⁺ or NADP⁺ could be demonstrated. The enzyme is located primarily in the mitochondria and is not removed from mitochondrial membranes by treatment with digitonin. Separation of digitonin-treated mitochondria on discontinuous sucrose gradients indicated that the enzyme is located on the mitochondrial inner membrane. The synthesis of both enzymes is under some form of catabolite repression since increased specific activities could only be observed in cells grown on acetate, but not glucose, sucrose, or xylose.     

Mechanism of nickel resistance in a cobalt-resistant wall-less mutant of Neurospora crassa (fz; sg; os-1)

A cobalt-resistant wall-less mutant of N. crassa (Cor-sl) characterized previously was also found to be 3-fold more resistant to nickel when compared to the parent wall-less mutant (W-sl). The Cor-sl strain accumulates relatively lower amounts of nickel when compared to W-sl. Sub-cellular fractionation showed significant quantities of nickel to be associated with nuclear and mitochondrial fractions in both the wall-less mutants. However significant differences were observed in vacuolar fractions of W-sl and Cor-sl strains. Fractionation of cell-free extracts on Sephadex G-10 column resolved nickel into two peaks, of which the peak II in Cor-sl constituted 70% of nickel, while the same in W-sl was about 30%. A 3-fold increase in histidine content was observed in case of Cor-sl as compared to W-sl strain, suggesting its role in Ni-resistance.     

The monensin-mediated transport of sodium ions through phospholipid bilayers studied by 23Na-NMR spectroscopy

The monensin-mediated transport of sodium ions through the walls of large unilamellar vesicles of egg phosphatidylcholine was studied using 23Na-NMR and aqueous shift reagents. The transport is dynamic on the NMR time-scale and is strictly first order in monensin over the concentration ranges studied indicating that transport occurs by a 1:1 Na+-ionophore complex. Transport appears to be inhibited by increasing concentrations of Na+.     

Chorismate synthase of Neurospora crassa: a flavoprotein

Chorismate synthase is purified from Neurospora crassa. The final step is accomplished by preparative electrophoresis. Its purity is estimated at ≥90% on the basis of analytical polyacrylamide gel electrophoresis. The enzyme appears to be active in at least two multimeric states, with a subunit molecular weight of ~55,000. The purified enzyme preparation is absolutely dependent on the presence of a reducing system, which can readily be provided under aerobic conditions by NADPH plus FMN or under stringent anaerobic conditions by dithionite. The following evidence implicates a physiological role for FMN in N. crassa chorismate synthase activity: (a) a preferential stimulation of activity by NADPH and FMN over other pyridine and flavin nucleotides, respectively, in both impure and purified enzyme preparations; (b) an alteration of the Chromatographic pattern of the enzyme on diethylaminoethylcellulose by the addition of FMN to the elution buffer; (c) an apparent binding of FMN to the enzyme as exhibited by gel filtration in the presence of the substrate, 3-enolpyruvylshikimate 5-phosphate; (d) a requirement for preliminary incubation with FMN, in concert with the substrate, to eliminate a reaction lag (i.e., to activate the enzyme); (e) a substrate-dependent diaphorase activity exhibited by purified enzyme preparations in the presence of FMN and NADPH. The observed activation and alteration of Chromatographic behavior of chorismate synthase by FMN suggest that the flavin nucleotide influences the conformation of the enzyme. The ability to replace NADPH and FMN with dithionite suggests that FMN mediates the flow of electrons from a source of reducing power (NADPH) to some enzymic site important to the function of the enzyme. Hence, the diaphorase activity which is observed as intrinsic to chorismate synthase of N. crassa may be significant from the standpoint of catalysis or may have importance as a regulatory mechanism.     

The modifying effects of strain and age on the mutagenic specificity of ultraviolet light in Neurospora crassa

Summary Two derivatives of K3/17 ad-3A 38701; inos 37401 of Neurospora crassa are described which show opposite specific reversional responses to UV. Both derivatives carry the same two auxotrophic alleles and appear to differ only in a single gene which influences the pattern of mutagen specificity. The differences between the derivatives only develop after the cultures have been aged for two to four weeks. Various possible explanations are considered.     

Unstable S-Adenosylmethionine synthetase in an ethionine-resistant strain of Neurospora crassa.

A pleitropic ethionine-resistant mutant of Neurospora contains an S-adenosylmethionine synthetase that is labile to heat and dialysis but exhibits normal kinetics for methionine and ethionine.     

Interrelationships in trace-element metabolism in metal toxicities in a cobalt-resistant strain of Neurospora crassa

A strain of Neurospora crassa was isolated by training the mould to grow on media containing high concentrations of Co(2+). This strain, the Co(R) strain, exhibited approximately tenfold the resistance of the parent strain to Co(2+) and Ni(2+) but not to Zn(2+) or Cu(2+). Co(2+) toxicity in the Co(R) strain was reversed by Mg(2+) but not by Fe(3+). Also, Co(2+) did not affect iron metabolism in this strain. It is suggested that the mechanism of resistance in the Co(R) strain involves an alteration in the pattern of iron metabolism such that the latter is no longer adversely affected by toxic concentrations of Co(2+). The Co(R) strain is genetically stable and is most probably a result of a resistance mutation in N. crassa induced by Co(2+).