Search

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pHcyt) and vacuolar pH (pHvac) values of 7.6 and 6.2, respectively, when the extracellular pH (pHex) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a ΔpH over the cytoplasmic membrane of 6.6–6.7 was reached (pHex 0.7–0.8). Maintenance of these large pH differences was possible without increased respiration compared to pHex 5.8. Perfusion in the presence of various hexoses and pentoses (pHex 5.8) revealed that the magnitude of ΔpH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger ΔpH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 µm CCCP), transient (2 µm CCCP) or permanent (10 µm CCCP) collapse of the vacuolar membrane ΔpH. Nonlethal levels of the metabolic inhibitor azide (N3–, 0.1 mm) caused a transient decrease in pHcyt that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pHex (1.8) and a high acid-secreting capacity, pHcyt and pHvac values were still well maintained (pH 7.5 and 6.4, respectively).

The Aspergillus niger hexokinase gene hxkA has been cloned by heterologous hybridisation using the Aspergillus nidulans hexokinase gene as a probe. The DNA sequence of the gene was determined, and the deduced amino acid sequence showed significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to those of the budding yeasts. The encoded protein was purified from a multicopy hxkA transformant, and extensively characterised. The hexokinase protein has a molecular mass of 54090, a pI of 4.9 and is a homodimer. D-Glucose, the glucose analogue 2-deoxy-D-glucose, D-fructose, D-mannose and D-glucosamine are phosphorylated by hexokinase, whereas the hexoses D-galactose, L-sorbose, methyl alpha-D-glucoside and the pentoses L-arabinose and D-xylose are not. The enzyme has high affinity for glucose (Km = 0.35 mM at pH 7.5) and for fructose (Km = 2.0 mM at pH 7.5) and is inhibited by ADP. The enzyme is strongly inhibited by physiological concentrations (0.1-0.2 mM) of trehalose 6-phosphate, which may be of importance for in vivo regulation of the enzyme. Inhibition of A. niger hexokinase by trehalose 6-phosphate is competitive towards the sugar substrate (Ki = 0.01 mM). Based on the kinetic constants of hexokinase and glucokinase their relative contribution to in vivo glucose phosphorylation was calculated and found to be strongly dependent on intracellular pH and glucose concentration. At pH 7.5 glucokinase is predominant, whereas at pH 6.5 hexokinase is predominant at glucose concentrations higher than 0.5 mM. Expression of the hexokinase and the glucokinase gene requires active carbon metabolism. Also on carbon sources which are not substrates for hexokinase or glucokinase, clear expression is observed. The hexokinase and glucokinase enzymes are quite stable in vivo. Even in the absence of transcription, active glucokinase and hexokinase remain present in the cells at almost the same level for at least 3-4 h after depletion of the carbon source.

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pH(cyt)) and vacuolar pH (pH(vac)) values of 7.6 and 6.2, respectively, when the extracellular pH (pH(ex)) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a Delta pH over the cytoplasmic membrane of 6.6-6.7 was reached (pH(ex) 0.7-0.8). Maintenance of these large pH differences was possible without increased respiration compared to pH(ex) 5.8. Perfusion in the presence of various hexoses and pentoses (pH(ex) 5.8) revealed that the magnitude of Delta pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger Delta pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 microm CCCP), transient (2 microm CCCP) or permanent (10 microm CCCP) collapse of the vacuolar membrane Delta pH. Nonlethal levels of the metabolic inhibitor azide (N3-, 0.1 mm) caused a transient decrease in pH(cyt) that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pH(ex) (1.8) and a high acid-secreting capacity, pH(cyt) and pH(vac) values were still well maintained (pH 7.5 and 6.4, respectively).

The Aspergillus niger glucokinase gene glkA has been cloned using a probe generated by polymerase chain reaction with degenerate oligonucleotides. The DNA sequence of the gene was determined, and the deduced amino acid sequence shows significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to the Saccharomyces cerevisiae glucokinase protein. The encoded protein was purified from a multicopy glkA transformant, and extensively characterised. The protein has a molecular mass of 54536 Da and a pI of 5.2. The enzyme has high affinity for glucose (K(m) 0.063 mM at pH 7.5) and a relatively low affinity for fructose (K(m) 120 mM at pH 7.5), and in vivo fructose phosphorylation by glucokinase is consequently negligible. The configurations at C1 and C4 of the substrate appear to be essential for substrate specificity. The A. niger glucokinase shows non-competitive inhibition by ADP towards ATP and uncompetitive inhibition by ADP towards glucose. The kcal (turnover number) decreases rapidly below pH 7.5 (56% at pH 7.0 and 17% at pH 6.5) and this may have important implications for the in vivo regulation of activity. In addition, proof is provided for the presence of a second hexosephosphorylating enzyme in A. niger. This enzyme is probably a hexokinase, since unlike glucokinase, this activity is inhibited by trehalose 6-phosphate.