Yoshiaki Nosoh

Studies on an ATPase of thermophilic bacteria: I. Purification and properties

Abstract ATPase (EC 3.6.1.3) from Bacillus stearothermophilus is purified to a state in which it is homogeneous, both ultracentrifugally and electrophoretically. The sedimentation constant and molecular weight are 11.9 S and 280 000, respectively, and the optimum pH is 8.0 or 8.5, in a Tris-acetate or Tris-maleate buffer. Divalent cations such as Mg2+, Mn2+ and Cd2+ activate the enzyme but the activity is strongly inhibited by I2, N3− and CN−. Sulfhydryl reagents and uncouplers have no effect on the activity. ADP inhibits the activity and the mode of inhibition appears to be different from that of ATPase from mesophiles. The proline content of the ATPase is low (4.3 g/100 g protein) and the average amount of proline contained in the protein of the thermophilic bacteria is also low and similar to that of mesophilic bacteria. The enzyme contains about 20% of the α-helical conformation. The activity increases gradually with increasing temperature, but above 50° the activity increases abruptly. The enzyme has an optimum temperature of 65° and exhibits a remarkable resistance to thermal inactivation; these results may indicate that the enzyme is not only thermostable but also thermophilic. Thermodynamic quantities for the enzyme, calculated in the temperature range 30–65°, suggest that a conformational change in the enzyme protein occurs at the transition temperature, 55°.

Change in chemical composition of membrane of Bacillus caldotenax after shifting the growth temperature

Membranes from Bacillus caldotenax contain neutral lipids and phospholipids such as phosphatidylethanolamine, phosphatidyl glycerol and cardiolipin. Each of the lipids has almost the same fatty acid composition. When the growth temperature decreases, not only the fatty acid composition but also the lipid composition changes such that the membrane fluidity increases, and the composition of membrane-bound proteins also changes. On shifting the growth temperature from 65° to 45°C, the bacterium grows immediately with a doubling time at 45°C, but the compositions of proteins and lipids in membranes gradually change and reach the compositions typical of cells growing at 45°C one doubling time after the temperature shift, respectively. It is concluded that the change in chemical composition of membrane of the bacterium on the temperature shift from 65° to 45°C is not prerequisite for growth at 45°C.

Purification and properties of esterase from Bacillus stearothermophilus

Abstract An enzyme, which hydrolyzes p-nitrophenyl and m-carboxyphenyl esters of n-fatty acids, is purified from Bacillus stearothermophilus. The enzyme reaction obeys the Michaelis-Menten theory. The Michaelis constant (Km) decreases with increasing the length of carbon number of the acids, but the maximum velocity (V) is maximum for n-caproate. The enzyme is inhibited by diisopropyl fluorophosphate (DFP),2 and 1 mole of DFP reacts with 1 mole of the enzyme of the molecular weight of 42,000–47,000. The enzyme is considered to be carboxylic ester hydrolase (EC 3.1.1.1). The effects of temperature on Km or V for p-nitrophenyl n-caproate and on the inhibitor constant (Ki) for n-laurate suggest a thermal transition in the conformation of the enzyme protein at 55 °C. The enzyme is strongly inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and 5,5′-dithiobis (2-nitrobenzoic acid) at 65 °C, but less at 30 °C. The relationship between the inhibition of the activity by p-chloromercuribenzoate and temperature may suggest that a thermal transition of the enzyme protein accompanies some structural change around sulfhydryl group.

Purification and characterization of glucose-6-phosphate isomerase from Bacillus stearothermophilus

Abstract Glucose-6-phosphate isomerase (EC 5.3.1.9) has been purified from Bacillus stearothermophilus to a state in which it is homogeneous, both ultracentrifugally and electrophoretically. The sedimentation coefficient ( s 20, w ) and molecular weight of the enzyme are 7.4S and 172,000. The enzyme exhibits a maximum activity at 70 °. The optimum pH in 40 m m Tris-acetate buffer is 8.0–9.0, both at 40 and 65 °. The activity, when glucose-6-phosphate is used as substrate, is competitively inhibited by P i and 6-phosphogluconate, both at 40 and 65 °. The activity is stable at 50 °, and only a slight inactivation is observed upon exposure of the enzyme to 55 °. Exposure to 65 ° results in an appreciable loss of the activity. The activity is considerably protected from the inactivation at 65 ° by glucose-6-phosphate or 6-phosphogluconate. Thermodynamic quantities for the enzyme suggest that a structural change of the enzyme molecule occurs at the transition temperature, 55 °.

Requirement of Na+ in flagellar rotation and amino-acid transport in a facultatively alkalophilic Bacillus

Abstract A facultatively alkalophilic Bacillus strain YN-2000 grew well between pH 6.8 and 10, although the available size of the protonmotive force for the cells was high around neutral pH, but considerably low at alkaline pH. The energy source for the cellular functions of this bacterium grown at different pHs was analyzed to test if the cells switch the energy source between the protonmotive force and another source, depending upon the available size of the protonmotive force. The flagellar motors and the transport system of glutamine in this bacterium showed optimal activities at an alkaline pH and absolutely required the presence of Na + in the medium, irrespective of the growth pH. These results are consistent with the idea that, like obligate alkalophiles, the flagellar motors and an amino acid transport system of this bacterium are designed to utilize only the electrochemical potential gradient of Na + and have no switch of the energy source, irrespective of the available size of the protonmotive force during cell growth.

Effect of potassium and sodium ions on the cytoplasmic pH of an alkalophilic Bacillus

Abstract A bacterium belonging to Bacillus and tentatively designated as a facultatively alkalophilic Bacillus grows optimally at pH 7.5–10.2 (Koyama, N., Takinishi, H. and Nosoh, Y. (1983) FEMS Microbiol. Lett. 16 213–216). The internal pH of the bacterium was higher than the external pH in the pH range below 8, in the presence of KCl, but in the pH range above 9 the internal pH was lower than the external pH, both in the absence and presence of KCl. The internal pH of the bacterium in the presence of KCl was thus kept at 8.2–9.2 in the external pH range from 7.5 to 10.0. At pH 7.5, an extrusion of H+ in specific exchange for K+ was observed. The membrane vesicles, when energized with ascorbate plus tetramethylphenylenediamine, exhibited the formation of a transmembrane pH gradient (acid, interior), the magnitude of which became greater on increasing the extravesicular pH above 8 in the presence of NaCl. The alkalinization and acidification of the cytoplasm by the specific exchange of H+ for K+ and Na+, respectively, were suggested for a pH homeostasis of this bacterium.

Thermal properties of fructose-1,6-diphosphate aldolase from thermophilic bacteria

Abstract 1. 1. Fructose-1,6-diphosphoate aldolase (EC 4.1.2.13) from Bacillus stearothermophilus is purified to a state in which it is homogeneous, both ultracentrifugally and electrophoretically. 2. 2. The enzyme has a molecular weight of 60 600 and contains two atoms of Zn. Its activity is strongly inhibited by EDTA at 65°. These results may indicate that the thermophile aldolase is of the yeast type. 3. 3. The enzyme is activated by K+ and, to a lesser extent, by Na+. The optimum pH is 8.5–8.6 in a Tris or borate buffer. 4. 4. The amino acid composition is almost similar to that of the muscle-type aldolase and contains 30% of the α-helical conformation. 5. 5. The enzyme exhibits a maximum activity at 70° and is stable on exposure up to 45°. Slight inactivation is observed on treatment of the enzyme at 55–65°, and exposure to 75° for 30 min results in almost complete inactivation. 6. 6. Thermodynamic quantities for the enzyme suggest that a structural change in the enzyme molecule occurs at the transition temperature, 50–53°. The relationship between the inhibition of the activity by EDTA and temperature may suggest that the change occurs around the active site of the enzyme.

Purification and properties of glutamine synthetase from Bacillus stearothermophilus

Abstract Glutamine synthetase (EC 6.3.1.2) has been purified from Bacillus stearothermophilus. The molecular weight of the enzyme was found to be 630 000, and that of a component obtained on treating the enzyme with 4 M urea or 1% sodium dodecyl-sulfate plus 10 mM 2-mercaptoethanol 540 000 or 500 000, respectively, suggesting that the enzyme consists of 12 subunits. The enzyme requires divalent cations for activity, Mg2+ being the most effective activator. The pH and temperature optima in the presence of Mg2+ or Mn2+ are 7.3 or 6.5 and 70 or 75 °C, respectively. The enzyme is inactivated on exposure to 70 °C, but the inactivation is partially protected by Mg2+ (Mn2+ or glutamate) and completely by Mg2+ (Mn2+), NH4Cl and glutamate. Thermodynamic quantities for the enzyme reaction show a conformational transition at 58 °C. Glycine, alanine, serine, tryptophan, histidine, AMP and CTP inhibit the enzyme in the presence of Mg2+ or Mn2+. The inhibition of the Mg2+- or Mn2+-activated enzyme by these compounds seems to be cumulative, except for the combined effects of amino acids on the Mn2+-activated enzyme. Circular dichroism analyses of the enzyme show an α-helix, s-structure and unfolded conformation. Addition of Mg2+ or Mn2+ results in an increase of the α-helix content accompanied by a decrease of the unfolded conformation content.

Purification and properties of NADH dehydrogenase from an alkalophilic bacillus

Abstract NADH dehydrogenase (NADH:2,6-dichlorophenolindophenol oxidoreductase, EC 1.6.99.3) in membranes of an alkalophilic Bacillus was solubilized by EDTA or easily detached from membranes during lysozyme treatment of the cells stored at −20°C. Cell-free extracts of the frozen cells were treated with EDTA, and the supernatant was fractionated by (NH 4 ) 2 SO 4 and subjected to 5′-AMP-Sepharose column chromatography. The enzyme thus purified was homogeneous as judged from polyacrylamide gel electrophoresis in the absence and presence of SDS. The enzyme has a molecular weight of 138 000 and consists of two subunits of equal molecular size (65 000). The chemical compositions, including amino acid composition, of the protein were determined. The enzyme contains 1 mol FAD per subunit, and seems not to possess an iron-containing prosthetic group and phospholipids. The protein is a composite of α-helix, β-structure and random coil. The enzyme is specific for NADH as electron donor, and can utilize 2,6-dichlorophenolindophenol, ferricyanide, menadione and cytochrome c as electron acceptors. The enzyme activity was remarkably stimulated by monovalent cations such as K + and Na + . The enzyme activity was slightly increased by phospholipids.

Purification and properties of ATPase from an alkalophilic Bacillus

Abstract ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (ϵ). The subunit structure is suggested to be α3β3γδϵ. The enzyme is activated by Mg2+ and Ca2+. The pH optima of the enzyme with 0.1 and 2.0 m m Mg2+ are 9 and 6, and those with 1 and 10 m m Ca2+ are 8–9 and 7, respectively. Ca2+-ATPase hydrolyzes only ATP, whereas Mg2+-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and Km of the enzyme with ATP in the presence of 10 m m Ca2+ or 0.6 m m Mg2+ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 m m , respectively. The enzyme with Mg2+ is appreciably activated by HCO−3. Relationship of the ATPase to the active transport system in the bacterium is suggested.