Electrophoresis of a Corynebacterium glutamicum membrane preparation in the presence of sodium dodecyl sulfate, followed by staining for peroxidase activity (heme staining), showed only one band at ...about 28 kDa. This 28 kDa protein was purified from C. glutamicum membranes by chromatography in the presence of decylglucoside using DEAE–Toyopearl and hydroxylapatite columns, as the sole c-type cytochrome in the bacterium. The cytochrome showed an alpha band at 551 nm, and its Em, 7 was about 210 mV. A QcrCAB operon encoding the subunits of a putative quinol cytochrome c reductase was found 3′-downstream of ctaE encoding subunit III of cytochrome aa3 in the C. glutamicum genome. The deduced amino acid sequence of qcrC, composed of 283 amino acid residues, contained two heme C-binding motifs and was in agreement with partial peptide sequences obtained from the 28 kDa protein after V8 protease digestion. We propose to name this protein cytochrome cc. The presence of cytochrome cc is a common feature of high G+C content Gram-positive bacteria, since we could confirm this protein by electrophoresis; homologous QcrCAB operons are also known in Mycobacterium and Streptomyces. QcrA and qcrB of C. glutamicum encode the Rieske Fe–S protein and cytochrome b, respectively, although these proteins were not co-purified with cytochrome cc. The phylogenetic tree of cytochromes b and b6 show that C. glutamicum cytochrome b, along with those of other bacteria in the high G+C group, is rather different from the Bacillus counterparts, but highly similar to the Deinococci and Thermus cytochromes. This indicates that there is a fourth group of bacteria in addition to the three clades: proteobacterial cytochrome b, cyanobacterial b6 and green sulfur-low G+C Gram-positive bacteria.
1. Five subunits (alpha, beta, gamma, delta, and epsilon) of an ATPase from a thermophilic bacterium PS3 were purified in the presence of 8 M urea by ion exchange chromatography. Then the ATPase ...activity was reconstituted by mixing the subunit solutions and incubating them at 20-45 degrees, at pH 6.3 to 7.0. 2. Mixtures containing beta + gamma or alpha + beta + delta regained ATP-hydrolyzing activity, but mixtures of alpha + beta and beta + delta did not. Combinations not including beta were all inactive. 3. The ATPase activity reconstituted from alpha + beta + delta was thermolabile and insensitive to NaN3, whereas the activities obtained from mixtures containing beta and gamma were thermostable and sensitive to NaN3, like the native ATPase. 4. The assemblies containing both beta and gamma subunits had the same mobility as the native ATPase molecule on gel electrophoresis, those without the gamma subunit moved more rapidly toward the anode. 5. Subunits epsilon and delta did not inhibit the ATPase activity of either the assembly (alpha + beta + gamma) or the native ATPase.
Cytochrome c oxidases, purified from bovine heart and the thermophilic bacterium PS3, were irradiated with a high‐energy electron beam. The proton transport activities of both preparations and their ...electron transfer activities decreased as single exponential functions of the radiation dosage. Applying the target theory with alkaline phosphatase as an internal standard, the following functional molecular weights were obtained for cytochrome c oxidation and H+ pumping: 63‐73 kd and 160‐220 kd, respectively, for the bovine enzyme, and 80‐100 kd and 190‐230 kd for the PS3 enzyme. The results suggest that a dimer structure is necessary for H+ pumping, while a core part of monomer (presumably the largest two subunits, i.e. subunits I and II) is sufficient for cytochrome c oxidation.
Cytochrome d was spectroscopically detected in membrane fractions of the amino-acid-fermenting, high-G+C gram-positive bacterium Corynebacterium glutamicum. Inhibition of NADH oxidase activity in the ...membranes by cyanide suggested that the main terminal respiratory oxidase during the stationary phase was a type of cytochrome bd. Cytochrome bd-type quinol oxidase, purified from the membranes, was composed of two subunits. Its reduced form showed absorption peaks at 627, 595, and 560 nm, which were due to haem d, high-spin protohaem, and low-spin protohaem, respectively. The air-oxidised form showed a peak at 645 nm, which might be due to oxygenated ferrous haem d. The spectral features and the size of subunit I are more similar to the properties of cytochromes bd from Proteobacteria, such as Escherichia coli, than to those of cytochrome bd from low-G+C gram-positive bacteria, such as Bacillus stearothermophilus. The menaquinol oxidase acitivity of the purified cytochrome bd was low, but was enhanced about fivefold by pre-incubating the enzyme with menaquinones. The order of effectiveness of quinols as oxidase substrates was clearly different from that of quinones as the activators of enzyme activity. Furthermore, activation was destroyed by ultraviolet irradiation of the pre-incubated enzyme and then restored by a second incubation with menaquinone. These results indicate that the enzymatic properties of this new oxidase are more similar to the properties of cytochromes bd from low-G+C gram-positive bacterial than to those of proteobacterial counterparts. They also suggest that the enzyme has a second quinone-binding site essential for full activity, in addition to the active centre for substrate oxidation. By using probes based on partial peptide sequences of the subunits, the genes for the two subunits of C. glutamicum cytochrome bd were cloned. The deduced amino acid sequence demonstrated that subunit I lacks the C-terminal half of the Q loop and that the primary structure of C. glutamicum cytochrome bd is more similar to that of other gram-positive bacteria than to proteobacterial cytochromes bd.
The proton-translocating ATPase of the thermophilic bacterium PS3 was incorporated into a planar phospholipid bilayer, and its electrogenicity was directly demonstrated. The enzyme (TF0F1) consists ...of a catalytic portion, F1, and a membrane-integrated portion, Fo. A short-circuit current of up to 1 nA/cm2 was generated upon the addition of ATP, and the direction of the current indicated the flow of positive charges from the TF1 side to the TF0 side. The generation of the electric current was progressively suppressed by the presence of an inhibitor of TF1 such as NaN3 or adenyl-5'-yl imidodiphosphate. An open-circuit membrane potential of 40-120 mV was also demonstrated (more negative on the TF1 side), which was inhibited by NaN3. Furthermore, an applied voltage of -180 mV (TF1 side negative) was sufficient to prevent the generation of electric current dependent on ATP hydrolysis, which indicated that the electrogenicity of TF0F1 is some 180 mV under the conditions studied. From these results it was tentatively concluded that the number of protons transported across the bilayer/mol of ATP is more than 3.
We prepared two β-lactoglobulin (β-LG)−carboxymethyl dextran (CMD) conjugates (Conj. 10A and Conj. 10B) by using a water-soluble carbodiimide to decrease the immunogenicity of β-LG. The molar ratios ...of β-LG to CMD in the conjugates were 5:1 (Conj. 10A) and 2:1 (Conj. 10B). The β-LG−CMD conjugates maintained the retinol-binding activity of native β-LG. Intrinsic fluorescence study indicated that shielding of the surface of β-LG by CMD occurred in each conjugate, which was eminent in Conj. 10B. A local conformational change around 125Thr−135Lys (α-helix) in each conjugate was detected by ELISA with monoclonal antibodies. The denaturation temperature of β-LG evaluated by differential scanning calorimetry was greatly enhanced in each conjugate. The anti-β-LG antibody response was markedly reduced after immunization with the β-LG−CMD conjugates in BALB/c, C57BL/6, and C3H/He mice. We determined the B cell epitopes of β-LG and each conjugate recognized in these mice and found that the linear epitope profiles of the β-LG−CMD conjugates were similar to those of β-LG, while the antibody response for each epitope was dramatically reduced. The reduced immunogenicity of β-LG was most marked in the case of Conj. 10B, which contained more CMD than Conj. 10A, and was effectively shielded by CMD. We concluded that masking of epitopes by CMD is responsible for the decreased immunogenicity of the β-LG in these conjugates.
PDF
