Biomedical subjects

K Hamaguchi

Publications and source records attributed to K Hamaguchi.

At least 271 records · Page 15Linked to original sources

PubMed1975

Binding of substrate analogues to hen egg-white lysozyme with 2-nitrophenylsulfenylated tryptophan 62.

The pH dependence of the extrinsic circular dichroic (CD) band at 375 nm of hen egg-white lysozyme [EC 3.2.1.17] in which Trp 62 had selectively 2-nitrophenylsulfenylated (NPS-lysozyme) was studied. This pH dependence was interpreted in terms of the participation of Glu 35 (pK 6.2), Asp 101 (pK 4.6), and Asp 66 (pK1.5). The fact that the ionization of Glu 35 affects the extrinsic CD band of the NPS chromophore attached to Trp 62 confirms the presence of a relation between the state of Trp 62 and the ionization state of one of the catalytic groups, Glu 35, in hen lysozyme, as proposed by Ikeda and Hamaguchi (J. Biochem., 74, 221-230 (1973)). The pH dependence of the binding constants of the dimer and trimer of N-acetylglucosamine (GlcNAc) and the beta-methyl glycoside of GlcNAc (beta-methyl-GlcNAc) to NPS-lysozyme were studied by measuring the changes in the extrinsic CD band. The changes in the CD spectrum on the binding of (GlcNAc)3 and (GlcNAc)2 were very similar to each other but were different from that on the binding of beta-methyl-GlcNAc. However, beta-methyl-GlcNAc competitively inhibited the binding of (GlcNAc)2 to NPS-lysozyme. The binding constants of the three saccharides to NPS-lysozyme were much smaller than those for intact lysozyme. The pH dependence of the binding constants of (GlcNAc)2 and (GlcNAc)3 were interpreted in terms of the participation of Glu 35 (pK 6.2), Asp 52 (pK 3.3), Asp 101 (pK 4.6), and Asp 66 (pK 1.5). These pK values are very similar to those for intact lysozyme, as determined by Kuramitsu et al. (J. Biochem., 76, 671-683 (1974); 77, 291-301 (1975). Comparison of the binding constants of Mn2qnd Co2'ons to the catalytic carboxyls of NPS-lysozyme with those to intact lysozyme also indicated that the catalytic site of NPS-lysozyme is scarcely affected by this modification. When (GlcNAc)2 or (GlcNAc)3 is bound to NPS-lysozyme, pK shifts of Glu 35, Asp 101, and Asp 66 occurred in the same directions as for intact lysozyme. In addition, a pK shift of Asp 52, which has not been observed for intact lysozyme, occurred. The participation of Asp 52 was also observed in the binding of beta-methyl-GlcNAc. However, the binding of the monomer to NPS-lysozyme produced no significant pK shifts of Glu 35 and Asp 101, in contrast to the situation for intact lysozyme. These facts indicate a small difference in the binding orientation of the saccharides between the modified and intact lysozymes.

Acetylglucosamine↗

PubMed1975

Binding of N-acetyl-chitotriose to human lysozyme.

The interaction of N-acetyl-chitotriose ((GlcNAc)3) with human lysozyme [EC 3.2.1.17] was studied at various pH values by measuring changes in the circular dichroic (CD) band at 294 or 255 nm and the data were compared with the results for hen and turkey lysozymes reported previously (Kuramitsu et al. (1974) J. Biochem.76, 671-683; Kuramitsu et al. (1975) J. Biochem. 77, 291-301). The pH dependence of the binding constant of (GlcNAc)3 to human lysozyme was different from those for hen and turkey lysozymes. The catalytic carboxyls of human lysozyme, Asp 52 and Glu 35, were not perturbed on binding of (GlcNAc)3. This is consistent with the previous findings that the macroscopic pK values of Asp 52 and Glu 35 of human lysozyme are 3.4 and 6.8 at 0.1 ionic strength and 25 degrees and were unchanged on complexing with (GlcNAc)3. An ionizable group with pK 4.5, which participates in the binding of (GlcNAc)3 to hen lysozyme and was assigned as Asp 101, did not participate in the binding of the saccharide to human lysozyme. Between pH 9 and 11, the binding constants of (GlcNAc)3 to hen lysozyme remained unchanged, whereas perturbation of an ionizable group with pK 10.5 to 10.0 was observed for human lysozyme. This group may be Tyr 62 in the active-site cleft. The binding constants of (GlcNAc)3 to human lysozyme molecules having different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated using the binding constants obtained in the present experiments and the microscopic ionization constants of the catalytic carboxyls obtained previously. All four species of human lysozyme had similar binding constants to (GlcNAc)3. This result is different from those for hen and turkey lysozymes.

Acetylglucosamine↗

PubMed1975

Effects of potassium chloride and temperature on the kinetics of recombination of heavy and light chains of a human IgGl myeloma protein.

The effects of KCl and temperature on the kinetics of the recombination reaction of alkylated H and L chains of a human myeloma protein were studied by means of circular dichroism (CD). The recombination rate was considerably reduced by the presence of KCl. The results of the difference spectra showed that tryptophyl residue(s) in L chains are perturbed by KCl. No changes in the CD and absorption spectra with KCl concentration were observed for H chains and recombined H2L2. The change in the rate constant with KCl concentration paralleled the changes in the CD at 235 nm and the difference in molar extinction coefficient at 293 nm. These facts suggest that the reduction of the recombination rate in the presence of KCl is due to a change in the conformation of L chains as a result of specific interaction with chloride ions or by a shift to the dimer form in the monomer-dimer equilibrium of L chains. The rate constant showed a temperature dependence with an activation energy of 17.4 kcal/mole.

Binding Sites↗

PubMed1975

Kinetics and equilibrium studies on autologous and heterologous recombinations of heavy and light chains of myeloma proteins.

1. The kinetics of the heterologous recombination reaction of alkylated H chains of a myeloma protein (Jo) with alkylated L chains of another myeloma protein (Ita) were studied by following changes with time in the circular dichroism at 235 nm and the results were compared with those for the autologous recombination of Jo-H chains with Jo-H chains reported previously (T. Azuma et al.(1975) J. Biochem. 77, 473-479 and the preceding paper). The heterologous reaction also followed second-order kinetics. The second-order rate constant (kapp) for heterologous recombination was about seven times smaller than that for autologous recombination at pH 5.5, while they were similar between pH 4.2 and 4.7. 2. The apparent association constants (Kapp) for the reaction, H2+L2=H2L2, were determined by measuring the ellipticities at 235 nm of mixtures of H and L chains in various ratios. The values of Kapp for the autologous and heterologous recombinations were both pH-dependent and changed from 10(6) M-1 at pH 3.9 to 108 M-1 at pH 4.3. Using these values of kapp and Kapp, the half-time for the dissociation of autologous H2L2 to H2 and L2 at pH 4.3 was estimated to be 80 hr.

Binding Sites↗

PubMed1975

pH dependence of the binding constants of N-acetylglucosamine monomers to hen and turkey egg-white lysozymes.

The binding constants of N-acetylglucosamine (G1cNAc) and its methyl alpha- and beta- glycosides to hen and turkey egg-white lysozymes [EC 3.2.1.17], in the latter of which Asp 101 is replaced by Gly, were determined at various pH values by measuring changes in the circular dichroic (DC) band at 295 nm. The binding of beta-methyl-G1cNAc to turkey and hen lysozymes perturbed the pK value of Glu 35 from 6.0 to 6.5, the pK value of Asp 52 from 3.5 to 3.9, and the pK value of Asp 66 from 1.3 to 0.7. In addition, perturbation of the pK value of Asp 101 from 4.4 to 4.0 was observed in the binding of this saccharide to hen lysozyme. The binding of alpha-methyl-GlcNAc to hen and turkey lysozymes perturbed the pK value of Glu 35 to the alkaline side by about 0.5 pH unit, the pK value of Asp 66 to the acidic side by about 0.5 pH unit, and the pK value (4.4) of an ionizable group to the acidic side by about 0.6 pH unit. The last ionizable group was tentatively assigned to Asp 48. The pK value of Asp 52 was not perturbed by the binding of this saccharide. The pH dependence curves for the binding of GlcNAc to hen and turkey lysozymes were very similar and it was suggested that Asp 48, in addition to Asp 66, Asp 52, and Glu 35, is perturbed by the binding of GlcNAc.

Acetylglucosamine↗

PubMed1975

Effects on tryptophyl absorption of the ionization of the catalytic carboxyls in hen and turkey lysozymes.

The difference spectra of hen and turkey egg-white lysozymes [EC 3.2.1.17] produced by acidification were measured. The difference spectra of both lysozymes had peaks at 295 and 301 nm which are characteristic of tryptophyl residues. The pH dependence curves of the extinction differences (delta eplision) at 301 nm and 295 nm for hen lysozyme were identical with the corresponding curves for turkey lysozyme. The pH dependence of delta eplision at 301 nm was analyzed assuming that the extinction at 301 nm is due to Trp 108 only, which interacts with the catalytic carboxyls, Glu 35 and Asp 52. The macroscopic pK values of Glu 35 and Asp 52 in both lysozymes thus determined were 6.0 and 3.3, respectively. These values were in excellent agreement with those determined by measuring the pH dependence of the circular dichroic band at 305 nm (Kuramitsu et al. (1974) J. Biochem, 76, 671-683; (1975) ibid. 77, 291-301). The pH dependence of delta eplision at 295 nm could not be completely explained in terms of the electrostatic effects of the catalytic groups on Trp 108.

Animals↗

PubMed1975