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K Hamaguchi

Publications and source records attributed to K Hamaguchi.

At least 235 records · Page 13Linked to original sources
PubMed1980

Ionization of the catalytic groups and tyrosyl residues in human lysozyme.

The pH difference absorption spectra of human lysozyme [EC 3.2.1.17] were measured. The difference spectra in the acidic region had a peak at 300 nm, as observed for hen and turkey lysozymes. The pH dependence curve of the extinction difference at 300 nm was well interpreted in terms of the pK values of the catalytic groups (3.4 for Asp 52 and 6.8 for Glu 35 at 0.1 ionic strength and 25 degrees C) determined from the pH dependence of the circular dichroism at 303.5 nm (Kuramitsu et al. (1974) J. Biochem. 76, 671--683) and the fluorescence excited at 305 nm (Kuramitsu et al. (1978) J. Biochem. 83, 159--170). The difference spectra of human lysozyme in the alkaline pH region were characteristic of tyrosyl ionization. The perturbation of tryptophyl residues, which had been observed for hen and turkey lysozymes (Kuramitsu & Hamaguchi (1979) J. Biochem. 85, 443--456), was not observed for human lysozyme. On the basis of the pH dependence curves of the extinction difference at 245 and and 295 nm, we roughly estimated the apparent pK values of the six tyrosyl residues as 9.2 9.2, 10.5, 10.9, 12.4, and 12.5. A time-dependent spectral change observed above pH 11 was not due to the exposure of buried tyrosyl residues on alkali denaturation but was due mainly to disulfide cleavage and exposure of buried tryptophyl residues.

Catalysis↗
PubMed1980

Hydrolysis of 4-methylumbelliferyl N-acetyl-chitotetraoside catalyzed by hen lysozyme.

Binding of 4-methylumbelliferyl chitotetraoside ((GlcNAc)4-MeU) to hen lysozyme [EC 3.2.1.17] was studied by measuring changes in fluorescence at 375 nm. Hydrolysis of (GlcNAc)4-MeU catalyzed by lysozyme was studied by measuring the release of 4-methylumbelliferone from (GlcNAc)4-MeU fluorimetrically, and the kinetic constants were determined in the pH range of 2 to 8 at 0.1 ionic strength and 42 degrees C. The binding and kinetic data showed that (GlcNAc)4-MeU binds to subsites A to E (productive binding) and subsites A to D with the nonreducing sugar residue extending beyond subsite A (nonproductive binding). The fraction of the productive complex was 0.77 at pH 8.5. The pH dependence of the kinetic constants was analyzed assuming that the molecular species with ionized Asp 52 and protonated Glu 35 is active and Asp 101 participates in the binding (Phillips (1966) Sci. Am. 215, 78-90; Blake et al. (1967) Proc. Roy. Soc. B167, 378-388), and the pK values of these groups were determined. The pK values of Asp 52, Glu 35, and Asp 101 were 3.60, 6.20, and 4.20, respectively, for free lysozyme, 3.40, 6.55, and 3.40, respectively, for the productive complex, and 3.95, 6.55, and 3.30, respectively, for the nonproductive complex. The pK values for free lysozyme were in excellent agreement with those obtained by analysis of the kinetic constants for (GlcNAc)3-MeU (Yang & Hamaguchi (1980) J. Biochem. 87, 1003-1014). The free energy of activation was 24 kcal mol-1 at pH 5.2. Comparison with the corresponding value obtained for hydrolysis of (GlcNAc)6 (Banerjee et all (1975) J. Biol. Chem. 250, 4355-4367) suggests that the interactions of GlcNAc residues with subsites E and F in the transition state are important in lysozyme catalysis. Hydrolysis of (GlcNAc)2-MeU catalyzed by lysozyme was also studied, and the Kcat/Km values for (GlcNAc)2-MeU, (GlcNAc)3-MeU, and (GlcNAc)4-MeU were compared.

Animals↗
PubMed1980

Analysis of the acid-base titration curve of hen lysozyme.

The acid-base titration curves of hen egg-white lysozyme [EC 3.2.1.17] obtained by three groups (Sakakibara & Hamaguchi (1968) J. Biochem. 64, 613--619; Tanford & Roxby (1972) Biochemistry 11, 2192--2198; Pfeil & Privalov (1976) Biophys. Chem. 4, 23--32) were analyzed using the empirical formula of Linderstrøm-Lang. Hen lysozyme has 32 ionizable groups including the alpha-amino and alpha-carboxyl groups. Of the 21 groups other than the 11 arginyl residues, the pK values of 17 ionizable groups have been determined by various methods. Using these pK values, the pK values of the other four ionizable groups were estimated. The apparent pK values obtained were 2.0 (pKmit = 3.4), 2.1 (pKint = 3.5), 2.5 (pKint = 3.8), and 7.9 (pKint = 8.5) at 0.1 ionic strength and 25 degrees C. The titration curves obtained by the above three groups were consistently explained in terms of the same set of pKint values. The results obtained also showed that no buried and untitratable groups are present in the native lysozyme molecule.

Animals↗
PubMed1980

Hydrolysis of 4-methylumbelliferyl N-acetyl-chitotrioside catalyzed by hen and turkey lysozymes. pH dependence of the kinetics constants.

The binding constants of 4-methylumbelliferyl-beta-glycosides of (GlcNAc)2 ((GlcNAc)2-MeU) and of (GlcNAc)3 ((GlcNAc)3-MeU) to hen lysozyme [EC 3.2.1.17] were determined by measuring changes in the fluorescence at 375 nm. It was shown that (GlcNAc)2-MeU and (GlcNAc)3-MeU bind mainly at subsites B, C, and D, and A, B, C, and D, respectively, with the terminal MeU group bound at subsite D. The rate of hydrolysis of (GlcNAc)3-MeU catalyzed by hen and turkey lysozymes was determined at 0.1 ionic strength and 42 degrees C in the pH range of 2 to 8. The release of 4-methylumbelliferone was followed fluorimetrically. The pH dependences of kcat, kcat/Km, and Km were analyzed assuming that that nonproductive binding occurs competitively and that the molecular species with ionized Asp 52 and protonated Glu 35 is active. Comparison of the pH dependences of the kinetic constants for hen lysozyme with those for turkey lysozyme, in which Asp 101 of hen lysozyme is replaced by Gly, made it possible to determine the pK values of Asp 52, Glu 35, and Asp 101. The pK values of Asp 52 and Glu 35 were 3.60 and 6.20, respectively, for hen and turkey lysozymes and 3.95 and 6.55, respectively, for their nonproductive complexes. The pK value of Asp 101 of hen lysozyme was 4.20 for the free enzyme, 3.30 for the nonproductive complex, and 3.95 for the productive complex. These pK values, except for the pK value of Asp 52 of the nonproductive complex, are in excellent agreement with those determined by spectroscopic methods in our laboratory (Kurasmitsu et al. (1974) J. Biochem. 76, 671--683; (1975) ibid. 77, 291--301; (1977) ibid. 82, 585--597; (1978) ibid. 83, 159--170). This demonstrates that lysozyme-catalyzed hydrolysis can be fully explained in terms of the proposal based on the X-ray data (Blake et al. (1967) Proc. Roy. Soc. B167, 378--388), with regard to the participation of Asp 52 and Glu 35.

Animals↗
PubMed1980

Binding of substrate analogs to hen lysozyme in which Trp 62 is modified to kynurenine.

The interactions of beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which Trp 62 is modified to kynurenine (Kyn 62-lysozyme) were studied by measuring the changes in the CD band and fluorescence due to the kynurenine at various pH values. The pH profiles of the binding constants of these saccharides to Kyn 62-lysozyme were very similar to those to intact lysozyme, although the binding constants were lower for the modified lysozyme than for intact lysozyme. The pK values of Asp 52, Glu 35, Asp 48, and Asp 101 in Kyn 62-lysozyme and in its complexes with beta-methyl-GlcNAc and with (GlcNAc)2 were in agreement with those of intact lysozyme and its complexes. The pK values of Asp 52 and Glu 35 in the modified lysozyme-(GlcNAc)3 complex were also in agreement with those of the complex with intact lysozyme, but the pK shift of Asp 101 was smaller for Kyn 62-lysozyme than for intact lysozyme. The significance of the decreased binding constants to Kyn 62-lysozyme is discussed. The pH dependence of the CD band due to the kynureinine in Kyn 62-lysozyme was interpreted in terms of the participation of the catalytic groups, Asp 52 (apparent pK 3.5) and Glu 35 (apparent pK 6.0), and the amino group of the kynurenine (apparent pK 0.75). This indicates that the ionization of the catalytic groups affects the state around Trp 62 and supports the previous proposal that there is a relation between the state around Trp 62 and the ionization of Glu 35 (Ikeda, K. & Hamaguchi, K. (1973) J. Biochem. 74, 221--230; (1975) ibid. 77, 1--16; Nakae et al. (1975) J. Biochem. 77, 993--1006). The intrinsic pK value of the amino group of the kynurenine at position 62 shifted from 2.1 to 0.8 on complexing with (GlcNAc)3, indicating between the kynurenine and the sugar residue at subsite B.

Animals↗
PubMed1979

Binding of N-acetyl-chitotriose to Asp 52-esterified hen lysozyme.

The pH dependence of the binding constant of (GlcNAc)3 to Asp 52-esterified lysozyme was determined by the fluorescence technique. The pK values of Asp 101 in the modified lysozyme and its complex with (GlcNAc)3 were determined to be 4.5 and 3.6, respectively, at 25 degrees C and 0.1 ionic strength. This result is different from that obtained by Parsons and Raftery ((1972) Biochemistry 11, 1633--1638), who observed no pK shift of Asp 101. The macroscopic pK value of Asp 52 in intact lysozyme determined by them using the pH difference titration data of Asp 52-esterified lysozyme relative to intact lysozyme ((1972) Biochemistry 11, 1623--1629) was 4.5, which is higher by about one pH unit than the pK value determined by our group (Kuramitsu et al. (1974) J. Biochem. 76, 671--683; (1977) ibid. 82, 585--597; (1978) ibid. 83, 159--170. We found that their pH difference titration data in the absence and presence of saccharides can be consistently interpreted in terms of our pK values of Asp 52, Glu 35, and Asp 101, if we assume that the pK value of another ionizable group (probably Asp 48) is perturbed on esterification of Asp 52.

Acetylglucosamine↗
PubMed1979

Difference absorption spectra, circular dichroism, and disulfide cleavage of hen and turkey lysozymes in the alkaline pH region.

The difference absorption spectra of hen and turkey lysozymes in the alkaline pH region had three maxima at around 245, 292, and 300 nm and had no isosbestic points. The ratio of the extinction difference at 245 nm to that at 295 nm changed with pH. These spectral features are quite different from those observed when only tyrosyl residues are ionized, and it was impossible to determine precisely the pK values of the tyrosyl residues in lysozyme by spectrophotometric titration. A time-dependent spectral change was observed above about pH 12. This is not due to exposure of a buried tyrosyl residue on alkali denaturation. The disulfide bonds and the peptide bonds in the lysozyme molecule were cleaved by alkali above about pH 11. The intrinsic pK value of Tyr 23 of hen lysozyme was determined to be 10.24 (apparent pK 9.8) at 0.1 ionic strength and 25 degrees C from the CD titration data. Comparison of the CD titration of turkey lysozyme with that of hen lysozyme suggested that Tyr 3 and Tyr 23 in turkey lysozyme have apparent pK values of 11.9 and 9.8, respectively.

Animals↗
PubMed1979

Refolding of the immunoglobulin light chain.

The kinetics of the refolding reactions of type lambda Bence Jones proteins from 4 M GuHCl were studied by CD, ultraviolet absorption, and fluorescence spectrophotometry. The kinetics were complex and consisted of at least three phases, an undetectable fast phase, a detectable fast phase, and a slow phase. The slow phase followed first-order kinetics and the three experimental methods used gave similar rate constants for all the Bence Jones proteins (about 3 X 10(-3) s-1). The refolding reaction of VL fragment was too fast to be measured in the present experiments. The refolding process of CL fragment was very similar to those of Bence Jones proteins except that the detectable fast phase was less significant. The rate constant of the slow phase observed for the CL fragment was similar to those of the slow phase observed for Bence Jones proteins. The activation energy of the slow phase was the same for a Bence Jones protein and its CL fragment. These results indicate that the refolding kinetics of the CL domain are very similar to those of isolated CL fragment and that refolding of the VL domain precedes refolding of the CL domain, even though both domains have similar immunoglobulin folds. However, the results of refolding experiments on Bence Jones proteins, and VL and CL fragments in the presence of ANS, as well as the other lines of evidence, indicate that the refolding kinetics of the Bence Jones protein molecule cannot be expressed as simple sum of the refolding reactions of isolated VL and CL fragments.

Bence Jones Protein↗
PubMed1979

The role of the intrachain disulfide bond in the conformation and stability of the constant fragment of the immunoglobulin light chain.

The conformation and stabilities of the CL fragment isolated from a type lambda Bence Jones protein and the fragment in which the intrachain disulfide bond had been reduced were studied by measuring CD, fluorescence, and ultraviolet absorption. The results indicated that no great conformational change occurs on reduction of the disulfide, unless the SH groups are alkylated. Intact CL was more resistant than reduced CL to guanidine hydrochloride. The denaturation curves were analyzed using an equation based on the binding of guanidine hydrochloride and the free energy changes of denaturation in the absence of the denaturant were estimated as about 6 kcal.mol-1 for intact CL and about 1.8 kcal.mol-1 for reduced CL. The difference in stability between intact CL and reduced CL was explained to a great extent in terms of the entropy change associated with reduction of the intrachain disulfide bond of the fragment in the denatured state.

Bence Jones Protein↗
PubMed1978

Monomer-dimer equilibria of a Bence Jones protein and its variable fragment.

The circular dichroic (CD) spectra of a type lambda Bence Jones protein (Tod), its variable (VL) fragment, and the constant (CL) fragment of a type lambda protein (Nag) were measured under various conditions. In the pH region from 5.5 to 7.5, the CD spectra of Tod protein with intact interchain disulfide bond (L(SS)) and and CL did not change with pH, while the spectra of Tod protein in which the interchain disulfide bond had been reduced and alkylated (L(RA)) and VL did not change with pH. The dimerization reactions of L(RA) and VL were studied by following the CD change with protein concentration. The CD spectrum of CL did not change with the protein concentration. The dimerization constant for L(RA) was 4 X 10(4) M-1 at at pH 7.5 and 25 degrees C, which was smaller than that for VL (1 X 10(5) M-1). The ellipticity at 278 nm for the L(RA) dimer was different from that for the L(SS) dimer and changed with pH. These findings indicate that the L(RA) dimer and L(SS) dimer have different conformations. The differences in the conformation and L-L interaction between the L(RA) dimer and L(SS) dimer are discussed on the basis of the conformations of VL and CL and the interactions between the paired domains.

Alkylation↗
PubMed1978

Formation of interchain disulfide bonds in Bence Jones proteins and Fab(t) fragments of immunoglobulin G through thiol-disulfide interchange.

The formation of interchain disulfide bonds from partially reduced Bence Jones protein (Nag, type lambda) and Fab(t) fragments of IgG1 myeloma proteins was studied in the presence of various disulfide reagents. The results could be well explained in terms of the scheme proposed previously (Kishida et al. (1976) J. Biochem. 79, 91-105). In this scheme, it was assumed that two kinds of intermediate, which form mixed disulfides with either of the paired thiol groups, are produced. For type lambda Bence Jones proteins, only one of the two intermediates can form the inter L-L disulfide bond. The fraction of intermediate having the ability to form the inter L-L disulfide bond was estimated to be 72% of the total Nag protein and was the same irrespective of the kind of disulfide reagent examined. For Fab(t), on the other hand, both intermediates equally can form the inter Fd-L disulfide bond. On the basis of the results with cystamine, it was shown that the formation of an inter Fd-L disulfide bond from the intermediate proceeds about 100 times as rapidly as that of an inter L-L disulfide bond.

Bence Jones Protein↗
PubMed1978

Ionization and reactivities of the thiol groups which participate in the formation of interchain disulfide bonds of Bence Jones proteins and an Fab(t) fragment.

The pK values and reactivities of the thiol groups which participate in the formation of interchain disulfide bonds in Bence Jones proteins and the Fab(t) fragment of a myeloma protein (Jo) (IgGl, kappa) were determined by means of the reactions with chloroacetamide and DTNB, and of spectrophotometric titration. The two thiol groups of partially reduced type kappa Bence Jones protein dimers had the same pK values (pK = 9.76 at 0.2 ionic strength and 25 degrees C) and the same true second-order rate constants (k) toward chloroacetamide (k = 18.8 x 10(-2) M-1 . S-1). The two thiol groups of partially reduced type lambda Bence Jones protein dimers had different pK values but the variation of the pK values among the specimens was small (pK1 = 8.5-8.6 and pK2 = 9.5-9.7 at 0.2 ionic strength and 25 degrees C). The spectrophotometric titration of partially reduced Nag protein (type lambda) also showed that the two thiol groups have different pK values. The pK values of two thiol groups of the partially reduced Fab(t) fragment were determined as 8.51 and 9.76 at 0.2 ionic strength and 25 degrees C. The effect of ionic strength on the pK values of the thiol groups of partially reduced Nag protein and the pK values of the thiol groups in partially reduced Ta protein (type kappa) and in a hybrid molecule formed between partially reduced Ta protein and partially reduced and alkylated H chains indicated that the difference in pK values did not arise from electrostatic interaction between the two thiol groups, but that the pK values are intrinsically different. The true rate constants, k1 and k2, of the two thiol groups of type lambda Bence Jones proteins varied with the specimen (k1 = 1.9-5.7 x 10(-2) M-1 . S-1 and k2 = 18.5-25.0 x 10(-2) M-1 . S-1). The k1 and k2 values for Jo-Fab(t) were 7.21 x 10(-2) and 23.1 x 10(-2) M-1 . S-1, respectively. On the basis of these pK values and reactivities, we discuss the reformation of the interchain disulfide bonds from partially reduced Bence Jones proteins and immunoglobulins in the presence of oxidized glutathione.

Acetamides↗