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Biomedical subjects

K Hamaguchi

Publications and source records attributed to K Hamaguchi.

At least 217 records · Page 12Linked to original sources
PubMed1983

Bindings of cobra venom phospholipases A2 to micelles of n-hexadecylphosphorylcholine.

Bindings of cobra venom phospholipases A2 to micelles of n-hexadecylphosphorylcholine were studied by the tryptophyl fluorescence method at 25 degrees C and ionic strength 0.1. The data were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of substrate (monomer) molecules, N = 10, 5 or 13 for N. naja atra apoenzyme and its Ca2+ complex, and N. naja kaouthia apoenzyme, respectively. The binding constant of the enzymes to the micelle, Kmic = 0.18-3.1 X 10(6) M-1, was 9-160 times greater than that to the monomeric substrate, Kmon = 2 X 10(4) M-1 (Teshima et al. (1981) J. Biochem. 89, 1163-1174). This was interpreted in terms of the presence of an additional substrate-binding site in the enzyme molecule. The binding constant of the enzyme-Ca2+ complex to the micelle was smaller than that for the apoenzyme over a wide range of pH. The pH dependence of the binding constant of the apoenzyme to the micelle was well interpreted in terms of pK shifts of two ionizable groups from 5.4 to 5.53 and 7.55 to 7.95. The pH dependence curve for the Ca2+ complex, which lacked the former transition, was interpreted in terms of the pK shift of only a single ionizable group from 7.25 to 7.55. The former ionizable group was assigned as Asp 49, to which Ca2+ can coordinate, and the latter as His 48 in the active site on the basis of the reported pK values of these ionizable groups in the apoenzyme and Ca2+ complex (Teshima et al. (1981) J. Biochem. 89, 13-20 and Teshima et al. (1982) J. Biochem. 91, 1777-1788). No participation of the alpha-amino group with a pK value of 8.55 was observed.

Animals↗
PubMed1982

Participation of an ionizable group with pK 8.55 in the reaction of p-bromophenacyl bromide with His 48 of cobra venom phospholipases A2.

The chemical reaction of p-bromophenacyl bromide (BPB) with His 48 of cobra venom phospholipases A2 (N. naja siamensis, N. naja kaouthia, and N. naja atra) was studied at 25 degrees C and ionic strength 0.1 by following the decreases in the fluorescence intensity of 8-anilino-1-naphthalene sulfonate (ANS) and/or in the enzymatic activity. The effect of the BPB concentration on the pseudo-first-order rate constant, chi obs, was studied for the N. naja atra enzyme and the dissociation constants of a noncovalent intermediate were determined to be 5.6 x 10(-4) and 1.8 x 10(-4) M at pH 8.4 and 9.3, respectively. The ph-dependence curve of chi obs, obtained at fixed concentration(s) of BPB was found to be biphasic for all three enzymes. The analysis showed that two ionizable groups with pK values of 7.3 and 8.55 participated in this reaction. The reaction of BPB with the modified enzyme of N. naja atra lacking the N-terminal octapeptide, which had been prepared by the CNBr-cleavage method, was followed by measuring the decrease in the tryptophyl fluorescence. Since this modified enzyme showed a monophasic pH-dependence curve lacking the latter transition, the former group was assigned to His 48 and the latter to the alpha-amino group located nearby in the active site. The pK value of His 48 determined at present was in good agreement with that estimated from the pH dependence of the binding constant of Ca2+ (Teshima et al. (1981) J. Biochem. 89, 13-20). The pK value of the alpha-amino group of this enzyme, 8.55, was found to be somewhat higher than that of the A. halys blomhoffii enzyme, 7.3 (Ikeda & Samejima (1981) J. Biochem. 90, 799-804 and Ikeda et al (1981) J. Biochem. 90, 1125-1130), but quite similar to that for the porcine pancreatic enzyme, 8.4 (van Dam-Mieras et al. (1976) Nobel Symp. 34, 177-197 and Slotboom et al. (1978) Biochemistry 17, 4593-4600).

Acetophenones↗
PubMed1982

Denaturation by guanidine hydrochloride of the Fc(t) and pFc' fragments of human immunoglobulin G.

The denaturation and renaturation by guanidine hydrochloride of Fc(t) fragment whose interchain disulfide bonds are reduced and alkylated (R.A.Fc(t)) and pFc' fragment of a human myeloma protein (IgGl, kappa) were studied using tryptophyl fluorescence. R.A.Fc(t) was found to consist of a slow-unfolding region and a rapid-unfolding region. The denaturation of pFc' was extremely slow. Comparison of the kinetic and equilibrium data of the denaturation of R.A.Fc(t) with those of pFcl indicated that the slow-unfolding region of R.A.Fc(t) corresponds to the CH3 region and the fast-unfolding region the CH2 domain. This was also confirmed by the analysis of the CD and fluorescence spectra for R.A.Fc(t) and pFc' at various concentrations of guanidine hydrochloride. Although the kinetic stability of the CH3 region was much higher than that of the CH2 region, the thermodynamic stabilities of these domains were almost the same; the free energy change of the denaturation in water being about 6 kcal . mol-1. This value is also the same as the value for the CL fragment (Goto, Y. & Hamaguchi, K. (1979) J. Biochem. 86, 1433--1441). It was suggested that the high kinetic stability of the CH3 region in R.A.Fc(t) is due to the strong tendency for the CH3 domains to form a dimer.

Circular Dichroism↗
PubMed1981

Hydrolysis of 4-methylumbelliferyl N-acetyl-chitooligosaccharides catalyzed by human lysozyme.

Binding of N-acetyl-chitotetraose, 4-methylumbelliferyl chitotrioside ((GlcNAc)3-MeU), and 4-methylumbelliferyl chitotetraoside ((GlcNAc)4-MeU) to human lysozyme [EC 3.2.1.17] was studied by fluorescence measurement. Hydrolysis of (GlcNAc)3-MeU and (GlcNAc)4-MeU catalyzed by human lysozyme was studied by measuring the release of 4-methylumbelliferone fluorimetrically and the kinetic constants were determined in the pH range of 2 to 8 at 0.1 ionic strength and 42 degrees C. On the basis of binding and kinetic data, it was shown that (GlcNAc)3-MeU binds mainly to subsites A to D with the terminal MeU group bound to subsite D (nonproductive binding) and 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 pH dependences of the kinetic constants for hydrolysis of (GlcNAc)3-MeU and (GlcNAc)4-MeU were analyzed assuming that nonproductive binding occurs competitively, that an ionizable group in addition to the catalytic groups (Asp 52 and Glu 35) participates in the catalysis, and that the molecular species with ionized Asp 52 and protonated Glu 35 is active. Analyses of the kinetic constants for (GlcNAc)3-MeU and (GlcNAc)4-MeU both gave the same pK values of the catalytic groups (pK52 = 3.6(3) and pK35 = 6.6(8) at 0.1 ionic strength and 42 degrees C). These pK values were very close to the values determined previously by spectroscopic methods in our laboratory (Kuramitsu et al. (1974) J. Biochem. 76, 671-683; (1978) ibid. 83, 159-170; (1980) ibid. 87, 771-778).

Chitin↗
PubMed1981

A large-scale preparation and some physicochemical properties of recA protein.

Pure recA protein was easily obtained from Escherichia coli harboring plasmid pTM-2 which carried the recA gene by two chromatographic steps on phosphocellulose and DEAE-cellulose. RecA protein was stable in the pH range of 6 to 9 at 25 degrees C. RecA protein was found to aggregate highly under these conditions. Lowering of the protein concentration, the presence of glycerol, and lowering of the pH in the pH stability region diminished the extent of aggregation. The spectroscopic properties of recA protein were measured in the presence of 10% (v/v) glycerol. RecA protein had an absorption maximum at 278 nm. The value of a1% 1cm at 278 nm was determined to be 5.7. The tryptophyl fluorescence spectrum excited at 295 nm had an emission maximum at 340 nm and the quantum efficiency of recA protein relative to N-acetyl-L-tryptophanamide was determined to be 0.65. The CD spectrum of recA protein had negative double maxima at 210 and 220 nm. The alpha-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250 nm. All three cysteinyl residues of recA protein were reacted with 5,5'-dithiobis(2-nitrobenzoic acid), and recA protein was found to have neither intramolecular nor intermolecular disulfide bond. The reactivities of the SH groups were changed by the presence of ATP or ADP. The denaturation of recA protein by guanidine hydrochloride was studied by measuring CD at 220 nm and tryptophyl fluorescence. The denaturation curve obtained by CD measurement consisted of two stages, one of which lies between 0 and 1.8 M and the other above 1.8 M guanidine hydrochloride. On the other hand, the denaturation curve obtained by fluorescence measurement consisted of a single transition in the concentration range of about 1 to 2.3 M guanidine hydrochloride.

Bacterial Proteins↗