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Y Arata

Publications and source records attributed to Y Arata.

At least 163 records · Page 9Linked to original sources
PubMed1986

Proton nuclear magnetic resonance study of human plasma alpha-2-macroglobulin.

A proton nuclear magnetic resonance (NMR) study is reported of human alpha-2-macroglobulin (alpha-2-M). It was observed that alpha-2-M, which consists of four identical subunits and has a molecular weight of 720,000, gives several sharp resonances. After cleavage of the "bait" region peptide with trypsin and subsequent removal of the peptide under a high salt condition, most of the sharp resonances disappeared, indicating that the sharp resonances observed in the native alpha-2-M originate from the amino acid residues in the bait region. Resonances due to the aromatic protons of the Tyr residue, which exists in the bait region, have been assigned on the basis of chemical shift. It was observed that the C3- and C5-H proton resonances for the Tyr residue are especially narrow, indicating that the side chain of the Tyr residue in the bait region is in a highly mobile state. Photochemically induced dynamic nuclear polarization experiments clearly show that the Tyr residue is actually exposed to the solvent. It was possible to identify resonances due to several His residues that are exposed to solvent. Other resonances, which probably originate from Arg residues in the bait region, were also observable in the conventional NMR spectra. On the basis of the present NMR data, we conclude that the bait region of the native alpha-2-M is highly flexible and exposed to solvent. On treatment of alpha-2-M with methylamine, no significant change has been detected in the NMR spectra observed in both the conventional and CIDNP mode.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Carbohydrate structures of acetylcholine receptor from Torpedo californica and distribution of oligosaccharides among the subunits.

The structure of carbohydrates in acetylcholine receptor (AChR) from Torpedo californica is reported. Oligosaccharides released quantitatively from the whole molecule by N-oligosaccharide glycopeptidase digestion were fractionated by thin-layer chromatography and further purified by high-performance liquid chromatography. We show that more than 70% of the total oligosaccharide chains in Torpedo AChR are of the high-mannose type with the structures (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2. The structure of these oligosaccharides were determined by proton nuclear magnetic resonance spectroscopy. These two types of oligosaccharides were shown to be distributed different proportions in all subunits of Torpedo AChR. We also show that several kinds of complex-type oligosaccharides comprising the rest of the carbohydrate in the protein exist mainly in the gamma and delta subunits. The structure of the carbohydrate moiety that is distributed on the four subunits of AChR was also examined by susceptibility to endo-beta-N-acetylglucosaminidase and sialidase and by binding affinity to lectins, e.g. concanavalin A, leucoagglutinating phytohemagglutinin, and wheat germ agglutinin.

Animals↗
PubMed1986

Mn2+-probe ESR method for the analyses of the dissociation of charged residues on the surface of immunoglobulins.

Dissociation of charged residues on the surface of immunoglobulins was analysed by an Mn2+ probe ESR method that has been developed in our previous work. Several kinds of IgG proteins and their Fab and Fc fragments were used for the experiments. The pH dependence of the intensity of ESR signals was analysed. It was shown that the number of Asp, Glu and His residues on the surface of Fc is about twice as many as that of Fab. The accessible surface area of amino acid residues calculated using X-ray crystallographic data is quite consistent with the present ESR experiments. This indicates that the number of the Asp, Glu and His residues on the surface of IgG molecules in solution is similar to that in the crystal. The Mn2+-probe ESR method was also applied to other classes of immunoglobulins, i.e. IgA and IgM. It was demonstrated that the IgA protein, which is known to lack the ability to bind Clq, has on the surface of it a smaller number of Asp, Glu and His residues as compared to IgG and IgM proteins. On the basis of these results obtained by the Mn2+-probe ESR method, we suggest that the Clq molecule, which is a basic protein, interacts favorably with the Fc portion whose surface is more negatively charged with Asp and Glu residues, compared to the Fab portion. Fine adjustment of fitting of the head of the Clq molecule into the CH2 domain of the Fc portion presumably follows for optimum binding. It was also demonstrated that Ser and Thr residues are much more abundant on the surface of Fab than in the case of Fc. We suggest that the Ser and Thr residues on the surface of Fab play an important role for binding of C4b upon activation of the complement system.

Asparagine↗
PubMed1985

Proton nuclear magnetic resonance study on the dynamics of the conformation of the hinge segment of human G1 immunoglobulin.

A proton nuclear magnetic resonance (NMR) study is reported for the dynamics of the conformation of the hinge segment of human G1 immunoglobulin. The hinge fragment (Thr223-His-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-Glu-Leu234)2 was obtained by tryptic digestion of F(ab')2, a peptic fragment of IgG1. Comparisons of the NMR results obtained for the hinge fragment with those for the intact IgG1 and its fragments led us to conclude that a significant change in conformation of the segment preceding the disulfide-linked Cys-Pro-Pro-Cys core is induced when the Fab portion is cleaved off and the presence or absence of the Fc portion affects very little, if any, of the conformation of this part of the hinge. On the basis of the present NMR results along with those which we have obtained previously using the intact IgG1 and its fragments, it was concluded that the conformation of the segment preceding the Cys-Pro-Pro-Cys core of the intact IgG1 can be maintained only when it is flanked by the Fab portion and the Cys-Pro-Pro-Cys core. An X-ray crystallographic study [Marquart, M., Deisenhofer, J., Huber, R., & Palm, W. (1980) J. Mol. Biol. 141, 369-392] showed that segment Cys-220-Thr-225 forms a one-turn helix with little inherent stability. Upon loss of Fab or Fc, residual segments of the hinge would become too short to form the helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Proton nuclear magnetic resonance study of the third component of complement: solution conformation of the carboxyl-terminal segment of C3a fragment.

A proton nuclear magnetic resonance (NMR) study is reported of des-Arg-C3a, which is a 76-residue fragment obtained from the N-terminal portion of the alpha chain of the third component of human complement. A method of carboxypeptidase digestion/difference spectroscopy [Endo, S., & Arata, Y. (1985) Biochemistry 24, 1561-1568] was used for the spectral assignments for Ala-76, Leu-75, Gly-74, His-72, His-67, and Ala-48. On the basis of the NMR results obtained for these residues, we conclude that in aqueous solution (1) the C-terminal segment Leu-73-Ala-76 is free from interactions with the rest of the C3a molecule and (2) the major part of the C-terminal segment takes an ordered conformation. We also suggest that the presence of a core, which is formed by segment Tyr-15-Tyr-59 [Huber, R., Scholze, H., Paques, E. P., & Deisenhofer, J. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1389-1399], is essential for the C-terminal segment in maintaining the ordered structure in aqueous solution. 1H NMR spectral data were also obtained for the intact C3 from human and porcine sources. The resonances for the C2-H protons of His-67 and His-72, which exist in the C3a part of the human C3 molecule, were assigned. Comparisons of the results obtained with those for des-Arg-C3a demonstrate that upon cleavage of C3a very little change, if any, is induced in microenvironments of His-67 and His-72 and a piece of segment that contains His-72 is exposed to solvent and highly flexible.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

pH-induced unfolding of the constant fragment of the immunoglobulin light chain: effect of reduction of the intrachain disulfide bond.

Circular dichroism and proton nuclear magnetic resonance measurements were made to clarify how reduction and reduction plus alkylation of the intrachain disulfide bond affect the conformation and stability of the constant (CL) fragment of the immunoglobulin light chain. The pH titration behavior of the two histidine residues in the intact CL, reduced CL, and reduced and alkylated CL fragments were followed by proton nuclear magnetic resonance spectroscopy. It was shown that reduction of the intrachain disulfide bond does not affect the solution conformation of the immunoglobulin fold, whereas reduction and alkylation results in extensive unfolding of the protein molecule. These results are consistent with the previous results obtained by Goto and Hamaguchi [1979) J. Biochem. 86, 1433-1441) using circular dichroism, fluorescence, and titration of SH groups. The pH-induced unfolding of the reduced CL fragment was compared with that of the intact CL fragment. The stability of the intact CL fragment to acid was much greater than that of the reduced CL fragment. The equilibria of the unfolding by acid could be explained by assuming that the same ionizable groups participate in the unfolding for the reduced CL fragment as for the intact CL fragment, and that the stability of the intact CL fragment is 100 times greater than that of the reduced CL fragment. In the alkaline pH region, the reduced CL fragment was unfolded above pH 8.5, while the intact CL fragment was not unfolded until pH 11.6. The kinetics of the unfolding by alkali of the reduced CL fragment was also studied. The unfolding by alkali of the reduced CL fragment could be explained by assuming that the reduced CL molecule can no longer adopt the folded conformation when either or both SH groups ionize.

Circular Dichroism↗
PubMed1984

Some evidence suggesting the existence of P2 and B3 sites in the active site of bovine pancreatic ribonuclease A.

In order to clarify the subsite structure of ribonuclease A (RNase A), the interactions of pdTp, pAp, dTpdAp, and pdTpdAp with RNase A were investigated by means of kinetic studies and 31P NMR spectroscopy. The pH profile of the 31P NMR spectrum of RNase A-pdTp complex indicated the interaction of the 3'- and 5'-phosphates with RNase A. The signal of 3'-phosphate of pdTp in the presence of RNase A gave a characteristic titration curve indicating the participation of more than 2 ionic groups in the P1 subsite. A similar 31P NMR titration was observed in the case of 5'-phosphate of pAp in the presence of RNase A. The results indicated that pAp interacted with RNase A at the P1, B2, and P2 sites. dTpdAp and pdTpdAp inhibited RNase A action more markedly than dTpdA, indicating the contribution of 3'- and 5'-terminal phosphate groups attached to dTpdA to the affinity of RNase A. The 31P NMR spectra of RNase A-dTpdAp and pdTpdAp complexes excluded the possible interaction of the monoester type phosphate of the inhibitors with the P1 site of RNase A, thus indicating the binding of the 3'-side phosphates with the P2 subsite of RNase A.

Animals↗
PubMed1984

Proton correlation NMR studies of metabolism in Rhodopseudomonas palustris.

A 1H correlation NMR study is reported, on the metabolism of a photosynthetic bacterium, Rhodopseudomonas palustris, in dark and light anaerobic conditions. Alkali treatment as well as sonication of the cells were employed to follow the process of accumulation and decomposition of poly-beta-hydroxybutyrate (PHB) which is the reserve material for the bacterium. It was shown that synthesis of PHB from trans-crotonate proceeds in the granules of the cells. It was also demonstrated that under anaerobic light conditions photometabolism and glycolysis generally compete with concomitant synthesis and decomposition of PHB, respectively, and that glycolysis gradually replaces photometabolism with aging of the cells. In contrast, glycolysis is always predominant in the dark and PHB is primarily used as the carbon source. It was observed that photo-induced transport of beta-hydroxybutyrate through the membrane occurs when photometabolism and glycolysis are equally active in the light. The implications of this observation are briefly discussed.

Anaerobiosis↗
PubMed1983

H NMR studies of the Fc region of human IgG1 and IgG3 immunoglobulins: assignment of histidine resonances in the CH3 domain and identification of IgG3 protein carrying G3m(st) allotypes.

A 1H NMR study of the Fc region of human IgG1 and IgG3 immunoglobulins is presented. 1H NMR data were collected for the Fc and pFc' fragments obtained from human monoclonal IgG1 and IgG3 and also from rabbit IgG. The C2-H proton signal of His-435 in the CH3 domain of IgG1 was assigned by comparing the spectra of the Fc fragment of IgG1 with that of IgG3 [G3m(g)] where there is a substitution of histidine by arginine at position 435. Chemical shifts and linewidths of the His-435 signal are quite different for the Fc and pFc' fragments. We suggest that His-435 is involved in the interdomain CH2-CH3 contact. Assignments of the C2-H proton signals of His-429 and His-433 in the CH3 domain were made on the basis of our previous 1H NMR results on the human light chain. NMR measurements clearly show that IgG3 Jir, which was isolated from a Japanese patient with cryoglobulinemia, has histidine at position 435 as in the case of IgG1. We also confirmed that IgG3 Jir reacts strongly with protein A. In marked contrast, IgG3 [G3m(g)] does not bind protein A. These results show that binding of protein A to the Fc region is not subclass-specific and the existence of His-435 is a necessary condition for the protein A binding. It has recently been demonstrated that IgG3 proteins carrying G3m(st) allotypes, which are relatively common in Mongoloid populations but quite rare in Caucasians, bind protein A strongly. We confirmed that, as expected, IgG3 Jir carries G3m(st) allotypes. The pH titration curve of His-435 observed for IgG3 Jir is quite different from that for IgG1. This result makes it possible to identify by 1H NMR IgG3 proteins carrying G3m(st) allotypes. In the case of the pFc' fragments, His-435 gives identical titration curves for IgG1 and IgG3 [G3m(st)]. This is consistent with the fact that no serological distinction can be made between the pFc' fragments obtained from these two types of proteins. We suggest that G3m(st)-specific antiserum differentiates IgG1 and IgG3 [G3m(st)] by recognizing the difference in the way in which the CH2 and CH3 domains make contact with each other.

Animals↗
PubMed1981

Hydrogen-tritium exchange and nuclear magnetic resonance titrations of the histidine residues in ribonuclease St and analysis of their microenvironment.

Ribonuclease St consists of 101 amino acid residues in a single polypeptide chain with one disulfide bond. It has two histidines located at positions 60 and 91 from the amino terminus. The pKa values of His-60 and His-91 were estimated by hydrogen-tritium exchange titration to be 8.0 and 6.3, respectively, and these values were confirmed by 1H NMR titration. The high pKa value of His-60 suggests that it interacts with a neighboring negative charge, presumably of a carboxylate. This is suggested by the presence of an inflection at pH 4.5 in the 1H NMR titration plot for His-60. The 1H-NMR titration plot for His-91 also suggests its interaction with a carboxylate, although the pKa of His-91 was close to that of unperturbed histidine residues. This suggests that a positively charged group is also located in the vicinity of His-91. It was concluded that His-91 is one of the active site residues of the enzyme. The pKa for His-91 was shifted to the alkaline side in the presence of 3'-GMP, a competitive inhibitor, in the titration plots observed by both hydrogen-tritium exchange and 1H NMR spectroscopy. The 31P NMR titration data suggest that in the 3'-GMP-RNase St complex the dianion form of the nucleotide participates in the interaction with the protonated form of His-91. The existence of another positively charged group with pKa of 7.0 was also suggested on the basis of the 31P NMR data.

Amino Acid Sequence↗
PubMed1980

An electrochemical study of the iron storage protein, ferritin.

The electrochemical behavior of iron storage protein, ferritin (molecular weight 700 000) was studied by pulse polarography, cyclic voltammetry and 'potentiometry' monitored by colorimetry. Comparison between in vivo and voltammetric redox reactions of the ferritin Fe(III) core and thermodynamic E'0 values for native ferritin are described.

Electrochemistry↗
PubMed1980

Proton nuclear magnetic resonance studies of human immunoglobulins: conformation of the hinge region of the IgG1 immunoglobulin.

The conformation of the hinge region of the human IgG1 immunoglobulin has been investigated by making use of His-224 in the hinge region as a built-in proton nuclear magnetic resonance (NMR) probe. Human myeloma IgG1 (kappa) proteins Ogo and Yot and human polyclonal IgG were used along with their Fab and F(ab')2 fragments for the assignment of the His-224 signals. The titration behavior of His-224 of the intact IgG and the fragments was compared. It was shown that the titration curves for the intact IgG and the F(ab')2 fragments are identical and quite similar to those for the histidine residue in small peptides. By contrast, the Fab fragments give titration curves which are quite different from those for the intact IgG and the F(ab')2 fragments. Conclusions derived may be summarized as follows: (1) in the intact IgG1, the hinge peptide is fully exposed to the solvent and exhibits internal motion which is much more rapid than the Fab segmental motion with respect to Fc: (2) at the loss of the Fc portion of the IgG, the conformation of the hinge peptide in the F(ab')2 fragments remains unchanged; (3) the heavy--heavy interchain interactions involving the two disulfide bridges do not play the primary role in determining the conformation of the hinge region in the intact IgG as well as in the F(ab')2 fragments; (4) the existence of a small stretch of peptide fragment Thr-225--Leu-234 is essential in maintaining the conformation of the hinge region of the intact IgG and the F(ab')2 fragments; (5) in the Fab fragments, as a result of cleavage of a major portion of the hinge peptide, the C-terminal part of the heavy chain including His-224 is partially folded back toward the globular portion of the polypeptide chains; and (6) the hinge peptide in the Fab fragments still retains a degree of flexibility which is similar to that in the intact IgG and the F(ab')2 fragments.

Histidine↗
PubMed1980

Proton nuclear magnetic resonance studies of human immunoglobulins. Solution conformation of the constant domain of the lambda light chains and identification of the isotypes.

Sixteen lambda-type Bence-Jones proteins and eight constant domain fragments of lambda chains were used to measure nuclear magnetic resonance (NMR) signal of the C2- AND C4-H protons of two histidine residues (His- 189 and His-198) which exist in the constant half of the lambda chains. The pH titration curves for the C2-H proton signals of His-189 and His-198 of six Bence-Jones dimers were compared with those for the corresponding constant fragments. It was concluded that the tertiary structure of the constant domain is well preserved even in the constant fragments which are known to exist as the monomer in solution. It was shown that the C2- and C4-H proton peaks of His-198 can be used to identify the Mcg isotype. It was also shown that the chemical shifts of the C4-H proton of His-189 and His-198 can be used to detect the amino acid substitutions at positions 153 and 190 which are the Kern and Oz markers, respectively. On the basis of the NMR measurements, it was suggested that the Kern and at least one of the Mcg markers are in close spatial proximity to His-189 and His-198, respectively. The NMR data were compared with the X-ray structure of the Fab fragment of human myeloma immunoglobulin, IgG1 (lambda) New [Poljak, R. J., Amzel, L. M., Avey, H. P., Chen, B. L., Phizacherley, R. P., & Saul, F. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3305-3310]. The NMR data are explained in terms of the crystal structure and are thus consistent with solution and crystal structure being quite similar in the constant domain of the lambda chains.

Bence Jones Protein↗