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Biomedical subjects

K Akasaka

Publications and source records attributed to K Akasaka.

At least 163 records · Page 9Linked to original sources
PubMed1985

Sodium dodecyl sulfate-bovine plasma albumin complex. Structural transition from native to relatively compact globule forms with mobile side chains in acidic region--N-F transition.

The acid-induced isomerization (the N-F transition) and expansion of sodium dodecyl sulfate-bovine plasma albumin complex (ADm; m, molar ratio of added sodium dodecyl sulfate to bovine plasma albumin; O less than or equal to m less than or equal to 12) were studied by measuring CD-resolved secondary structure, fluorescence polarization and life-time of tryptophyl fluorophors, acid-titration with the electrostatic correction for the surface potential, 1H-n.m.r. spectra and cross relaxation time between irradiated and observed protons. The immobilization of tryptophyl fluorophors observed in the F-form of AD0 was suppressed in the F-form of AD10. The acidtitration analysis of AD12 showed non salt-bonding between carboxylate groups and cationic side chains in the F-form, as in the case of AD0, indicating charged side chains being presumably mobile. 1H-n.m.r. spectra and cross relaxation times between irradiated and observed protons in the F-form of AD10 indicated the increase in the local motion. On the other hand, AD10 and AD12 did not show any significant change in the CD-resolved secondary structure in the N-F transition region. The F-form of AD10 or AD12 may therefore be the moltenglobule state which has secondary structure similar to the N-form of the complexes with fluctuating tertiary structure (side chains).

Animals↗
PubMed1984

1H NMR studies of aspartate aminotransferase. Histidyl residues of cytosolic and mitochondrial isoenzymes.

200 MHz proton nuclear magnetic resonance spectra were compared between the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase (EC 2.6.1.1) from pig heart. The pattern of signal distribution in the whole spectral region differed considerably between the two isoenzymes, reflecting the difference in their amino acid sequences. A group of distinct signals were resolved at elevated temperatures (50 to 70 degrees C) in the low field region (9.0 to 7.5 ppm) of the spectra of both isoenzymes in the pyridoxal form. Most of these signals were also observable at 28 degrees C although some showed considerable line broadening. Among resonance lines in this spectral region, cAAT in the pyridoxal form showed four pH-titratable resonances with pKa of 9.54, 6.72, 5.69, and 4.87 at 28 degrees C. Variation in pK and line width of these signals indicated differences in the microenvironment of histidyl residues. On the other hand, mAAT showed six pH-titratable resonances with pKa of 6.73 (peak 2), 6.77 (peak 3), 6.07 (peak 4), 4.71 (peak 5), 4.54 (peak 6), and 4.33 (peak 7). Peaks 2, 3, and 4 were narrow and others were considerably broad. Thus, only part of the histidyl residues present in each isoenzyme (8 and 10 His/monomeric unit of cAAT and mAAT, respectively) appeared on the spectra as pH-titratable resonances. With both isoenzymes, chemical shift and pKa values of these signals obtained for the pyridoxal form were indistinguishable from those for the pyridoxamine form and the borohydride-reduced form. None of the observable signals were affected upon the interaction of cAAT with glutarate. By contrast, peaks 2 and 4 in mAAT showed subtle but distinct chemical shift changes upon complex formation with succinate, suggesting that these two resonances are due to histidyl residues located at the part of the enzyme molecule which undergoes a conformational change upon the interaction with the dicarboxylate.

Animals↗
PubMed1984

A proteoglycan fraction isolated from the EDTA extract of sea urchin (Hemicentrotus pulcherrimus) gastrulae stimulates reaggregation of dissociated embryonic cells.

A sulfated proteoglycan fraction was prepared from mid-gastrulae Hemicentrotus pulcherrimus by chromatographing the EDTA extract of embryos on DEAE-cellulose and then on Sepharose 6B. A peak fraction (with MW greater than 10(6] of the Sepharose eluates stimulated in a concentration-dependent manner the reaggregation of cells dissociated from Hemicentrotus blastulae by EDTA treatment, the activity being detected at a concentration of as low as 5.8 micrograms protein per ml or 2.5 micrograms fucose per ml. The proteoglycan fraction appeared to contain sulfated fucan and glycosaminoglycan chains linked to a common protein. The glycan moiety obtained by digesting the fraction with protease failed to stimulate the cellular reaggregation.

Animals↗
PubMed1983

Sulfated glycan present in the EDTA extract of Hemicentrotus embryos (mid-gastrula).

Light microscopical observations of the Alcian blue-stained gastrulae of Hemicentrotus pulcherrimus together with the scanning electron microscopical observations of the embryos revealed the presence of highly acidic glycans in the invaginating archenteron (inside surface), the surrounding of secondary mesenchyme cells (pseudopodial protrusions and filamentous structures) and the hyaline layer. In the embryos grown in sulfate-free sea water and thus with arrested gastrulation it was found that the dye stainability in the above regions was markedly reduced. The glycosaminoglycan fraction prepared from the whole embryos (mid-gastrulae) was found to contain various kinds of acidic glycans as analysed by chromatography on DEAE-cellulose. Among these glycan components, the "F" component was mainly recovered in the EDTA extract of the embryos, and was shown to be specifically deleted in the embryos grown in sulfate-free sea water, suggesting that the "F" component may be related to the Alcian blue-stainable material in Hemicentrotus embryos. The component "F" was found to consist of sulfated fucan and acid mucopolysaccharide (unidentified) chains, which are probably linked to a common peptide core, forming macromolecules with larger than 10(6) molecular weights.

Animals↗
PubMed1982

Dynamic states of the three methionyl residues of Streptomyces subtilisin inhibitor. 1H NMR studies.

High resolution 1H NMR spectroscopy at 360 and 400 MHZ was used to study the dynamic states of the three methionyl residues (Met 70, 73, and 103) of Streptomyces subtilisin inhibitor (SSI) in a neutral aqueous (2H2O) solution. Chemical modification and nuclear Overhauser effect studies show that one of the methyl groups exists in the interior of the protein and is surrounded by phenylalanyl residues. With reference to the crystal structure, this methyl group is assignable to that of Met 103 in the hydrophobic core. The other two methionyl residues (Met 70 and 73) are equally susceptible to chemical modifications, but their methyl signals are affected in a different manner by the alpha-chymotrypsin-cleavage of the peptide bond between Met 73 and Val 74 at the reactive site. The observed linewidths of the methyl groups are consistent with the assumption that the whole side-chain of both Met 70 and 73 are already undergoing rapid internal motions at 20 degrees C, whereas for Met 103 only the rotation of the methyl group is allowed, even above approximately 40 degrees C. The high-field shift of the methyl proton resonance of Met 103 by about 0.3 ppm is attributable to the ring current effect from the surrounding phenylalanyl residues. This shift is invariant in the temperature range of 40-90 degrees C, showing that the native environment of the methyl group of Met 103 persists up to 90 degrees C at neutral pH. Destruction of the native methionyl environments occurs only above 90 degrees C, and the rate of conversion between the native and the denatured conformations is slower than 10 s-1. When SSI is mixed with subtilisin BPN' in a one-to-one molar ratio, at least one of the methyl signals of Met 70 and 73 is completely broadened, showing that the side-chain of this methionine is directly involved in the interaction with the enzyme.

Bacterial Proteins↗
PubMed1981

Proton magnetic resonance study of Streptomyces subtilisin inhibitor. pH titration and assignments of individual tyrosyl resonances.

This paper reports the pH titration at 25 degrees C and the assignment of aromatic proton resonances of three tyrosyl residues of each identical subunit of Streptomyces subtilisin inhibitor (Mr 23000) by high-resolution 1H NMR spectroscopy. The complete assignments of the specific tyrosyl resonances were made based on the results of the differential chemical modification of the tyrosyl residues with tetranitromethane followed by peptide analysis, independently of the knowledge of the crystal structure. pKa values of Tyr-7, -75, and -93 were determined in a 2H2O solution to be 10.95, 11.8, and greater than or equal to 12.6, respectively, at 25 degrees C, whereas pKa values of nitrated Tyr-7 and -75 were determined to be 7.3 and 7.9, respectively. Tyr-93 was not nitratable under normal conditions. The strong resistance to nitration, together with the extremely high pKa value and the high-field shifted positions of the ring proton resonances of Tyr-93 at neutral pH, strongly suggests that Tyr-93 takes part in a hydrogen bonding as a proton donor. Tyr-7 is more easily modified with tetranitromethane than Tyr-75, although in the crystal Tyr-75 is more exposed than Tyr-7. The result, together with the pKa value of Tyr-75 significantly higher than that of a normal tyrosine, indicates that the microenvironment of Tyr-75 is more restricted in solution than in solid. These results imply that structural details of a protein may be quite similar in solution to those in the crystal in the rigid hydrophobic region of the protein but that in the surface region of the protein local structures may well differ between the solution and the crystal.

Amino Acids↗
PubMed1981

Exposure of aromatic residues of Streptomyces subtilisin inhibitor. A photo-CIDNP study.

Exposure of aromatic residues, Tyr 7, Tyr 75, Tyr 93, His 43, His 106, and Trp 8, was studied by laser-induced photo-CIDNP in the 1H NMR spectrum of Streptomyces subtilisin inhibitor at 360 MHz. Only Tyr 7 and Tyr 75 gave strong CIDNP signals, whereas the rest of the aromatic residues gave no detectable signals in the temperature range 25-55 degrees C. From the temperature dependence data, it is concluded that Tyr 7 is well exposed at all temperatures, whereas the exposure of Tyr 75 increases with temperature, in agreement with the conclusion obtained by other methods. Agreements and discrepancies between the conclusions derived from the CIDNP data and the results so far obtained by other methods are compared for all the aforementioned aromatic residues.

Bacterial Proteins↗
PubMed1980

Acid denaturation steps of Streptomyces subtilisin inhibitor. A proton magnetic resonance study of individual histidine environment.

Resonance positions and intensities of the C(2) protons of histidyl residues (His 43 His 106) were followed in the NMR spectrum of a protein, Streptomyces subtilisin inhibitor (MW 23,000), in the pH range 2-9 at 30 degrees C, in order to clarify the microenvironment of the individual histidyl residues and the acid denaturation processes of the protein. The large difference in the 1H-2H exchange rate between the C(2) protons of the two histidyl residues indicates that, whereas His 106 is well exposed to the solvent, His 43 is highly shielded from the solvent. Protonation of His 106 occurs in the pH range 5-9, giving a pKa of 6.0 +/- 0.1. In contrast, His 43 can not be protonated in the native conformation, but is protonation occurs simultaneously with the acid denaturation of the protein in a narrow pH range 2.7-3.6, with pHmid of transition at 3.25. On the other hand, the denaturation of the His 106 environment starts at pH 5.0 and finishes at pH 2.7 with pHmid of transition at 3.7. The denaturation transitions are reversible with pH for both His 43 and His 106, but the rates of these transitions are slow (less than 25 s-1). The Hill coefficient for the His 106 transition was found to be 0.9 +/- 0.1 in the pH range 3.5-5.0, but to be distinctively larger than unity below pH 3.5. On the other hand, the Hill coefficient for the His 43 transition was found to be 4.4 +/- 0.6 for the whole range of transition (pH 2.7-3.6). These results combined with available information about the crystal structure lead us to conclude that the transition of His 106 in the pH range 3.5-5.0 represents a local denaturation caused by the protonation of one neighboring residue, probably Glu 102, whereas the transition of His 43 occurs as a result of cooperative protonation of several residues probably including Phe 113 and Asp 52, and is a denaturation step accompanied by the destruction of the major hydrophobic core.

Bacterial Proteins↗
PubMed1979

Conformation of adenosine 3',5'-monophosphate in solution as studied by the NMR-desert method. II. Self-association and temperature-dependent glycosidic isomerization at pH 7.

A study has been made of the association and the temperature-dependent conformation of adenosine 3',5'-monophosphate (cyclic AMP) in a neutral aqueous (2H2O) solution by means of proton magnetic resonance chemical shift and relaxation. The concentration and temperature-dependent chemical shifts of H(1'), H(2), and H(8), have enabled us to estimate the self-association constant, Ka = 1.1 +/- 0.3 M-1 at 25 degrees C and thermodynamic parameters delta H = -5.8 +/- 1.5 kcal/mol and delta S (25 degrees C) = -19.0 +/- 3 cal/mol per degree. The NMR-DESERT (Deuterium Substitution Effect on Relaxation Times) method has been utilized for the determination of the syn-anti conformational equilibrium in the monomeric state and for the determination of the mutual orientation of the two adenine rings in the dimeric state of cyclic AMP. The molecules were found to coexist with nearly equimolarity or syn-anti conformers and thermal activation of the molecules perturbs the syn-anti conformational equilibrium to comprise the syn form in preference at higher temperature. The glycosidic isomerization (from anti to syn) was found to be characterized both by a positive enthalpy change and by a positive entropy change. The cyclic AMP molecules prefer to take a 'trans-stacking' conformation in the dimeric state where the two molecules are arranged in such a way that the H(2) of one molecule is close to the H(8) of the other.

Chemical Phenomena↗
PubMed1979

Internal motions in myosin.

High-resolution proton nuclear magnetic resonance (1H NMR) measurements were made on myosin, heavy meromyosin (HMM), myosin subfragment 1 (S1), light meromyosin (LMM), and actin. A strong signal from amino acid side chains undergoing motions too fast to be accounted for by simple rotations of groups on a rigid backbone was obtained from myosin. Comparison of myosin, HMM, S1, and LMM showed that the mobile region is located almost entirely in S1 and accounts for approximately 22% of its structure. Adenosine triphosphate (ATP) and ATP analogues had no measurable effect on the S1 spectrum. Actin, on the other hand, quenched the internal motions of S1. When S1 was titrated with actin, an association was obtained which was in agreement with other measured values. The actin effect was reversed by adding magnesium pyrophosphate (MgPPi) or adenyl-5'-yl imidophosphate (MgAMPPNP). Quantitative treatment of the broad signals from myosin and its subfragments substantiated the existence of two flexible regions in myosin. The highly mobile portion of myosin may be located in the "swivel" between S1 and the rest of myosin or in the actin binding site or in both. These possibilites are discussed, and a new possible mechanism for muscle cross bridge elasticity is proposed.

Actins↗
PubMed1978

Solubilization of 6-oxybenzo(a)pyrene radical by caffeine and DNA as studied by magnetic resonance. Observation of intermolecular charge transfer.

Crystals of 6-oxybenzo(a)pyrene free radical, formed chemically from the hydroxy derivative of the carcinogen benzo(a)pyrene, can be solubilized in aqueous solutions of DNA and of caffeine. ESR spectral evidence indicate that the radicals exist as dispersed monomers associated with DNA and with caffeine. Comparison of NMR spin-lattice and spin-spin relaxation times in the protons of caffeine has given direct evidence that a part of the unpaired electron (at least 10(-4)) is transferred from the radical to the associated caffeine molecule. Simple consideration of Mulliken's charge transfer theory, however, leads to the conclusion that the intermolecular charge transfer is not likely to be a major source of stabilization energy of the complex.

Benzopyrenes↗
PubMed1978

31P-NMR study on nucleotides and intracellular pH of hereditary spherocytes.

As determined by 31p-NMR spectroscopy, intracellular pH of hereditary spherocytes was lower (pH 6.7-6.9) than that of normal red cells. The level of adenosine diphosphate in hereditary spherocytes was found to be persistently high. The metabolism of nucleotides and other phosphoryl compounds in human red blood cells have been studied in detail by 31p-MNR spectroscopy. However, to our knowledge, there seems to be no report describing the result of 31p-NMR spectroscopy on red blood cells from hereditary spherocytosis.

Adenosine Diphosphate↗