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Biomedical subjects

K Hiromi

Publications and source records attributed to K Hiromi.

At least 55 records · Page 3Linked to original sources
PubMed1984

Light scattering stopped-flow studies on the time course of shape changes induced in human erythrocytes by several amphiphilic lipids.

The light scattering stopped-flow method was used to study the time course of shape changes in human erythrocytes induced by two series of lipids (saturated and unsaturated free fatty acids and lysophosphatidylcholine (lysoPC) species, both with various acyl chain lengths (C12-C18] at pH 7.4 and 23 degrees C. The decrease in the scattered light intensity as a function of time suggested that these lipids induce crenation of erythrocytes, the rate constant of which is largely dependent on the nature of the respective lipids. For both series of lipids, an increase in chain length was found to decrease the rate of the induction of shape changes. The results were discussed in relation to hydrophobicity of lipid molecules.

Erythrocytes↗
PubMed1983

Subsite structure and ligand binding mechanism of glucoamylase.

1. The basic concept and outline of the subsite theory were described, which correlates quantitatively the subsite structure (the arrangement of subsite affinities) to the action pattern of amylases in a unified manner. 2. The subsite structures of several amylases including glucoamylase were summarized. 3. In parallel with the theoretical prediction obtained therefrom, the binding subsites of glucose, gluconolactone and linear substrates to Rhizopus glucoamylase were investigated experimentally, by using steady-state inhibition kinetics, difference absorption spectrophotometry, and fluorometric titration. 4. From several lines of evidence, it was concluded that gluconolactone, a transition state analogue, is bound at Subsite 1 (nonreducing end side) where a tryptophan residue is located. 5. The stopped-flow kinetic studies have revealed that all the ligand bindings studied consist of two-step mechanism in which a bimolecular association between the enzyme and a ligand to form a loosely bound complex (EL) followed by the unimolecular isomerization process in which EL converts to the final firmly bound EL complex. For substrates the EL may be the productive complex and the fluorescence of the tryptophan located at Subsite 1 is quenched in their isomerization process, most probably a relocation of ligand to occupy this subsite.

Binding Sites↗
PubMed1983

Equilibrium study on the binding between thermolysin and Streptomyces metalloprotease inhibitor, talopeptin (MKI).

The binding between thermolysin and its specific inhibitor, talopeptin (MKI), was found to show a fluorescence increase when excited at 280 nm and 295 nm, and a difference spectrum characterized by two peaks at 294 nm and 285 nm with a shoulder around 278 nm, indicating a microenvironmental change in tryptophan residue(s) of thermolysin and/or talopeptin. The inhibitor constant of talopeptin against thermolysin, Ki, was determined over the pH range 5-9 from the inhibition of the enzyme activity towards 3-(2-furylacryloyl)-glycyl-L-leucine amide (FAGLA) as a substrate. The dissociation constant of thermolysin-talopeptin complex, Kd, determined directly from fluorometric titration was in good agreement with the inhibitor constant, Ki, between pH 6 and 8.5. The pH dependence of Ki and Kd suggested that at least two ionizable groups of thermolysin in their protonated forms are essential for the binding between thermolysin and talopeptin. The temperature dependence of K1 at pH 5.5 indicated that the binding is largely exothermic (delta H degree = -12 kcal/mol) and essentially enthalpy-driven.

Glycopeptides↗
PubMed1983

Kinetics of binding between thermolysin and Streptomyces metalloprotease inhibitor, talopeptin (MKI).

The mechanism of binding between thermolysin with its specific inhibitor, talopeptin (MKI), was studied kinetically with the stopped-flow method by monitoring the enhancement of tryptophan fluorescence caused by the complex formation. Only one relaxation obeying first-order kinetics was observed. The dependence of the apparent first-order constant, kapp, on the inhibitor concentration is consistent with a minimum two-step mechanism, including a fast bimolecular binding step followed by a slow unimolecular step. It was found that the increase in tryptophan fluorescence occurs solely in the slow unimolecular step. The apparent second-order rate constant, (kon)app, in the low inhibitor concentration range, was determined over the pH range between 5 and 8.5 and decreases with increasing pH. The activation parameters for the overall binding process were obtained from the temperature dependence of (kon)app.

Glycopeptides↗
PubMed1983

Binding between thermolysin and talopeptin (MKI) in which the tryptophan residue was converted into kynurenine.

The tryptophan residue of talopeptin, which is a specific inhibitor for thermolysin, was converted into kynurenine by ozonization followed by acid-catalyzed hydrolysis, and (Trp leads to Kyn) talopeptin (Kyn-talopeptin) thus obtained was purified with gel-chromatography. The inhibitor constant of Kyn-talopeptin, K1, and the dissociation constant of thermolysin-Kyn-talopeptin complex, Kd, directly obtained by fluorometric titration were in good agreement with each other. These values were found to be about 10 times larger than those of intact talopeptin, but both inhibitors showed a similar pH dependence. Upon the binding of Kyn-talopeptin with thermolysin, the protein fluorescence of thermolysin decreases by about 60%, while the kynurenine fluorescence (measured at 450 nm when excited at 360 nm) of the inhibitor increases about 14 times. The measurements of the excitation and fluorescence spectra of EI complex strongly indicated the energy transfer from tryptophan residue(s) (the donor) of the enzyme to kynurenine residue (the acceptor) of the inhibitor. The distance between the donor and the acceptor was roughly estimated to be 18 A. This value is in good agreement with the one expected from the crystallography of phosphoramidon-thermolysin complex. The binding process was studied kinetically with the stopped-flow method over the pH range between 4.5 and 8.5, by monitoring the decrease in the fluorescence intensity of the enzyme tryptophan caused by the complex formation. Comparison of the data with those previously obtained for talopeptin-thermolysin system revealed that the replacement of the tryptophan residue by kynurenine of the inhibitor does not affect the apparent second-order association rate constant, kon, seriously.

Biotransformation↗
PubMed1983

Studies on the chemical modification of tryptophan residues in thermolysin and in talopeptin (MKI) with N-bromosuccinimide.

Tryptophan residues in thermolysin (3 Trp/molecule) and in its specific inhibitor, talopeptin (1 Trp/molecule), were modified with N-bromosuccinimide (NBS). The decrease in the absorption at 280 nm and the fluorescence intensity above 310 nm (excited at 280 nm) accompanying the modification were followed by the stopped-flow method as a function of time. When the sole tryptophan residue of talopeptin was modified with NBS, its inhibitory activity against thermolysin was almost completely destroyed. For thermolysin, the decrease in molar absorptivity corresponds to the modification of one of its three tryptophan residues, and the enzyme activity does not decrease significantly with the modification (remaining activity was 96% at [NBS]/[E] = 6). The results obtained for the modification of EI complex suggested that the formation of EI complex remarkably reduces the rate constant for the modification of the tryptophan residue in talopeptin, but does not affect that of the tryptophan residue(s) in thermolysin.

Bromosuccinimide↗
PubMed1983

Kinetic studies on acid denaturation and renaturation of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Fast reversible conformational changes induced by down and up pH-jumps of Streptomyces subtilisin inhibitor, a protein proteinase inhibitor (abbreviated to SSI), was studied kinetically with the stopped-flow method by monitoring the change in tryptophan fluorescence. Both the acid denaturation and the renaturation proceeded in two phases. The pH-dependence of the apparent first-order rate constants and of the magnitudes of the fluorescence changes examined in the range of pH 1.7-7.0 were essentially consistent with a two-step sequential mechanism involving a common intermediate for denaturation and renaturation with rapid protonation equilibria, as follows: (Formula: see text) where N, N', and D denote the native, the intermediate, and the denatured states of the SSI molecule, respectively. The parameters were evaluated as follows: pK1 = 6.0, pK2 = 4.8, pK3 = pK4 = 2.5, k1 = 1.0 s-1, k2 = 0.13 s-1, k3 = 0.05 s-1, and k4 = 60 s-1 (at 25 degrees C). The contributions of Step I and Step II to the total fluorescence change were estimated to be 20.5% and 79.5%, respectively. It is noteworthy that the overall renaturation is faster than the denaturation. The rate constant of restoration of binding ability of SSI to alpha-chymotrypsin (43 s-1), measured by using proflavine as indicator, was of the same order of magnitude as that of the faster step of the renaturation (k4 = 60 s-1), which indicated that the inhibitory activity was lost in the N' leads to D process in Step II.

Bacterial Proteins↗
PubMed1983

Continuous production of maltooligosaccharides from glucose 1-phosphate by amylase-phosphorylase reactor.

Using an ultrafiltration cell, the continuous production of maltose and a mixture of other oligosaccharides was achieved by the combined action of alpha- or beta-amylase and phosphorylase. For the beta-amylase-phosphorylase system, a steady production of maltose was obtained until the beta-amylase was inactivated. For the alpha-amylase-phosphorylase system, it was difficult to maintain a stable production of oligosaccharides, probably due to the destruction of substrate glycogen by random cleavage by alpha-amylase.

Amylases↗
PubMed1980

Binding of Streptomyces pepsin inhibitor (acetyl-pepstatin) with chymosin (Rennin).

Chymosin (Rennin) was effectively purified using an AH-Sepharose 4B column. Binding of Streptomyces pepsin inhibitor (acetul-pepstatin) with chymosin was studied spectroscopically. The binding caused ultraviolet difference and CD spectral changes suggesting microenvironmental changes around tryptophan and/or tyrosine residue(s) in chymosin. The fluorescence intensity of a hydrophobic probe, 2-p-toluidinylnaphthalene-6-sulfonate, increased in the presence of chymosin and was further amplified when Streptomyces pepsin inhibitor was added to the chymosin-2-p-toluidinylnaphthalene-6-sulfonate solution. The binding and dissociation-rate constants between chymosin and the inhibitor were determined using 2-p-toluidinylhnaphthalene-6-sulfonate as a probe. The binding constant was determined from the binding and dissociation-rate constants, to be 3.1 . 10(7) M-1 at 25 degrees C, pH 5.5.

Chymosin↗
PubMed1980

Stopped-flow studies on the chemical modification with N-bromosuccinimide of model compounds of tryptophan residues.

The aim of this work was to study the selective modification of tryptophan residues with NBS. To accomplish this, a specific method to determine tryptophan was required initially. NBS reacts with both tryptophan and tyrosine residues, which are found in most enzyme proteins, and static spectrophotometric observation, which is usually employed to follow the progress of modification, is not selective for tryptophan. However, discrimination of tryptophan from tyrosine was achieved by the kinetic method with a stoppeed-flow apparatus. The rate of modification of tryptophan residues is 10(3) times larger than that of tyrosine, so rapid stopping of the reaction of NBS brings about the selective modification of tryptophan residues. Using fluorescence-spectrophotometric and kinetic methods, the modification with NBS of model compounds of the tryptophan residue could be simply followed as a single phase, even though the reaction is complex when followed by the static spectrophotometric method.

Bromosuccinimide↗