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K Inouye

Publications and source records attributed to K Inouye.

At least 145 records · Page 8Linked to original sources
PubMed1979

The interaction of a tyrosyl residue and carboxyl groups in the specific interaction between Streptomyces subtilisin inhibitor and subtilisin BPN'. A chemical modification study.

An ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7 leads to greater than 11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3.4.21.14] at alkaline pH, ionic strength 0.1 M, at 25 degrees C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6 M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction. Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitromethane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' and alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule. Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.

Amino Acids↗
PubMed1978

The determination of molecular weights of Streptomyces subtilisin inhibitor and the complex of Streptomyces subtilisin inhibitor and subtilisin BPN' by sedimentation equilibrium.

The molecular weight of Streptomyces subtilisin inhibitor (SSI), a protein proteinase inhibitor, and that of the complex of SSI and subtilisin BPN' [EC 3.4.21.14] were determined by a sedimentation equilibrium method in 25 mM phosphate buffer, at pH 7.0, ionic strength 0.1 M (NaCl), 25.0 degrees C. The molecular weight of SSI was found to be 23,000 over a wide concentration range, 0.01-10 mg/ml, the range used for inhibitory, spectrophotometric, and kinetic measurements. Based on the amino acid sequence, the molecular weight of SSI has been calculated to be 11,500 (Ikenaka, T., et al. (1974) J. Biochem. 76, 1191-1209); therefore, the molecular weight of 23,000 obtained above suggests that SSI is in a dimeric form under usual conditions in the concentration range of 5 X 10(-7)-5 X 10(-4) M. The molecular weight of the subtilisin BPN'-SSI complex was determined to be 78,000 in the concentration range of 0.03-5.0 mg/ml by sedimentation equilibrium of the crystallized preparation and by that of a mixture of subtilisin BPN' and SSI treated as a multicomponent-polydisperse system. The molecular weight obtained here, combined with the results of binding stoichiometry (Inouye, K., et al. (1977) J. Biochem. 82, 961-967) that showed that one mol of SSI (molecular weight, 11,500) and one mol of the enzyme (molecular weight, 27,500) are tightly bound (Kd less than 1 nM), demonstrate that one mol of dimeric SSI binds two mol of the enzyme to form a stable complex, E2I2.

Bacterial Proteins↗
PubMed1977

Oscillations of membrane potential in L cells. I. Basic characteristics.

The membrane potentials and resistances of L cells were measured using a standard electrophysiological technique. The values obtained in physiological media were around--15 mV and 37Momega, respectively. Almost all the large nondividing L cells (giant L cells) showed spontaneous oscillations of the membrane potential between around -15 and -40 mV. Application of an appropriate electrical or mechanical stimulus was also capable of eliciting responses but such were usually induced only once. The total membrane conductance increased significantly and in parallel with such a hyperpolarizing response. Cooling of the cells and application of metabolic inhibitors to the cells completely blocked the spontaneous oscillation despite the fact that the electrically induced hyperpolarizing response remained. Intracellular K+, Na+ and Cl- concentrations were measured by means of a flame photometer and a chloridometer, and the equilibrium potential for each ion was estimated.

Acetylcholine↗
PubMed1977

Interaction of thiolsubtilisin with Streptomyces subtilisin inhibitor, SSI.

Subtilisin BPN' was chemically converted to thiolsubtilisin and the interaction of this modified enzyme with Streptomyces subtilisin inhibitor (SSI) was examined. SSI competitively inhibited the esterolytic activity of thiolsubtilisin toward p-nitrophenyl acetate with a K1 value of 1.3 X 10(-5) M at pH 7.5 Spectrophotometric analysis of the interaction between SSI and the modified enzyme yielded a Kd value of 4 X 10(-5) M at pH 9.7. These values are about 10(5)-fold greater than the Kd value (less than 10(-9) M at pH 7.5) for the native enzyme. This indicates that the small change in the active site structure of subtilisin (Ser221 to Cys221) leads to a considerable decrease in the binding affinity (by about 6-7 kcal/mol) to SSI.

Acetates↗
PubMed1977

The stoichiometry of inhibition and binding of a protein proteinase inhibitor from Streptomyces (Streptomyces subtilisin inhibitor) against subtilisin BPN'1.

The stoichiometry of inhibition and binding of Streptomyces subtilisin inhibitor, a protein proteinase inhibitor produced by Streptomyces albogriseolus S-3253 (Sato, S. and Murao, S. (1973) Agric. Biol. Chem. 37, 1067) against subtilison BPN' [EC 3.4.21.14] was studied. The inhibition of the hydrolysis of p-nitrophenyl acetate by subtilisin BPN' was measured both at the pre-steady state with a stopped-flow apparatus and at the steady state. The stopped-flow study demonstrated the disappearance of the initial burst of the enzyme reaction. The ultraviolet absorption difference spectra observed on mixing the inhibitor and the enzyme suggested changes in the environment of tryptophyl and tyrosyl residues in the proteins. Titration by means either of the degree of inhibition at the steady state or of the magnitude of the ultraviolet difference absorbance revealed that the inhibitor (dimer, MW: 23,000) bound and inhibited two molecules of subtilisin BPN'. The inhibitor constant, Ki, against subtilisin BPN' was estimated to be less than 10(-9)M at pH 8.50. The type of inhibition of this inhibitor is discussed.

Bacterial Proteins↗
PubMed1977

The states of tyrosyl and tryptophyl residues in a protein proteinase inhibitor (Streptomyces subtilisin inhibitor.

The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.

Bacterial Proteins↗
PubMed1976

Effects of the stereo-configuration of the hydroxyl group in 4-hydroxyproline on the triple-helical structures formed by homogenous peptides resembling collagen.

To explore further the recent demonstration that hydroxyproline stabilizes the triple-helical structure of collagen, two peptides containing allohydroxyproline, (aHyp-Pro-Gly)10 and (Pro-aHyp-Gly)10, were synthesized by a modified Merrifield technique which yields products of defined molecular weight. Examination of the peptides by optical rotation and circular dichroism showed that neither of them formed triple-helical structures in aqueous solution. Since the peptides had less tendency than (Pro-Hyp-Gly)10 to become helical, the results demonstrated that the trans-4-hydroxyl group of hydroxyproline makes a specific contribution to stability of the triple helix formed by (Pro-Hyp-Gly)10. Since the peptides also had less tendency than (Pro-Pro-Gly10 to become helical, the results further demonstrated that the cis-4-hydroxyl group on allohydroxyproline decreases the stability of the triple helix. The observations provided direct support for previous data indicating that incorporation of proline analogues such as allohydroxyproline into pro-alpha chains during procollagen biosynthesis prevents the polypeptides from becoming triple helical.

Binding Sites↗
PubMed1975

Calcium-binding of synaptosomes isolated from rat brain cortex, III. Binding with some divalent heavy metal ions and calcium-binding sites.

Free ion concentration of some divalent heavy metal ions such as Mn2+, Co2+, Ni2+, Cd2+ and Zn2+ in the synaptosomal suspension was measured to determine binding with synaptosomes isolated from rat brain cortex. A dual wavelength spectrophotometer was utilized to monitor the absorbance changes of murexide raised by stepwise addition of these ions (as chloride salts). Such titration experiments of the synaptosomal suspension revealed that a part of the added divalent cation such as Mn2+, Co2+ or Ni2+ was almost instantaneously bound to synaptosomes in isotonic NaCl media. Our previous study (Kamino, Uyesaka & Inouye, J. Membrane Biol. 17:13, 1974) demonstrated that raised external K+ resulted in a specific noncompetitive inhibition of synaptosomal Ca-binding. Just like the Ca-binding, Mn-, Co- or Ni-binding was almost completely depressed by high external K+ or ruthenium red when the free concentration of the cations was 10 mum or less, while at higher concentrations the binding was not affected. The present results indicate that tested divalent cations bind with both "Ca-binding sites" and "non-Ca-binding sites" of synaptosomal membrane, the nature of the binding sites of both being quite different: the former is sensitive to high external K+ and to ruthenium red but the latter is not.

Animals↗