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K Inouye

Publications and source records attributed to K Inouye.

At least 73 records · Page 4Linked to original sources
PubMed1996

Cellular uptake and biological effects of antisense oligodeoxynucleotide analogs targeted to herpes simplex virus.

In this study, we synthesized antisense oligodeoxynucleotides (ODNs) with phosphodiester, phosphorothioate (S-ODNs), or methylphosphonate linkages complementary to the splicing acceptor site of immediate-early pre-mRNA 5 of herpes simplex virus type 1 (HSV-1). The antiviral activity of each analog on cytopathic effect in cells infected with HSV-1 or HSV-2 was assessed and compared with the cellular uptake of the analog. We found that antisense S-ODNs showed the most potent antiherpetic activity, with 50% inhibitory concentrations of 5 microM for HSV-1 and 0.25 microM for HSV-2. The antiviral effect of antisense S-ODNs was stronger and longer acting than that of acyclovir. Cell association of S-ODNs was the highest and paralleled antiviral activity. Furthermore, some fluorescein isothiocyanate (FITC)-labeled S-ODNs were recognized in the nuclei in HSV-1 infected cells by confocal laser scanning microscopy. S-ODNs located in the nucleus could access the targeted mRNA, which might be responsible for the antiviral activities. Although our study also showed non-sequence-specific activity, which implies that multiple mechanisms are involved, S-ODNs are a promising novel anti-herpetic agent.

Animals↗
PubMed1995

Effects of alcohols on the hydrolysis of colominic acid catalyzed by Streptococcus neuraminidase.

Regulation of the activity of neuraminidase of Streptococcus sp. (group K) was evaluated by examining the effects of alcohols on the hydrolysis of colominic acids catalyzed by the neuraminidase. Two kinds of alcohol binding site, activation and inhibition sites, were proposed to exist. Competitive inhibition was observed with alcohols smaller than polyethylene glycol #300 (average molecular weight: 300), so the inhibition site is considered to be the substrate binding site, the size of which was estimated to be 10 A in diameter. On the contrary, polyethylene glycols larger than this size activated the enzyme by 1.5-1.8 times. The activity could be raised by binding of the polyethylene glycols to the activation site. This activation was shown to be due solely to the decrease in the Michaelis constant, Km. The smaller polyethylene glycols (#200 and #300) were also considered to bind to the activation site, although activation was not clearly observed due to compensation with inhibition. Strong substrate inhibition by colominic acid was also observed. The activity of Streptococcus neuraminidase was shown to be regulated intricately by the substrate colominic acid and alcohols contained in the reaction medium.

Alcohols↗
PubMed1994

New hapten-protein conjugation method using N-(m-aminobenzoyloxy) succinimide as a two-level heterobifunctional agent: thyrotropin-releasing hormone as a model peptide without free amino or carboxyl groups.

The use of a two-level heterobifunctional agent N-(m-aminobenzoyloxy)succinimide (m-ABS) allowed us to develop a new method for preparing hapten-protein conjugates. This was demonstrated by a conjugation between thyrotropin-releasing hormone (TRH) and bovine or human serum albumin (BSA or HSA). The conjugation is based on the principle that the succinimidyl ester group of m-ABS immediately acts on an epsilon-amino group of lysine residues of carrier protein BSA (or HSA) and a m-aminobenzoyl group incorporated into the protein is then activated by diazotization to a functional m-diazobenzoyl group (m-DB) acting on a histidyl group of TRH. The TRH-BSA containing about 3.5 mol of TRH per BSA molecule, elicited the production of TRH antibody in rabbits. A new type of enzyme-linked immunosorbent assay (ELISA) for TRH was developed using the antiserum, the solid-phase antigen TRH-HSA and the commercially available horseradish peroxidase-labeled goat anti-rabbit IgG/Fab' as a marker, revealing that the ELISA was monospecific to the hormone and measured as low as 50 pg of the hormone reproducibly. Also, using the antiserum by the indirect immunoperoxidase method the distribution of immunoreactive TRH in the rat brain was demonstrated in neurons of the paraventricular nucleus and neuronal processes of the median eminence. These results strongly suggested that the use of m-ABS provided a simple and efficient new method for preparing immunogens not only for the previously reported haptens with a primary amino group(s) (J. Immunol. Methods 134 (1990) 227), but also for haptens with an imidazole, phenolic, or indole group(s) in the molecule.

Amino Acid Sequence↗
PubMed1994

Preparation of F(ab')2 mu fragments from rat IgM monoclonal antibodies and their application to the enzyme immunoassay of mouse interleukin-6.

F(ab')2 fragments, herein designated as F(ab')2 mu, were prepared from rat IgM monoclonal antibodies (mAbs). The IgM was digested at a pepsin-to-IgM ratio of 1:200 (w/w) in 100 mM citrate buffer (pH 4.5) at 37 degrees C for 2 h. During digestion, the light (L) chain (27 kDa) of IgM remained undegraded, whereas the heavy (H) chain disappeared and two new bands of 44 and 48 kDa appeared. The digests were fractionated by means of hydrophobic interaction HPLC with TSKgel Phenyl-5PW. The fraction containing F(ab')2 mu was homogeneous and the recovery of antigen-binding activity was 41-52%. The molecular mass of F(ab')2 mu was estimated to be 147-153 kDa, and we concluded that the fragment was composed of two truncated H chains and two intact L chains. F(ab')2 mu was used in an enzyme immunoassay of mouse interleukin-6 and the interaction of IgM with non-specific proteins was greatly reduced, when it was converted to F(ab')2 mu fragments.

Animals↗
PubMed1994

Dissociation of dimer of bovine erythrocyte Cu,Zn-superoxide dismutase and activity of the monomer subunit: effects of urea, temperature, and enzyme concentration.

Cu,Zn-superoxide dismutase (SOD) of bovine erythrocytes is a dimeric enzyme of identical subunits associated through unusually strong noncovalent interactions. It is known not to be dissociated into subunits even in 8 M urea for 72 h at 25 degrees C [Malinowski, D.P. & Fridovich, I. (1979) Biochemistry 18, 5055-5060]. Effects of urea, temperature, and SOD concentration on the inactivation and dissociation into subunits were examined. The activation energy of the inactivation of SOD (at 0.05 mg/ml) was 64 kcal/mol at pH 7.8, and was decreased to 40 kcal/mol in the presence of urea (2.0-7.3 M). The apparent first-order rate constant (kapp) of the inactivation by urea was dependent on the SOD concentration [SOD] during the urea treatment, and SOD showed a higher resistance to the inactivation with increase in the concentration. Dissociation of SOD was monitored by gel filtration HPLC. When SOD solutions of various concentrations were incubated in 6 M urea at 45 degrees C, two monomer species (M1 and M2) were observed in addition to dimer (D). The dimer maintained full activity, while the monomers did not show the activity. The peak areas of these species were changed depending on the SOD concentration during urea treatment; at over 15 mg/ml, almost all SOD was eluted as D, and with a decrease in the SOD concentration, the peak area of D decreased and concomitantly the monomers appeared. M2 could be the sole form in infinitely diluted SOD solution, and D was considered to be converted to M2 through M1. The SOD concentration giving 50% D ([SOD]1/2) was 1.0 mg/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A spectrophotometric study on the interaction of thermolysin with chloride and bromide ions, and the state of tryptophyl residue 115.

The activity of thermolysin is greatly enhanced in the presence of high concentrations of neutral salts [Holmquist, B. and Vallee, B.L. (1976) Biochemistry 15, 101-107; Inouye, K. (1992) J. Biochem. 112, 335-340]. NaBr and NaCl are the most effective for the activation. An absorption difference spectrum with a peak around 293 nm, which is characteristic of the red-shift of a tryptophyl residue caused by charge effects, was observed on mixing of thermolysin with NaCl. As the peak disappeared in the presence of competitive inhibitors of the enzyme (phosphoramidon and zincov), it was considered to be derived from a tryptophyl residue (Trp 115) located in the active site of the enzyme. On the other hand, this peak was not observed on the mixing of thermolysin and NaBr, indicating that the slight difference in size between chloride and bromide ions is critical for the interaction with the tryptophyl residue. NaCl and NaBr exhibit comparable effects on the activation of thermolysin regardless of the considerable discrepancy in their effects on the absorptivity difference around 293 nm. This suggests that the interaction of salts with Trp 115 is not necessarily correlated with the activation of thermolysin.

Binding Sites↗
PubMed1993

Paradoxical structure and function in a mutant human insulin associated with diabetes mellitus.

The solution structure of a diabetes-associated mutant human insulin (insulin Los Angeles; PheB24-->Ser) was determined by 13C-edited NMR spectroscopy and distance-geometry/simulated annealing calculations. Among vertebrate insulins PheB24 is invariant, and in crystal structures the aromatic ring appears to anchor the putative receptor-binding surface through long-range packing interactions in the hydrophobic core. B24 substitutions are of particular interest in relation to the mechanism of receptor binding. In one analogue ([GlyB24]insulin), partial unfolding of the B chain has been observed with paradoxical retention of near-native bioactivity. The present study of [SerB24]insulin extends this observation: relative to [GlyB24]insulin, near-native structure is restored despite significant loss of function. To our knowledge, our results provide the first structural study of a diabetes-associated mutant insulin and support the hypothesis that insulin undergoes a change in conformation on receptor binding.

Amino Acid Sequence↗
PubMed1993

Purification of monoclonal antibodies with light-chain heterogeneity produced by mouse hybridomas raised with NS-1 myelomas: application of hydrophobic interaction high-performance liquid chromatography.

Monoclonal antibodies (mAbs) of IgG1 class produced by hybridomas raised with NS-1 myelomas, which were purified homogeneously by anion-exchange high-performance liquid chromatography (HPLC), contained two types of immunoglobulin light (kappa) chain. Since NS-1 myeloma synthesizes the light (kappa) chain, the mAb was presumed to be the mixture of hybrid mAbs formed by the random association of heavy (gamma l) and light chains from antigen-immunized spleen cells and light chain from NS-1 cells. Hydrophobic interaction HPLC using TSKgel Phenyl-5PW was applicable to separate 3 species of hybrid mAb from mAb fractions obtained by anion-exchange HPLC. mAbs in the fractions were adsorbed onto the gel equilibrated with phosphate-buffered saline containing 1 M ammonium sulfate and eluted by reducing the concentration to 0 M. The hybrid mAbs were purified separately. The hydrophobic interaction HPLC could discriminate a small difference in hydrophobicity between kappa chains from spleen and NS-1 cells. The immunoreactivities of hybrid mAb bearing light chains only from spleen cells and that bearing those from both spleen and NS-1 cells were almost the same, and hybrid mAb bearing light chains derived only from NS-1 cells showed a relatively lower immunoreactivity than the others. The method described here could be useful for purification of hybrid mAbs.

Animals↗
PubMed1993

Single-step purification of F(ab')2 mu fragments of mouse monoclonal antibodies (immunoglobulins M) by hydrophobic interaction high-performance liquid chromatography using TSKgel ether-5PW.

A procedure is described for preparation and single-step purification of F(ab')2 fragments, herein designated as F(ab')2 mu' from mouse monoclonal antibodies of the IgM class. Hydrophobic interaction high-performance liquid chromatography (HPLC) using TSKgel Ether-5PW was well applicable to the purification. The IgM was digested with pepsin at the pepsin-to-IgM ratio of 1:200 (w/w) in 100 mM citrate buffer (pH 4.2) at 37 degrees C for 2 h. The digests were applied to the gel equilibrated with the buffer containing 1 M ammonium sulfate. F(ab')2 mu fragments were adsorbed onto the gel with the same buffer, and eluted by reducing the ammonium sulfate concentration to 0 M. The fraction containing F(ab')2 mu fragments was homogeneous (purity higher than 97%) by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration HPLC. The recovery of the antigen-binding site was 55-72%. The cycle time of the Ether-5PW HPLC was 40 min, and up to 98 mg F(ab')2 mu fragments. The molecular mass of F(ab')2 mu was estimated to be 144-146 kDa. In comparison with IgM, F(ab')2 mu lost entirely the complement C1q binding activity, and the sugar content was greatly reduced. The binding of IgM with non-specific proteins turned to be negligible, when IgM was converted to F(ab')2 mu, suggesting that the fragments are useful for immunological application.

Animals↗
PubMed1993

Analysis of three tissue-specific elements from the wheat Cab-1 enhancer.

The genes encoding the major light-harvesting antenna chlorophyll a/b-binding protein (Cab) of higher plants are regulated by light at the transcriptional level. In addition, their expression is largely restricted to photosynthetically competent organs such as leaves. A 268 bp fragment of the Cab-1 promoter from wheat functions as a light-responsive and organ-specific enhancer in transgenic tobacco. Using DNase I footprinting, four different regions have been mapped (Cab1-A, Cab1-B, Cab1-C and Cab1-D) in this fragment that bind to protein factors in tobacco nuclear extracts. Three of these regions (A, B and C) coincide with sequences that have been found to be functionally important from previous cis-element analyses. Synthetic tetramers of these three sites interact with different proteins in gel retardation assays. In addition, cross-competition analyses demonstrate that Cab1-C is likely to interact with ASF-2, a tobacco DNA-binding activity that binds to a conserved GATA element found in many dicot Cab promoters. In transgenic tobacco, a 95 bp fragment of the Cab-1 enhancer containing the A, B and C regions can confer leaf expression when fused upstream of a truncated derivative of the cauliflower mosaic virus (CaMV) 35S promoter. However, expression observed with this enhancer fragment in the promoter context of these studies does not appear to be significantly dependent on light. Similar results were obtained with synthetic tetramers of Cab1-A, -B or -C. These data thus suggest that the wheat Cab-1 enhancer contains at least three distinct elements that contribute to leaf-specific expression in transgenic tobacco. Interaction between factors binding to these positive elements and those that bind elsewhere in the Cab-1 enhancer may be necessary for light-responsive transcriptional activation.

Base Sequence↗
PubMed1993

Interaction of subtilisin BPN' and recombinant Streptomyces subtilisin inhibitors with substituted P1 site residues.

Kinetic analysis was performed on the interaction between subtilisin BPN' and recombinant species of a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), of which the P1 site amino acid residue, Met73, was replaced by site-directed mutagenesis. The inhibitor constant, Ki, was determined from the residual enzyme activity by using a peptide substrate. The rate constant of binding, kon, and the rate constant of dissociation, koff, were determined from a progress curve of the substrate hydrolysis in the presence of the inhibitor by using newly derived equations. A recombinant SSI in which Met73 was replaced by Ile showed an affinity (1/Ki) toward subtilisin BPN' of only about 7% of that of the wild-type SSI, and the kinetic analysis revealed that the increase of koff was responsible for this difference. The affinity of other SSI mutants in which Met73 was replaced by Glu or Asp decreased significantly as pH became increasingly alkaline. The decrease in the affinity of these recombinants was due to the decrease of kon rather than the increase of koff. Stopped-flow studies revealed that the binding reaction was reconcilable with a two-step mechanism, and the kinetic parameters for each step were obtained for the binding of the enzyme and recombinant SSIs.

Bacterial Proteins↗
PubMed1993

Identification of amino acid residues responsible for the changes of absorption and fluorescence spectra on the binding of subtilisin BPN' and Streptomyces subtilisin inhibitor.

An ultraviolet absorption difference spectrum characteristic of the ionization change of a tyrosyl residue was observed on the binding of subtilisin BPN' with Streptomyces subtilisin inhibitor (SSI) at alkaline pH. This difference spectrum was considered to be induced by a pKa shift (from 9.7 to > or = 11.5) of a tyrosyl residue of subtilisin BPN' in the interaction with carboxyls of SSI [Inouye et al. (1979) J. Biochem. 85, 1115-1126]. In the present paper, the tyrosyl residue in subtilisin BPN' and the carboxyls in SSI were identified by analyzing the difference spectrum using mutants of subtilisin BPN' and SSI: naturally occurring mutants and those prepared by site-directed and cassette mutagenesis. The difference spectrum disappeared on the binding of a mutant subtilisin BPN' of which Tyr104 was replaced by Phe (S-BPN'Y104F) and SSI at pH 9.8. The magnitude of the absorption difference was much smaller when subtilisin BPN' was bound with a mutant SSI of which both Glu67 and Asp68 were replaced by Gly than with the wild-type SSI. These lines of evidence indicated that the difference spectrum was caused by Tyr104 of subtilisin BPN' interacting with Glu67 and Asp68 of SSI. The binding of subtilisin BPN' and SSI is accompanied by an increase of tryptophan fluorescence, which is pH-dependent in the range of pH 7-11 [Uehara et al. (1978) J. Biochem. 84, 1195-1202]. In the present study, this pH-dependence of the fluorescence diminished when SSI bound with S-BPN'Y104F. This suggested that the fluorescence increase was due to Trp106 of subtilisin BPN' and was influenced by the ionization of Tyr104.

Amino Acids↗
PubMed1992

Single-step purification of F(ab')2 fragments of mouse monoclonal antibodies (immunoglobulins G1) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW.

Hydrophobic interaction high performance liquid chromatography (HPLC) using TSKgel Phenyl-5PW was applicable to single-step purification of F(ab')2 fragments from pepsin digests of mouse monoclonal antibodies of IgG1 class. The digests were applied to the gel equilibrated with phosphate-buffered saline containing 1 M ammonium sulfate. F(ab')2 fragments were adsorbed onto the gel using the same buffer, and eluted by reducing the ammonium sulfate concentration to 0 M. The fraction containing F(ab')2 fragments was homogeneous (purity: higher than 98%) by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration HPLC. The recovery of the antigen binding site was 42-58%. The cycle time of the Phenyl-5PW HPLC was 45 min, and F(ab')2 of up to 2200 mg was purified in a cycle. This method could be useful especially for large scale purification of F(ab')2 fragments.

Animals↗
PubMed1992

Effects of salts on thermolysin: activation of hydrolysis and synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester, and a unique change in the absorption spectrum of thermolysin.

It has been reported that neutral salts such as NaCl activate the thermolysin-catalyzed hydrolysis of substrates containing glycine at the P1 position (carboxylic side of the cleavage bond) [Holmquist, B. & Vallee, B.L. (1976) Biochemistry 15, 101-107]. In this paper, we demonstrate that high concentrations (1-4 M) of neutral salts greatly enhance the thermolysin activity in both hydrolysis and synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZAPM), a precursor of a peptide sweetener, aspartame, in which the L-aspartyl residue is the P1 residue. The enzyme activity is enhanced with an increase in salt concentration in a pseudo-exponential fashion. The degree of activation by salts was in the order LiCl > NaCl > KCl. The rate of ZAPM hydrolysis in the presence of 3.8 M NaCl was 6-7 times higher than that in its absence, and 50 times or more activation is expected in saturated NaCl solution. The activation is brought about solely through an increase in the catalytic constant (kcat), and the Michaelis constant (Km) is not affected at all by the presence of NaCl. On mixing thermolysin with NaCl, a unique absorption difference spectrum suggesting a conformational change of the enzyme was observed. The intensity increased in a pseudo-exponential fashion with increase of NaCl concentration up to 3 M, and this dependence is similar to that of the enzyme activity.

Bacillus↗