Biomedical subjects

H Nagamune

Publications and source records attributed to H Nagamune.

At least 37 records · Page 2Linked to original sources

PubMed1994

Identification of targeting proteinase for rat alpha 1-macroglobulin in vivo. Mast-cell tryptase is a major component of the alpha 1-macroglobulin-proteinase complex endocytosed into rat liver lysosomes.

The alpha 1-macroglobulin-proteinase complex endocytosed into rat liver lysosomes was purified by a series of column chromatographic steps on concanavalin A-Sepharose, Sephacryl S-300, DEAE-cellulose and TSK gel DEAE-5PW columns. The complex contained no detectable alpha 2-macroglobulin. Studies on the substrate specificity indicated that the complex had tryptase-like activities towards various synthetic substrates, but no elastase, chymotrypsin, cathepsin-B and cathepsin-L activities. The proteinase activity was completely inhibited by di-isopropyl fluorophosphate, leupeptin and antipain, indicating that the proteinase bound to alpha 1-macroglobulin is a serine proteinase. Two protein bands (62 and 59 kDa) of the complex were labelled with [3H]diisopropyl fluorophosphate and both bands cross-reacted with anti-(mast-cell tryptase)antibody. These results suggest that mast-cell tryptase is a major targeting proteinase for alpha 1-macroglobulin in vivo. The main alpha-macroglobulin-proteinase complex in the adjuvant-treated rats was also the alpha 1-macroglobulin-tryptase complex, even though the plasma level of alpha 2-macroglobulin was elevated.

Amino Acid Sequence↗

PubMed1994

Lysosomal enzyme replacement using alpha 2-macroglobulin as a transport vehicle.

Improvement of the delivery of exogenous enzymes is essential to achieve effective enzyme replacement therapy in lysosomal storage diseases. To test whether alpha 2-macroglobulin, an endogenous plasma protein, could serve as a transport vehicle of therapeutic agents to cells, alpha 2-macroglobulin and acid alpha-glucosidase or alpha-galactosidase A were coupled using two heterobifunctional cross-linking reagents. The alpha-glucosidase-alpha 2-macroglobulin conjugate was internalized and transported into lysosomes of acid alpha-glucosidase-deficient fibroblasts. The enzyme activity was stable after being taken up by the cells. Uptake of the conjugate resulted in the degradation of glycogen accumulated in lysosomes. The alpha-galactosidase A-alpha 2-macroglobulin conjugate was also internalized into the lysosomes of alpha-galactosidase A-deficient fibroblasts. Internalized alpha-galactosidase A-conjugate degraded globotriaosylceramide accumulated in lysosomes. The endocytosis of both conjugate was inhibited by alpha 2-macroglobulin-trypsin complex, indicating that the conjugates were endocytosed by an alpha 2-macroglobulin receptor system. These results showed the usefulness of alpha 2-macroglobulin as a transport vehicle of lysosomal enzymes for effective enzyme replacement.

Animals↗

PubMed1993

The lipophilic weak base (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421) is a potent protonophore type cationic uncoupler of oxidative phosphorylation in mitochondria.

The lipophilic weak base AU-1421 acts as a simple protonophoric uncoupler of oxidative phosphorylation in rat liver mitochondria judging from the following observations. In the absence of any carrier lipophilic anions or P(i), AU-1421 stimulated the rate of state 4 respiration maximally about 7-fold at a concentration of 30 nmol/mg mitochondrial protein. At the same maximum effective concentration, it also inhibited ATP synthesis, released oligomycin-inhibited state 3 respiration, dissipated the proton motive force in the energized state, and activated latent H(+)-ATPase. AU-1421 also allowed proton conduction in both mitochondrial membranes and liposomes. These actions of AU-1421 resemble those of the typical anionic uncoupler SF6847. A marked difference between the two was, however, that ATPase activation by AU-1421 was not suppressed at higher concentrations of AU-1421, whereas ATPase activated by SF6847 was suppressed on increase of the SF6847 concentration. The finding that this simple protonophoric cation acts as an uncoupler at a micromolar concentration is significant, because all true (i.e., protonophore type) uncouplers known so far are anionic not cationic. Thus, AU-1421 is a unique uncoupler of the protonophore type.

Adenosine Triphosphate↗

PubMed1990

A monoclonal antibody against a native conformation of the porcine renal Na+/K(+)-ATPase alpha-subunit protein.

A monoclonal antibody (mAb50c) against the native porcine renal Na+/K(+)-transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na+/K(+)-ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to Na+/K(+)-ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na+, ATP or Mg2+ (E1 form), slightly less in the presence of K+ (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na+, Mg2+ and ATP. The antibody inhibited both the Na+,K(+)-ATPase activity and the K(+)-dependent p-nitrophenylphosphatase activity by 25%, but it had no effect on Na(+)-dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5'-isothiocyanatofluorescein after the addition of orthophosphate and Mg2+, and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the alpha-subunit.

Animals↗

PubMed1990

Cross-reactive polysaccharide antigens (types a, d, and h) of the mutans group of streptococci: different molecular forms of the type as distinguishable by monoclonal antibodies.

As compared to the previous precipitin inhibition tests differences were found in the reactivities of monoclonal antibodies (MAbs), a-4 and a-84 with Streptococcus cricetus (serotype a) in an enzyme immunoassay using whole cells, purified cell wall antigen and haptenic sugars coated onto microtitre wells. Investigation into the differences led to the finding that the purified antigen from S. cricetus cells consisted mainly of three forms with different molecular weights and sugar contents. MAb a-4 reacted with a high molecular weight form (AgI, molecular weight of 46,000) and low molecular weight forms (AgII and AgIII, molecular weights of 9,800 and 20,000, respectively) whereas MAb a-84 reacted only with the high-molecular form. Gas chromatographic analysis revealed that all antigens contained rhamnose, galactose and glucose but in different ratios of the sugars. Although the binding site of AgII/AgIII with MAb a-4 seemed to be slightly different from that of AgI with MAb a-84, the predominant immunodeterminant of the antigens was considered to be the same. On the basis of these results, the chemical structures of the antigenic determinants are suggested. The nature of the antigen-antibody reactions is discussed.

Antibodies, Monoclonal↗

PubMed1990

Enzyme immunoassay and monoclonal antibodies for immunological studies of the mutans group of streptococci.

Monoclonal antibodies (MAbs) were prepared from myeloma cells and splenocytes of mice immunized with Streptococcus cricetus HS1 or Streptococcus downei MF25 cells. Various makes of immunoplates were coated with whole cells or purified polysaccharide antigens of oral streptococcal strains and tested for reactivity in enzyme immunoassay (EIA) using the Mabs. It was revealed that different immunoplates had different reactivities. It was also shown that the EIA system and MAbs were useful for the immunological study and identification of oral streptococci.

Antibodies, Monoclonal↗

PubMed1989

A comparison of competitive enzyme immunoassay and precipitin inhibition tests in the analysis of polysaccharide antigenic determinants of oral streptococci.

A rapid and convenient method of competitive enzyme immunoassay (EIA) was explored for the immunological analysis by MAbs of polysaccharide antigens prepared from the mutans group of streptococci, and optimal conditions for it were established. These included the concentrations of antigen, monoclonal antibodies (MAbs) and labelled antibody, temperature, pH and reaction time. The results were compared with those obtained by precipitin inhibition tests using the classical quantitative precipitin reaction, and they were found to be consistent with the latter method. It was concluded that the EIA system can be extended to the immunological study of polysaccharide antigens of oral streptococci using MAbs which perform poorly in precipitation reactions.

Animals↗

PubMed1989

Directional immobilization of sodium- and potassium-activated ATPase to expose its cytoplasmic part to the liquid phase on microtiter plates by wheat germ agglutinin.

A convenient method for highly efficient and directional immobilization of intact sodium- and potassium-activated ATPase (Na,K-ATPase) using wheat germ agglutinin linked on microtiter plates was developed. Wheat germ agglutinin, which bound tightly to the beta-subunit of Na,K-ATPase and had no effect on the Na,K-ATPase activity, the potassium-activated p-nitrophenylphosphatase activity, or the inhibitory action of ouabain, was covalently linked to microtiter plates and used as an immobilizer of the enzyme. The amount of Na,K-ATPase coupled to microtiter plates in this immobilizing system was more than 10-fold greater than that used in the direct immobilizing system (O. Urayama, M. Nakao, H. Nagamune, and H. Sugiyama, (1984) Anal. Biochem. 141, 194-198). Also in this system, the cytoplasmic domain of Na,K-ATPase was exposed to the liquid phase. This technique was useful for investigating the reactivities of monoclonal antibody specific for the cytoplasmic domain of the enzyme. Moreover, because this technique was used successfully in the immobilization of periodic acid--Schiff positive staining glycoprotein 1 prepared from human erythrocytes and human alpha 2-macroglobulin, the technique should also be useful for other membrane or secreted proteins that possess N-linked sugar chains containing bisecting N-acetylglucosamine or a high amount of sialic acid.

Animals↗

PubMed1989

Type-specific antigen of Streptococcus rattus strains (KAY1 and FA1). I. Characterization.

A serotype-specific antigen was extracted from Streptococcus rattus KAY1 strain isolated first in Japan from human dental plaque and purified on an ion exchange column to compare it chemically and immunologically with that of FA1 strain which had been examined extensively by previous workers. Antigens of both strains reacted in a double diffusion test specifically with anti-FA1 serum which had previously been demonstrated specific for the strains in the same test, agglutination reactions and/or radioimmunoassay using whole cells. After separation on a DEAE-Sephadex A-25 ion exchange column the antigen was found to be resistant to various enzymatic treatments with pronases, lipase and nucleases and produce a single precipitin band against absorbed anti-FA1 serum in immunoelectrophoresis. Chemical analysis of this antigen revealed that it composed of carbohydrate, protein and a few percentages of glycerol and phosphorus. Hapten inhibition tests between antigen and antibody showed that galactose as well as glucose were the most potent inhibitors, suggesting their involvement in the antigenic determinant. Involvement of the sugars was also supported by gas chromatographic analysis and abolishment of reactivity with antiserum after the treatment of antigens with NaIO4. Moreover, protein does not seem to be involved since after SDS-PAGE analysis an enzyme immunoassay gave a negative reaction with immunoblotted antigens.

Antigens, Bacterial↗

PubMed1989

Type-specific antigen of Streptococcus rattus strains (KAY1, FA1 and BHT). II. Ultrastructural localization.

Streptococcus rattus strains (KAY1, FA1 and BHT) were examined by electron microscopy in whole cell-mounted samples and ultrathin sections for the localization of the type-specific antigen, using specific antiserum and ferritin-labelled anti-rabbit IgG as secondary antibody. In thin sections as well as whole cell-mounted samples the antigen was observed as irregular masses over the entire surface of the cells, in particular in the septal region between cells. The localization of the antigen was also examined in thin sections of a cell wall fraction prepared by treating the whole cells with glass beads in a disintegrator. Ferritin particles were observed around the outer surface, but not as many in number as those in whole cell-mounted or sectioned samples and rarely on the inner surface of the cell wall.

Antigens, Bacterial↗

PubMed1988

The third type of alpha-subunit of Na,K-ATPase.

The cDNA encoding the third type of alpha-subunit of NaK-ATPase was found in rat brain cDNA library. The deduced amino acid sequence was very similar to those of alpha- and alpha plus-subunits. The third one was suggested to be highly ouabain-sensitive from its aminoacid sequence. From Northern blot analysis the third type of the subunit was not expressed in adult rat kidney and heart.

Animals↗

PubMed1987

The hydrophobic cationic cyanine dye inhibits oxidative phosphorylation by inhibiting ADP transport, not by electrophoretic transfer, into mitochondria.

The effect of the divalent cationic cyanine dye tri-S-C4(5) on oxidative phosphorylation in rat liver mitochondria was examined. The dye at about 100 n mols per mg mitochondrial protein inhibited state 3 respiration and ATP synthesis almost completely. However, it had no effect on submitochondrial particles, like other hydrophobic cations. The dye inhibited the transport of ADP into mitochondria mediated by the adenine nucleotide translocator. Thus, the inhibition of oxidative phosphorylation by the cationic dye was concluded to be due to its action on the adenine nucleotide translocator, not to its electrophoretic transfer into the inner space of mitochondria according to the inside-negative electrochemical potential.

Adenosine Diphosphate↗

PubMed1987

Primary structures of two types of alpha-subunit of rat brain Na+,K+,-ATPase deduced from cDNA sequences.

A rat brain cDNA library was screened by using as a probe a fragment of cDNA encoding the alpha-subunit of human Na+,K+-ATPase. Two different cDNA clones were obtained and analyzed. One of them was concluded to be a cDNA encoding the alpha-subunit of the weakly ouabain-sensitive rat kidney-type Na+,K+-ATPase. The deduced amino acid sequence consists of 1,018 amino acids. The alpha-subunit of the rat kidney-type Na+,K+-ATPase shows 97% homology in amino acid sequence with the alpha-subunit of human, sheep, or pig enzyme and 87% with that of Torpedo. Based on a comparison of the amino acid sequence at the extracellular domain of the alpha-subunit between weakly ouabain-sensitive rat kidney-type enzyme and the ouabain-sensitive human, sheep, pig, or Torpedo enzyme, it was proposed that only two significant amino acid replacements are unique to the rat kidney-type alpha-subunit. Another cDNA clone obtained showed 72% homology in nucleotide sequence with the former cDNA coding the alpha-subunit of the rat kidney-type Na+,K+-ATPase and the deduced amino acid sequence exhibited 85% homology with that of the alpha-subunit of rat kidney-type Na+,K+-ATPase.

Amino Acid Sequence↗

PubMed1986

An Escherichia coli mutant exhibiting temperature-sensitive ATP synthesis.

A mutant strain SM434 (ttr-3) of Escherichia coli that exhibits a temperature-sensitive Unc(succinate-nonutilizing) phenotype was characterized. The mutant allele ttr-3 was not linked to the ilvA gene, but was complemented by Fill carrying 81 min-91 min of the E. coli chromosome. The mutant strain SM434 exhibited resistance to N,N'-dicyclohexylcarbodiimide (DCCD) and a temperature-sensitive phenotype at the level of ATP synthesis, compatible with that of cell growth. These findings indicate that the mutant strain SM434 could carry a mutation (ttr-3) in an unknown gene responsible for the energy-transduction system.

Adenosine Triphosphate↗

PubMed1986

Characterization of ouabain-sensitive phosphatase activity in the absence of potassium ion in purified pig kidney Na,K-ATPase.

The ouabain-sensitive phosphatase activity of purified pig kidney Na,K-ATPase preparation in the absence of potassium ion ((-K)phosphatase) was examined precisely. During the preparation procedures, the (-K)3-O-methylfluoresceinphosphatase ((-K)3-OMFPase) activity or the (-K)p-nitrophenylphosphatase ((-K)pNPPase) activity appeared to be purified in parallel with the Na,K-ATPase activity. The (-K)phosphatase activity was competitively inhibited by ATP and by ADP, with the K1 values of 0.25 microM and 1.4 microM, respectively. These values are consistent with their Kd values for the high-affinity ATP binding site of the Na,K-ATPase (Hegyvary, C. & Post, R.L. (1971) J. Biol. Chem. 246, 5234-5240). The substrate, pNPP, apparently competed with covalently bound fluorescein-5'-isothiocyanate (FITC), which is known to bind in the neighborhood of the high-affinity ATP binding site of the Na,K-ATPase, in both the (-K)phosphatase and the (+K)phosphatase reactions. The FITC-fluorescence intensity of FITC-labeled enzyme at the maximal steady-state activity of the (-K)phosphatase reaction was at a similar level to that of the E2 species. However, the FITC-labeled enzyme in the presence of only magnesium ion or only pNPP gave a fluorescence level similar to that of the E1 species. Oligomycin inhibited the (-K)phosphatase activity by at most 46%. On the basis of these results, it is strongly suggested that the (-K)phosphatase reaction is catalyzed at the high-affinity ATP binding site of Na,K-ATPase, and the (-K)phosphatase reaction proceeds in a cyclic manner (E1----E2----E1).

Animals↗

PubMed1985

Uncoupling of oxidative phosphorylation by divalent cationic cyanine dye. Participation of phosphate transporter.

The trinuclear cationic cyanine dye tri-S-C4(5) was found to be an uncoupler of oxidative phosphorylation. Its uncoupling required inorganic phosphate (Pi) or arsenate, which is transported into mitochondria via the Pi transport system, and was abolished by the Pi-transport inhibitor N-ethylmaleimide or mersalyl. The dye stimulated Pi uptake into mitochondria, and its uncoupling action was accompanied by swelling of the mitochondria. The adenine nucleotides ADP and ATP protected mitochondria from uncoupling by the dye. The dye taken up by mitochondria was released into the incubation medium on induction of uncoupling. In the absence of Pi, the dye did not cause uncoupling, but its uptake was much greater than in the presence of Pi. The cyanine dye is suggested to induce uncoupling by acting on the membrane, rather than after its electrophoretic transfer into the mitochondria.

Adenine Nucleotides↗

PubMed1985

Isolation and characterization of a monoclonal antibody against pig kidney sodium- and potassium-activated ATPase.

A hybridoma cell line producing mouse monoclonal antibody against pig kidney Na,K-ATPase was established. The antibody, named 38 (mAb38, IgG1), was purified from mouse ascites fluid by chromatography on a protein A-Sepharose column. Antigens immobilized on microplate wells with p-benzoquinone were used for titer assays. mAb38 cross-reacted with both dodecyloctaethyleneglycol monoether (C12E8)-solubilized enzyme and membranous sodium dodecyl sulfate (SDS)-treated enzyme from kidney with high affinity (50% binding = 0.6 nM). However, the antibody bound to neither alpha- nor beta-subunit separated by preparative SDS-polyacrylamide gel electrophoresis (PAGE). The stoichiometry of antibody binding to the purified enzyme was estimated to be about 0.86 mol of IgG per mol of alpha beta-protomer. Na,K-ATPase proteins were recovered from a column of mAb38-coupled Affi-Gel by elution with pH 3 buffer when C12E8-solubilized kidney enzyme or detergent extracts of brain microsomes were applied to it, confirming that the mAb is directed to Na,K-ATPase. mAb38 at saturation level concentrations had no effect on kidney Na,K-ATPase activity or on ouabain-sensitive Rb uptake in erythrocytes. In an immunofluorescence study, the antibody bound to intact erythrocytes much more strongly than control IgG1 (mAb50c), but the extent of the antibody binding to inside-out vesicles under hypotonic conditions was lower than that of the control. Most of the antibody binding activity remained when the kidney enzyme was treated with sialidase. These results suggest that this mAb38 was raised against an intact conformation of a cell-surface-exposed site of Na,K-ATPase.

Animals↗

PubMed1984

Coupling of membrane-bound and detergent-solubilized sodium- and potassium-activated ATPase to p-benzoquinone-treated microtiter plates.

Membrane-bound and dodecyloctaoxyethyleneglycol monoether-solubilized Na,K-ATPases from pig kidney were covalently attached to microtiter plate wells pretreated with p-benzoquinone (plus collodion for some plates). The immobilized enzymes were detected with the mouse monoclonal antibody (named 38) specific to Na,K-ATPase and a perioxidase-conjugated rabbit IgG anti-mouse IgG. When the two Na,K-ATPase preparations were applied to each well at the same protein concentration, the color intensity of the peroxidase reaction for determination of antibody was two to three times stronger with the solubilized enzyme than with the membrane-bound enzyme. Similar titer values were obtained from the graphical analysis of titration curves of both enzymes. Red cell membrane proteins as well as Na,K-ATPase were covalently attached to the plastic. p-Benzoquinone should be generally useful for coupling membrane proteins, even in detergent solutions, to microtiter plate wells.

Animals↗