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Y Kanaoka

Publications and source records attributed to Y Kanaoka.

At least 19 recordsLinked to original sources
PubMed1992

A site-directed antibody that inhibits phosphorylation of the rat-brain sodium channel by cyclic-AMP-dependent protein kinase.

Antibodies were raised against three peptides corresponding to the potential protein phosphorylation sites of rat-brain sodium channels by the cAMP-dependent protein kinase (PKA). One of the antibody against sequence (C561-575) reacted to the channel molecule. This immunoreaction occurred in a sequence-specific manner, as it was inhibited by the antigen peptide itself but not inhibited by two other peptides. Although PKA phosphorylates two synthetic peptides, C561-575 and C681-689, of the three, anti-(C561-575) antibody can only inhibit the phosphorylation of peptide (C561-575). PKA catalyzed the incorporation of 3.1-3.5 mol of phosphates into the alpha subunit of the purified sodium channel. The anti-(C561-575) antibody inhibited the channel phosphorylation by 40%. Digestion of the phosphorylated sodium channel with lysyl endoproteinase yielded four major phosphorylated fragments of 3.5, 5.0, 7.0, and 10 kDa. However, similar digestion of the channel that was phosphorylated in the presence of anti-(C561-575) antibody did not yield the phosphorylated fragment of 3.5 kDa and gave the 7.0 kDa fragment in reducing yield. Inspection of these phosphorylated fragments by the predicted sizes of the peptide fragments containing the five potential phosphorylation sites gives a conclusion that anti-(C561-575) antibody inhibits the phosphorylation on Ser-573 completely, and on either Ser-610 or Ser-623 partially, probably due to their proximity orientation in the tertiary structure.

Amino Acid Sequence↗
PubMed1992

Photolabeled sites with a tetrodotoxin derivative in the domain III and IV of the electroplax sodium channel.

Forty three percent of the labeled sites, at least, in the electroplax sodium channel with a photoactivable tetrodotoxin derivative were identified by probing protease-digested labeled fragments with several sequence-directed antibodies. They are located in the loop between segments S5 and S6 of domain IV, as well as the region containing transmembrane segment S6 and adjacent extracellular and cytoplasmic sequences in domain III. No photolabeled fragments were detected in the corresponding region of domain I. These results suggest that C-11 of tetrodotoxin where the photoreactive moiety is attached orients to the region between S5 and S6 in domain III and IV. Probable orientation of the tetrodotoxin molecule in sodium channels is considered by taking together with the recent report of the site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1992

Anhydrosubtilisin-catalyzed peptide synthesis.

Anhydrosubtilisin was found to be a good catalyst for the peptide synthesis though its hydrolytic activity was fully inhibited. Amino acid (peptide) p-chlorophenyl ester (acyl donor) was coupled with amino acid (peptide) amide in the presence of the modified enzyme. The method did not afford by-products resulting from the hydrolysis of the acyl donor. This is the most advantageous characteristic of the method, since the hydrolysis of the acyl donor is unavoidable for the methods using catalytically active proteases. Anhydrosubtilisin was further shown to be useful for the fragment condensation of peptides.

Amidohydrolases↗
PubMed1992

Structural characterization of the dihydropyridine receptor-linked calcium channel from porcine heart.

Ca(2+)-channel was purified 230-fold from digitonin extracts of the porcine cardiac sarcolemmal membranes by means of a four-step procedure. Two antibodies, a site-directed antibody against the sequence 1691-1707 of the rabbit cardiac alpha 1 subunit (anti-CCP5) and a monoclonal antibody directed to rabbit skeletal muscle alpha 2 delta subunit-complex (MCC-1), effectively immunoprecipitated the 125I-labeled cardiac Ca(2+)-channel complex in 0.2% digitonin. SDS-PAGE analysis of the immunoprecipitates under reducing conditions revealed that the cardiac channel is mainly composed of two large polypeptides of 190 and 150 kDa, and five smaller polypeptides of 60, 55, 35, 30, and 25 kDa. An additional polypeptide of either 79 or 55 kDa is crosslinked with the 190 kDa component to form 250-270 kDa (approximately 270 kDa) to the extent of 15-20% through disulfide bond(s). The 190 kDa component (alpha 1) is responsible for photoaffinity labeling with [3H]diazepine, since minor photolabeled approximately 270 kDa was converged to the major labeled 190 kDa component when electrophoresed under reducing conditions. The 150 kDa component (alpha 2) was derived by reduction of disulfide bonds from another 190 kDa component of glycopolypeptide which was separated from the channel complex in 1% Triton X-100 and capable of binding to WGA-Sepharose. The four smaller components of 60, 35, 30, and 25 kDa were not covalently associated with the large components through disulfide bonds, whereas the 55 kDa polypeptide was suggested to be a mixture of two kinds of peptides with respect to the disulfide bond: one was crosslinked with alpha 1 through disulfide linkage and the other was not covalently associated with any other component.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Characterization of saccharide moiety in the electroplax sodium channel.

Carbohydrate chains on the large peptide of the voltage-sensitive sodium channel from Electrophorus electricus electroplax have been partially characterized by the lectin-blotting technique combined with digestion using three glucosidases: neuraminidase, endo-beta-N-acetylglucosaminidase H, and peptide: N-glycosidase F. The results show that both N-linked oligosaccharides and O-linked (mucin-type) oligosaccharides are present. In N-linked oligosaccharides, the results suggest the presence of complex- and hybrid-type oligosaccharides which contain bisecting N-acetylglucosamine(s), as well as the complex-type oligosaccharides with the alpha-Fuc-GlcNAc-(Asn) residue(s). In O-linked oligosaccharides, they must carry Gal beta1----3GalNAc- moieties which contain NeuNAc residues in the terminal.

Animals↗
PubMed1991

Identification of 1,4-dihydropyridine binding regions within the alpha 1 subunit of skeletal muscle Ca2+ channels by photoaffinity labeling with diazipine.

To identify regions that are involved in the formation of the dihydropyridine receptor site of skeletal muscle L-type Ca2+ channels, the alpha 1 subunit of the channel complex was specifically labeled with the 1,4-dihydropyridine-receptor-selective photoaffinity probe [3H]diazipine. Photoaffinity-labeled regions were identified by probing labeled proteolytic fragments with several anti-peptide antibodies recognizing different segments of the alpha 1 sequence. Forty to 50% of the alpha 1-associated [3H]diazipine label was contained in the tryptic fragment between Arg-988 and Ala-1023 derived from the loop between segments S5 and S6 in domain III. This region corresponds to a portion of the channel that is believed to contribute to formation of the transmembrane pore. Twenty to 30% of the labeling occurred in a V8 protease fragment between Glu-1349 and Trp-1391. This fragment contains transmembrane segment S6 of domain IV and has previously been shown to form part of the drug receptor for phenylalkylamine Ca2+ antagonists. Our data suggest that the dihydropyridine receptor is formed by close apposition of two discontinuous regions of the alpha 1 subunit sequence in domains III and IV. In light of previous work localizing this receptor site to the extracellular surface of the lipid bilayer, it is proposed that amino acid residues at the extracellular surface in the loop connecting segments IIIS5 and IIIS6 and at the extracellular end of segment IVS6 contribute to formation of the dihydropyridine receptor site.

Affinity Labels↗
PubMed1991

A new hereditary abnormal protein C (protein C Yonago) with a dysfunctional Gla-domain.

A familial abnormal protein C most probably with the dysfunctional Gla domain was found in a 60-year-old man with recurrent thrombosis. Namely, the anticoagulant activity as measured by the APTT method and the antigen level by an ELISA utilizing a calcium-dependent antibody were reduced to nearly half of normal, 43.5% and 2.1 micrograms/ml (normal range: 2.8-5.0 micrograms/ml), respectively. On the other hand, the amidolytic activity determined on a synthetic chromogenic substrate, S-2366, and the total antigen measured by an ELISA utilizing a polyclonal antibody were both in the normal range, 74.1% and 83% of normal, respectively. Crossed immunoelectrophoresis showed more anodal migration than the normal control in the presence of calcium ions, and adsorption of protein C to barium citrate was insufficient. These data altogether indicated that a half population of protein C in the patient's plasma was dysfunctional in the Gla domain or its related structures. Four other members of his immediate family were found to have the same abnormality of protein C, although they had been all asymptomatic. We thus conclude that the dysfunctional protein C is hereditary, and that the abnormalities noted in several tests are most likely due to a structural defect residing in the Gla or its related regions. We hereby designate this abnormal protein C as protein C Yonago.

Blood Coagulation Tests↗
PubMed1991

Diazipine, a novel photoaffinity probe for dihydropyridine receptors of calcium channels.

A new 1,4-dihydropyridine photoaffinity ligand, [3H]diazipine, has been assessed by binding and photolabeling, and compared with a currently used [3H]azidopine. [3H]Diazipine reversibly binds to skeletal muscle Ca2+ channels with a similar affinity to [3H]azidopine, but [3H]diazipine labels the channel two times more efficiently and no release of the incorported amount is observed after dithiothreitol treatment.

Affinity Labels↗
PubMed1991

Application of bimane-peptide substrates to spectrofluorometric assays of metalloendopeptidases.

A spectrofluorometric method for sensitive determination of metalloendopeptidase activity has been developed by using a bimane-peptide containing a tryptophan residue, i.e. 1,7-dioxo-2,5,6-trimethyl-1H,7H-pyrazolo[1,2-alpha]pyrazol-3-yl-methyl- thiomethylcarbonyl-phenylalanyl-tryptophanyl-leucine (Bim-SCH2CO-Phe-Trp-Leu-OH). Such an "intramolecularly quenched" substrate was originally designed for a sensitive assay of angiotensin I converting enzyme (ACE) [Sato, E. et al. (1989) Chem. Pharm. Bull. 37, 145-147]. All the typical metalloendopeptidases tested, such as thermolysin, Pseudomonas aeruginosa (Ps.) elastase, Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), and alkinonase A, a metalloendopeptidase from Streptomyces violaceorectus, cleaved this substrate strictly at a Phe-Trp bond, leading to a marked increase in fluorescence. Kinetic parameters of the enzymatic hydrolyses of five kinds of analogous bimane substrates were compared to examine how the nature of neighboring amino acid residues on either side of the cleavable bond affects the catalytic efficiency of each of the metalloendopeptidases. Bim-SCH2CO-Phe-Trp-Leu-OH was most efficiently hydrolyzed by all of these enzymes. The use of this substrate made it possible to determine minute amounts of metalloendopeptidases, especially those originating from Streptomycetes (for example, as little as 10 fmol of SGMPII).

Amino Acid Sequence↗
PubMed1991

Fluorogenic bimane substrates with dabsyl group for endopeptidases; chymotrypsin, collagenase and thermolysin.

It was found that the fluorescence of 9,10-dioxa-syn-3,4,6,7-tetramethylbimane (bimane) can be quenched in the presence of dimethylaminoazobenzensulfonyl (Dabsyl) group. New combination of bimane (fluorophor) and dabsyl group (quencher) was applied to the syntheses of intramolecularly quenched fluorogenic substrates for hydrolytic enzymes. Bimane peptides containing dabsyl group were prepared, and were shown to be useful fluorogenic substrates for the assay of endopeptidases such as chymotrypsin, collagenase and thermolysin.

Bridged Bicyclo Compounds↗
PubMed1991

Synthesis of diazipine and [3H]diazipine: novel dihydropyridines as photoaffinity probes of calcium channels.

Diazipine and [3H]diazipine were synthesized as new 1,4-dihydropyridine photoaffinity ligands containing a phenyldiazirine group. After simple high performance liquid chromatography separation, both compounds were purified in good overall yields. [3H]Diazipine (21.2 Ci/mmol) was synthesized in two steps from commercially available [3H]ethanolamine. Diazipine competitively inhibited [3H]PN200-110 binding to the calcium channel of cardiac membranes with high affinity.

Affinity Labels↗
PubMed1991

Bimane fluorogenic substrates for microdetermination of angiotensin converting enzyme level in serum.

The fluorescence of 9,10-dioxa-syn-3,4,6,7-tetramethylbimane (bimane) was found to be quenched in the presence of nitrated aromatic amino acid. Bimane peptides containing nitrated amino acid (1a, b) were shown to be useful fluorogenic substrates for the assay of angiotensin I converting enzyme (ACE) from rabbit lung, similar to bimane substrate containing tryptophan (3) previously reported. Among these bimane substrates, substrate 3 was shown to be a potent fluorogenic substrate for microdetermination of the ACE level in human serum.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1991

[Effect of malignant ascitic fluids and OK-432 on the induction of LAK activity in peripheral blood mononuclear cells of gynecological cancer patients].

Recently, successful treatment of peritonitis carcinomatosa by intraperitoneal administration of lymphokine-activated killer (LAK) cells followed by intraperitoneal recombinant interleukin 2 (rIL-2) has been reported by several authors, in spite of the well documented results of the immunosuppressive activity of malignant ascitic fluid. We investigated the effect of malignant ascitic fluid on in vitro induction of LAK cells obtained from patients' peripheral blood mononuclear cells. We also examined whether Streptococcal preparation OK-432, which has been instilled into the peritoneal cavity to treat malignant ascites, is able to synergize with rIL-2 to induce LAK activity, and the following results were obtained; 1. A little augmentation of LAK activity was observed at a lower concentration of malignant ascitic fluid, while the results were diversified at a concentration higher than 10%. In two cases out of six, a severe suppressive effect was observed at a concentration higher than 40%. At the higher concentration, fewer cells were recovered after a 5 day culture in all cases. 2. No augmentation of LAK activity following the combination of OK-432 with rIL-2 was observed. At a lower concentration of OK-432 (ranging from 0.01-0.04KE/ml), the number of cells increased in comparison with rIL-2 alone. These results suggest that the potential of adoptively transferred LAK cells followed by rIL-2 was not effectively suppressed by malignant ascitic fluid in vivo and that the administration of OK-432 followed by rIL-2 could induce a larger number of various killer cells than rIL-2 alone.

Ascites↗
PubMed1991

[The effect of intraperitoneal administration of OK-432 with recombinant interleukin-2 to patients with peritonitis carcinomatosa of recurrent gynecological cancer and changes in ascitic lymphocyte subsets].

The effect of intraperitoneal administration of OK-432 followed by intraperitoneal instillation of recombinant interleukin-2(rIL-2) was examined in tumor bearing animals and in four recurrent gynecological cancer patients with peritonitis carcinomatosa which had been resistant to chemotherapeutic drugs. Seven days after intraperitoneal inoculation of tumor cells (10(6) cells/body of MH134 hepatoma into C3H/He mice and 10(5) cells/body of Meth-A fibrosarcoma into BALB/C mice, respectively), OK-432 was administered intraperitoneally. Two days later, a 14 day course of daily intraperitoneal instillation of rIL-2 followed. The survival time for animals treated with OK-432 combined with rIL-2 was significantly prolonged. Three of the 8 C3H/He mice and one of the 8 BALB/C mice in this group survived more than 150 days without forming ascites. However, the group treated by rIL-2 alone did not survive for more than 40 days. The group treated by OK-432 alone as well as the untreated group did not survive for more than 20 days. Ascitic fluid disappeared clinically in two of four cases and decreased in the rest. Ascitic cancer cells disappeared in one case and decreased in three cases. The serum CA125 level declined significantly in all cases. The surface markers of ascitic lymphocytes were analyzed by flow cytometry on day 8. The CD4+ subset accounted for 70-90% whereas the CD8+ subset accounted for only 7-17%. In three cases in which two color analysis was performed, the CD4+, CD29+ helper inducer T cell was dominant. We could conclude that LAK cells were not the main effector cells.

Adult↗
PubMed1990

[Intraperitoneal administration of OK-432 and rIL-2 in a case of peritonitis carcinomatosa with chronic renal failure].

Recombinant interleukin-2 (rIL-2) was administered intraperitoneally for 15 days, 2 days after intraperitoneal administration of Streptococcal preparation OK-432 to a patient of peritonitis carcinomatosa occurred eight months after second look operation, in which residual tumor could not be removed completely. The patient had been maintained by hemodialysis three times a week for over ten years. Combination chemotherapy using CDDP and Ifosfamide, or CDDP and THP-Adriamycin had not been effective to control rapidly increasing ascites. Negative cytological exam, was achieved on day 7 and ascites disappeared by day 15. No severe side effects including fluid retention were observed. Fever up was controllable by Indomethacin. Flow cytometric analysis revealed dominant (73%) CD4+, CD29+ helper inducer subset, while CD4+, CD45RA+ was 6%, in the lymphocytes in ascites on day 8. It was suggested that intraperitoneal administration of rIL-2 after OK-432 was safe and effective for peritonitis carcinomatosa with chronic renal failure.

Adenocarcinoma↗