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Biomedical subjects

M Kuwada

Publications and source records attributed to M Kuwada.

At least 37 records · Page 2Linked to original sources
PubMed1994

[Investigations of the use of IABP during open heart surgery].

Since 1977, IABP has been employed in 180 cases. We studied 94 adult patients who underwent open heart surgery and were treated with IABP procedure for the past 10 years, and investigated the following items, the timing of IABP initiation, preoperative left heart functions, aortic clamping time, period of IABP use, and mortality. Additional, P-V loop was measured during the operation. The following conclusions were drawn. When IABP was used preoperatively in cases with deteriorated cardiac functions, the incidence of in cases in which IABP was used during or after the operation. P-V loop is useful to obtain the detail of cardiac function which provides sufficient patient management during and after the operation, and could be useful for establishing the criteria of IABP use.

Adult↗
PubMed1993

Purification from adult pig testicular P-450 and 17 alpha-hydroxylase activity of P-450 containing liposomal membranes.

A cytochrome P-450 from adult pig testicular microsomes was purified to a specific content of 12 nmol P-450/mg protein. P-450 has a minimum molecular weight of 46000 +/- 1000, as judged on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Adult testicular P-450 is prepared in the low-spin form with an absorbance maximum at 417 nm. The substrate-induced difference spectrum with progesterone is a typical I difference spectrum. P-450 was incorporated into liposomal membranes composed of phosphatidylcholine, and 17 alpha-hydroxylase activity was shown to amount to 15.5 nmol product/min/nmol of P-450. Furthermore, testicular cytochrome b 5 did not increase the 17 alpha-hydroxylase activity, and the activity was largely inhibited by the addition of sodium cholate, Emulgen 913 and testicular lipid.

Animals↗
PubMed1993

A novel peptide from funnel web spider venom, omega-Aga-TK, selectively blocks, P-type calcium channels.

In the course of purification of omega-Aga-IVA, a specific P-type calcium channel blocker, from the venom of Agelenopsis aperta we discovered a novel peptide. This peptide, named omega-agatoxin Tsukuba (omega-Aga-TK), also blocked P-type channels and was twelve times more abundant in the venom than omega-Aga-IVA. omega-Aga-TK was purified to homogeneity by a two-step reverse-phase HPLC procedure. Its amino acid sequence is 71% identical to that of omega-Aga-IVA. omega-Aga-TK has a negatively charged N-terminus, whereas omega-Aga-IVA has a positively charged one. Electrophysiological data indicate that omega-Aga-TK is a potent and selective inhibitor of P-type channels.

Agatoxins↗
PubMed1991

Purification and properties of cytochrome P-450 (SCC) from pig testis mitochondria.

Cytochrome P-450 was purified from pig testis mitochondria to a specific content of 13.1 n mol/mg of protein. The purified preparation was found to contain a single species of P-450, on sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular weight of about 53000 +/- 2000. The cholesterol side chain-cleavage system could be reconstituted by mixing the purified cytochrome P-450, adrenodoxin reductase, adrenodoxin, cholesterol and NADPH. The rate of conversion of cholesterol to pregnenolone was 6.2 n mol/min/n mol of P-450 under the conditions employed. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 416, 530 and 568 nm. The reduced CO-complex of the cytochrome P-450 exhibited an absorption maximum at 448 nm. The purified P-450 was subjected to microsequence analysis and its NH2-terminal amino acid sequence was found to show considerable homology with that of bovine adrenal P-450 (SCC).

Amino Acid Sequence↗
PubMed1991

Comparison of the multiple molecular forms of bovine adrenocortical P450scc with those of corpus luteum P450scc.

1. Bovine adrenocortical P450scc was resolved into several fractions by chromatography on AH-Sepharose 4B followed by gel filtration on Toyopearl HW55S. All fractions contained P450scc of the same molecular size and the P450scc could be resolved into 3-4 major and more than 10 minor isoelectric point forms by isoelectric focusing on polyacrylamide gel in the presence of Emulgen 913. 2. Both the AH-Sepharose chromatography profile and the isoelectric focusing pattern of the adrenocortical P450scc were more complex than those of the corpus luteum P450scc. The corpus luteum P450scc was practically devoid of the neutral to acidic isoelectric point forms. 3. Three to four P450scc subfractions with different isoelectric focusing pattern were obtained from a purified preparation of adrenocortical P450scc by ion-exchange chromatography on DEAE-Toyopearl 650S or DEAE-Sephadex A25. These P450scc subfractions showed essentially the same spectral properties, catalytic activity, molecular weight and N-terminal amino acid sequence. 4. The most acidic (the latest eluting) subfraction was composed mostly of the neutral to acidic isoelectric point forms. The sedimentation characteristics of this subfraction was also studied. 5. The structural basis of the multiple molecular forms was discussed.

Adrenal Cortex↗
PubMed1990

Four disulfide bonds' allocation of Na+, K(+)-ATPase inhibitor (SPAI).

We have recently reported the primary structures of the three unique peptide inhibitors (SPAI-1, -2, and -3) against Na+, K(+)-ATPase which contained four disulfide bridges in common (Biochem. Biophys. Res. Commun. 164, 496 (1989)). The disulfide connectivities of SPAI were determined by the combination of amino acid analyses with the direct application to a gas-phase sequencer of its proteolytic fragments. The disulfide bond was identified by detection of phenylthiohydantoin derivatives of cystine and its decomposed product dehydroalanine. The four cysteine pairs were disclosed to be Cys20 to Cys49, Cys27 to Cys53, Cys36 to Cys48, and Cys42 to Cys57, all linked by disulfide bridge formation. The allocation pattern of these disulfide bonds was the same as that recently reported for human mucous proteinase inhibitor (EMBO J. 7, 345 (1988], though SPAI showed no proteinase inhibitory activity at all.

Amino Acid Sequence↗
PubMed1989

Novel peptide inhibitor (SPAI) of Na+, K+-ATPase from porcine intestine.

Three unique inhibitors (SPAI-1, -+2, and -3) were first purified from porcine duodenal extract based on the Na+, K+-ATPase inhibitory activity. These peptide inhibitors had four disulfide bridges in common. The sequencing results of their S-carboxymethyl derivatives, lysilendopeptidase fragments, and chymotryptic peptides disclosed their entire primary structures. Both SPAI-2 and -3 consisted of 61 amino acids, respectively, and had almost the same sequences except for two amino acid substitutions, while SPAI-1 was found to lack the N-terminal twelve amino acid sequence of SPAI-2. The kinetics study revealed that SPAIs inhibited Na+, K+-ATPase by the competitive mode against Na+ and were uncompetitive with K+.

Amino Acid Sequence↗
PubMed1988

Plasma and bone osteocalcin levels in rats with type II collagen-induced arthritis.

Osteocalcin levels in plasma and bone were measured by enzyme immunoassay in rats with arthritis induced by immunization with type II collagen and in untreated control rats. Compared with levels in control rats, the plasma levels of osteocalcin in arthritic rats were markedly decreased 1-3 weeks after immunization; during weeks 8-14, these levels were significantly increased. The osteocalcin content of tarsal bones changed in parallel with the plasma levels. These data suggest that osteocalcin levels in the plasma of arthritic rats reflect alterations in bone formation activity.

Animals↗
PubMed1987

Amino acid sequence of sialic acid binding lectin from frog (Rana catesbeiana) eggs.

The complete amino acid sequence of sialic acid binding lectin from frog (Rana catesbeiana) egg is presented. The 111-residue sequence was determined by the analysis of peptides generated by digestion of the S-carboxymethylated protein with Achromobacter protease I, chymotrypsin, or cyanogen bromide. The sequence is unique and not homologous to any known protein sequence. The protein may represent a new type of lectin.

Amino Acid Sequence↗
PubMed1986

An enzyme immunoassay for osteocalcin.

A sensitive and specific enzyme immunoassay for bovine osteocalcin was developed with the use of enzyme-labeled antigen. Bovine osteocalcin was conjugated with beta-D-galactosidase for use in the immunoassay. The minimum amount of osteocalcin detectable by this method was 5-10 pg/assay. The enzyme immunoassay could detect immunoreactive osteocalcin levels in normal human sera, giving a value of 6.58 +/- 0.72 ng/ml (mean +/- SE of 16 human subjects). The correlation coefficient between the levels measured by this method and radioimmunoassay was 0.94.

Animals↗
PubMed1985

Purification of NADPH-cytochrome c reductase from swine testis microsomes by chromatofocusing and characterization of the purified reductase.

A purified NADPH-cytochrome c reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was prepared from swine testis microsomes by detergent solubilization followed by a procedure including chromatofocusing. The reductase was eluted at an isoelectric point of 4.8 from the chromatofocusing column. 730-fold purification was achieved with an overall yield of 1.2%. The preparation was found to be homogeneous upon polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (SDS). Upon SDS-polyacrylamide gel electrophoresis, however, the purified preparation resolved into one major band (Mr 78 000) and two minor bands (Mr 60 000 and 15 000). The enzyme contained about 1 mol each of FMN and FAD, which were both extractable with trichloroacetic acid and also boiling water. The oxidized form of the enzyme showed the absorption spectrum of a typical flavoprotein. Aerobic reduction with NADPH resulted in conversion of the spectrum into one of an air-stable semiquinone form. The activity of the purified preparation was 26 mumol cytochrome c reduced/min per mg protein under the standard assay conditions at 22 degrees C. The enzyme catalyzed the reaction through a ping-pong mechanism.

Animals↗
PubMed1984

Partial purification and characterization of a rat kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide.

The activity for the hydrolysis of succinyl trialanine-4-nitroanilide was higher in kidney homogenates of female rats and mice than in those of male rats and mice. An enzyme hydrolyzing the above substrate was extracted from female rat kidney homogenate and partially purified by means of gel filtration on Sepharose 4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme cleaved the bond between succinyl dialanine and alanine-4-nitroanilide of the substrate and showed a Km value of 3.3 mM at the optimal pH of 7.5. The activity was increased by Ca2+ and Mg2+, but inhibited by EDTA. With oxidized insulin B chain as a substrate, the enzyme cleaved the carbonyl bonds of Ala-14, Tyr-16 and Gly-23 efficiently, and those of His-5 and His-10 less efficiently.

Amino Acid Sequence↗
PubMed1984

Differentiation of endopeptidases and aminopeptidases by high-performance liquid chromatography of reaction products from chromogenic peptide p-nitroanilines as substrates.

A method for differentiating endopeptidases and aminopeptidases on the basis of substrate specificity is presented. Various synthetic chromogenic substrates, succinyl-(Ala)3-p-nitroaniline, succinyl-(Ala)2-p-nitroaniline, (Ala)3-p-nitroaniline, and (Ala)2-p-nitroaniline, were incubated with various peptidases and the incubation mixtures were directly analyzed by high-performance liquid chromatography to determine the splitting patterns of these substrates by the enzymes. The substrates and hydrolyzed products containing the chromophore were separated on a reverse-phase column under isocratic conditions, and the chromophore was specifically detected in the effluent fractions by absorbance measurement at 314 nm. Endopeptidases, leucine aminopeptidase, and dipeptidyl aminopeptidase showed different patterns of cleavage of the substrates. This simple and rapid high-performance liquid chromatographic procedure is suitable for identifying the above activities in different fractions obtained during separation and purification studies. The same approach was applied to the simultaneous determination of three types of endopeptidase activities in rat tissues based on the ability of the enzymes to hydrolyze different sites in succinyl-(Ala)3-p-nitroaniline.

Aminopeptidases↗
PubMed1983

An improved method for the determination of gamma-carboxyglutamic acid in proteins, bone, and urine.

A method of high-performance liquid chromatographic separation of the fluorescence derivative of gamma-carboxyglutamic acid (Gla) is presented. Alkaline hydrolysates of protein samples were reacted with o-phthalaldehyde in the presence of ethanethiol for 2 min, and the fluorescence derivative of gamma-carboxyglutamic acid was resolved from the other amino acids by a short column packed with silica-based anion exchanger under isocratic conditions. By this method, as low as 200 fmol of gamma-carboxyglutamic acid can be quantitatively analyzed within 10 min. The method presented here shortened the analysis time for Gla and was at least 10 times more sensitive than the method we described previously (Anal. Biochem. 117, 259-265, 1981). The application of this method to the formic acid-soluble or insoluble gamma-carboxyglutamic acid-containing proteins in chicken bone and the concomitant increase of gamma-carboxyglutamic acid content in chicken bone with age are reported.

1-Carboxyglutamic Acid↗