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Biomedical subjects

Y Kawata

Publications and source records attributed to Y Kawata.

At least 127 records · Page 7Linked to original sources
PubMed1991

[Analyses of creatine kinase isoforms to detect early diagnosis of reinfarction and reperfusion after acute myocardial infarction].

The detection of reinfarction and reperfusion of the acute myocardial infarction (AMI) in the early phase was studied by monitoring the 34 AMI patients of their changes in creatine kinase (CK, EC 2.7.3.2) isoforms level. CK isoform was assayed by immunoinhibition method using monoclonal antibody. The increased ratio of serum CK-MM3/CK-MM1 after the onset was observed earlier than conventional serum parameters such as CK-MB, CK-MM3, PK and LD and it was almost the same as myoglobin in both reperfused and non-reperfused patients. The increased ratio of CK-MM3/CK-MM1 in reperfusion group was gradually reduced after 5 hours of onset earlier and lowered to reference level two to three times more rapidly than in non-reperfusion group. The ratio of serum CK-MM3/CK-MM1 with AMI increased much more sharply compared to that of after hard exercise. Therefore, monitoring the ratio of patients serum CK-MM3/CK-MM1 with AMI (at serial times, or periodically, or every 3 hours, etc.) is significant for clinical diagnosis.

Aged↗
PubMed1990

Role of tryptophan 248 in the active site of tryptophanase from Escherichia coli.

Tryptophan 248, located in the active site of tryptophanase from Escherichia coli, has been replaced with phenylalanine by site-directed mutagenesis. Judging from CD and fluorescence spectra, the global structure of the mutant enzyme was found to be the same as that of the wild-type enzyme. The binding affinity of the mutant enzyme for the coenzyme pyridoxal 5'-phosphate (PLP) was reduced tenfold compared to the wild-type enzyme. Kinetic analyses under PLP-saturated conditions indicated that the Km values of the mutant enzyme for substrates are the same as those of wild-type enzyme but the kcat values are decreased to about 85%, which accounts for the overall activity decrease. These findings suggest that tryptophan 248 interacts closely with PLP and plays an important role in the catalytic reaction.

Animals↗
PubMed1990

The role of an invariant tryptophan residue in alpha-bungarotoxin and cobrotoxin. Investigation of active derivatives with the invariant tryptophan replaced by kynurenine.

Ozone oxidation converted the single, invariant, tryptophan residue to N2-formylkynurenine in alpha-bungarotoxin and cobrotoxin. Upon this modification, the lethal toxicity was significantly reduced in cobrotoxin but mostly retained in alpha-bungarotoxin. Each neurotoxin containing kynurenine instead of tryptophan retained the same antigenicity as the native toxin. Fluorescence and CD spectroscopy revealed that, although the environment and state of the kynurenine residue were similar, [Kyn29]cobrotoxin was much more sensitive to pH change than alpha-[Kyn28]bungarotoxin. In terms of lethal toxicity and conformational stability, the invariant tryptophan residue appears to play a more important role in cobrotoxin, imparting a higher lethal toxicity than that in alpha-bungarotoxin, which has a disulfide bond at Cys29-Cys33.

Animals↗
PubMed1990

Chemical modification of tryptophan residues and stability changes in proteins.

The role of tryptophan residues in the stability of proteins was studied by ozone oxidation, which causes a small change in the tryptophan side chain. Trp 187 of the constant fragment of a type lambda immunoglobulin light chain, Trp 59 of ribonuclease T1, and Trp 62 of hen egg white lysozyme were oxidized specifically by ozone to N'-formylkynurenine or kynurenine. Judging from their circular dichroic and fluorescence spectra, these modified proteins were found to be the same as those of the respective intact proteins. However, even the slight modification of a single tryptophan residue produced a large decrease in the stability of these proteins to guanidine hydrochloride and heat. The smaller the extent of exposure of the tryptophan residue, the greater the effect of the modification on the stability. The formal kinetic mechanism of unfolding and refolding by guanidine hydrochloride of the CL fragment was not altered by tryptophan oxidation, but the rate constants for unfolding and refolding changed. The thermal unfolding transitions were analyzed to obtain the thermodynamic parameters. The enthalpy and entropy changes for the modified proteins were larger than the respective values for the intact proteins.

Animals↗
PubMed1990

Conformations of isolated fragments of pancreatic polypeptide.

In spite of its short polypeptide chain, the pancreatic polypeptide molecule consists of a polyproline II type helix and alpha-helix. To understand the stability and formation of the alpha-helical region, we prepared some peptide fragments including the helical segment of chicken pancreatic polypeptide and studied their conformations by circular dichroism (CD). PP7-36 (a peptide fragment corresponding to residues 7-36 of chicken pancreatic polypeptide) showed a CD spectrum characteristic of the helix at pH 4.6 and at peptide concentrations as low as 1 microM. PP11-36 was able to form a helical conformation only at high peptide concentrations and not at concentrations lower than 10 microM. However, acetyl PP11-36 (in which the alpha-amino group is acetylated so that no positive charge exists at the N terminus) was able to form the helical conformation at pH 4.6 and at the peptide concentrations where PP11-36 could not. Succinyl PP12-36 (in which the alpha-amino group is succinylated to introduce a negative charge) was also able to form the helical conformation. The CD spectra of PP12-36 and PP13-36 were not characteristic of the helical conformation at all the pH values and peptide concentrations studied. Acetyl PP13-36, which has no charge at the N terminus, did not form the helix. On the other hand, succinyl PP13-36, which has a negative charge at the N-terminal end, did form the helix at pH 4.6. These findings indicate that the presence of the negative charge of carboxylate at the N-terminal region of a peptide fragment is important for helix formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1990

Identification of two essential histidine residues of ribonuclease T2 from Aspergillus oryzae.

Ribonuclease (RNase) T2 from Aspergillus oryzae was modified by diethyl pyrocarbonate and iodoacetic acid. RNase T2 was rapidly inactivated by diethyl pyrocarbonate above pH 6.0 and by incorporation of a carboxymethyl group. No inactivation occurred in the presence of 3'AMP. 1H-NMR titration and photo-chemically induced dynamic nuclear polarization experiments demonstrated that two histidine residues were involved in the active site of RNase T2. Furthermore, analysis of inactive carboxymethylated RNase T2 showed that both His53 and His115 were partially modified to yield a total of one mole of N tau-carboxymethylhistidine/mole enzyme. The results indicate that the two histidine residues in the active site of RNase T2 are essential for catalysis and that modification of either His53 or His115 inactivates the enzyme.

Amino Acid Sequence↗
PubMed1990

Global fluctuations of the immunoglobulin domains under physiological conditions.

Hydrogen-exchange rates of the indole NH proton of a tryptophan residue, buried fully in the interior of each of the constant (CL) and variable (VL) fragments of a type-kappa-immunoglobulin light chain, were studied at various pH values and at 25 degrees C under 1H-nuclear magnetic resonance. The activation energies for the exchange reactions were determined also and compared with those for the unfolding reactions of these fragments induced by guanidine hydrochloride. The pH profiles of the exchange rates of the CL(kappa) and VL(kappa) fragments were very similar to that for a CL (lambda) fragment reported previously. It was found that the CL (kappa) and VL (kappa) fragments as well as the CL (lambda) fragment undergo a global unfolding transition with a conformation very similar to that of the fully unfolded state induced by guanidine hydrochloride even under physiological conditions.

Bence Jones Protein↗
PubMed1990

Effects of low dose infusion of atrial natriuretic factor on acute inhibition of angiotensin converting enzyme in normal man.

1. We have studied the effects of low dose infusions of atrial natriuretic factor (human ANF (99-126), 1.95 pmol/min per kg) on angiotensin converting enzyme (ACE) inhibitor-induced haemodynamic and hormonal changes in healthy subjects. 2. ACE inhibitor (captopril 25 mg, administered orally) was given against a background infusion of physiological saline (placebo day) or ANF (experimental day). 3. Compared with the placebo observations, ANF enhanced the fall in plasma aldosterone concentrations induced by captopril (P less than 0.05). 4. The rise of plasma renin activity following administration of ACE inhibitor which was observed during placebo infusion was abolished by ANF (P less than 0.05). 5. The responses of systemic blood pressure and heart rate to the converting enzyme inhibition were not affected by the infusion of ANF. 6. These results suggest that variations in endogenous circulating ANF may influence, in part, the response of these hormonal levels during ACE inhibition.

Adult↗
PubMed1990

Overproduction and crystallization of tryptophanase from recombinant cells of Escherichia coli.

We have cloned the tryptophanase structural gene from Escherichia coli B/1t7-A into E. coli K-12 MD55 with a vector plasmid, pBR322. The cloned cells produced a large amount of the enzyme corresponding to more than 30% of the total soluble protein. With the enzyme obtained by this overproduction system, we have prepared three different crystals of tryptophanase, apo-enzyme, holo-enzyme, and a complex of holo-enzyme and L-alanine, by using polyethylene glycol 4000 or potassium phosphate as a precipitant and the hanging drop method. These single crystals appeared to be suitable for X-ray diffraction analysis.

Alanine↗
PubMed1990

[Early detection of successful reperfusion by analysis of serum myoglobin in patients with acute myocardial infarction].

We examined the changes of serum myoglobin (Mb) levels after reperfusion therapy (PTCR, PTCA) in 24 patients with acute myocardial infarction by latex agglutination turbidimetry. In the cases of 13 patients successfully reperfused, significantly higher Mb levels was observed and they disappeared rapidly from blood after the therapy. On the other hand, serum Mb levels disappeared slowly in the cases of patients unsuccessfully reperfused. The disappearance rate of Mb (23.4 +/- 6.7%) in successful reperfused patients was higher than that in unsuccessful reperfusion (5.3 +/- 6.8%) (p less than 0.001), the cut-off value of serum Mb levels in the decision regarding success or failure of reperfusion was set at 17% of disappearance rate, the predictive values for successful and unsuccessful reperfusion were 92% and 91% respectively. The maximum peak time of CK, CK-MB activities and Mb levels after onset of chest pain were also observed. Only the maximum peak time of CK-MB was significantly fasten than others in the cases of patients sera with successful reperfusion (p less than 0.05). We conclude that the measurement of serum Mb levels was useful and suitable for rapid judging on success or failure of reperfusion therapy by use of the disappearance rate of it.

Evaluation Studies as Topic↗
PubMed1989

Extrinsic 33-kilodalton protein of spinach oxygen-evolving complexes: kinetic studies of folding and disulfide reduction.

The 33-kDa protein is one of the three extrinsic proteins in the oxygen-evolving photosystem II complexes. The protein has one intrachain disulfide bond. On reduction of this disulfide bond, the protein was unfolded and lost its activity. On the basis of the unfolding equilibrium curve obtained by using guanidine hydrochloride, the free energy change of unfolding in the absence of guanidine hydrochloride was estimated to be 4.4 kcal/mol using the Tanford method [Tanford, C. (1970) Adv. Protein Chem. 24, 1-95] and 2.8 kcal/mol using the linear extrapolation method. The unfolding of the 33-kDa protein caused by reduction was explained in terms of the entropy change associated with reduction of the intrachain disulfide bond. The kinetics of the reduction of the disulfide bond using dithiothreitol were studied at various concentrations of guanidine hydrochloride at pH 7.5 and 25 degrees C. The disulfide bond was reduced even in the absence of guanidine hydrochloride. The unfolding and refolding kinetics of the 33-kDa protein using guanidine hydrochloride were also studied under the same conditions, and the results were compared with those for the reduction kinetics. It was shown that the reduction of the disulfide bond proceeds through a species in which the disulfide bond is exposed by local fluctuations.

Chemical Phenomena↗
PubMed1989

[Ruptured nonparasitic liver cyst caused by blunt abdominal trauma].

Three patients with ruptured nonparasitic liver cyst (two with suspected multiple liver cysts and one with polycystic disease) following blunt abdominal trauma were described. Ultrasonographic examinations and computed tomography (CT) scans were useful in evaluating the presence and degree of liver injury, the amount of hemoperitoneum and the need for operative repair. Two patients with intraperitoneal bleeding were treated by transcatheter hepatic arterial embolization and all three patients were successfully managed nonoperatively.

Abdominal Injuries↗
PubMed1988

Amino-acid sequence of ribonuclease T2 from Aspergillus oryzae.

The amino acid sequence of ribonuclease T2 (RNase T2) from Aspergillus oryzae has been determined. This has been achieved by analyzing peptides obtained by digestions with Achromobacter lyticus protease I, Staphylococcus aureus V8 protease, and alpha-chymotrypsin of two large cyanogen bromide peptides derived from the reduced and S-carboxymethylated or S-aminoethylated protein. Digestion with A. lyticus protease I was successfully used to degrade the N-terminal half of the S-aminoethylated protein at cysteine residues. RNase T2 is a glycoprotein consisting of 239 amino acid residues with a relative molecular mass of 29,155. The sugar content is 7.9% (by mass). Three glycosylation sites were determined at Asns 15, 76 and 239. Apparently RNase T2 has a very low degree of sequence similarity with RNase T1, but a considerable similarity is observed around the amino acid residues involved in substrate recognition and binding in RNase T1. These similar residues may be important for the catalytic activity of RNase T2.

Amino Acid Sequence↗
PubMed1988

Hydrogen-exchange kinetics of the indole NH proton of the buried tryptophan in the constant fragment of the immunoglobulin light chain.

The constant fragment of the immunoglobulin light chain (type lambda) has two tryptophyl residues at positions 150 and 187. Trp-150 is buried in the interior, and Trp-187 lies on the surface of the molecule. The hydrogen-deuterium exchange kinetics of the indole NH proton of Trp-150 were studied at various pH values at 25 degrees C by 1H nuclear magnetic resonance. Exchange rates were approximately first order in hydroxyl ion dependence above pH 8, were relatively independent of pH between pH 7 and 8, and decreased below pH 7. On the assumption that the exchange above pH 8 proceeds through local fluctuations of the protein molecule, the exchange rates between pH 7 and 8 through global unfolding were estimated. The exchange rate constant within this pH range at 25 degrees C thus estimated was consistent with that of the global unfolding of the constant fragment under the same conditions as those reported previously [Kikuchi, H., Goto, Y., & Hamaguchi, K. (1986) Biochemistry 25, 2009-2013]. The activation energy for the exchange process at pH 7.8 was the same as that for the unfolding process by 2 M guanidine hydrochloride. The exchange rates of backbone NH protons were almost the same as that of the indole NH proton of Trp-150 at pH 7.1. These observations also indicated that the exchange between pH 7 and 8 occurs through global unfolding of the protein molecule and is rate-limited by the unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)

Bence Jones Protein↗
PubMed1988

Low molecular weight RNA of Drosophila cells which is induced by heat shock--I. Synthesis and its effect on protein synthesis.

When Drosophila cells were heat shocked, low molecular weight RNA of a novel type was synthesized and accumulated in the cytoplasm. This RNA, which is about 320 nucleotides long, inhibited the translation of Drosophila mRNAs in rabbit reticulocyte lysate. It remains to be clarified whether this RNA is involved in the regulation mechanism of heat shock at the translational level.

Animals↗