Biomedical subjects
Publications and source records attributed to S Aibara.
Single crystals of NADPH-adrenodoxin oxidoreductase were grown in 50 mM potassium phosphate (pH 7.4) containing 5% glycerol and ammonium sulfate. The crystals are monoclinic, belong to space group P21 and have dimensions of a= 83.4 A, b = 62.6 A, c = 59.3 A, alpha = gamma = 90 degrees, and beta = 107.1 degrees. There is one molecule per asymmetric unit.
Kinetic parameters for each reaction step of the peroxidase-catalyzed reaction were determined by the stopped-flow technique on three distinct isoenzymes: acidic A2, neutral C1, and basic E5. The pH dependence of the reaction for the formation of compound I with hydrogen peroxide was examined. The three isoenzymes had a common ionizing group at about pK 4 which affects the rate constant for the formation of compound I. The heat of ionization determined from the temperature dependence of the dissociation constant of the group strongly suggested that it is of carboxyl nature. The rate constant for isoenzyme A2 was a tenth of those for the other two isoenzymes over the whole range of pH. Furthermore, the thermodynamic parameters of isoenzyme A2 were found to be different from those for the other two isoenzymes. These difference as well as the different behavior in alkaline transition of the isoenzymes are discussed in relation to the sixth ligand of the heme. The rate constant of the reduction of compound I and compound II by ferrocyanide were also determined. In both reduction steps, the pH profiles of the apparent rate constant for isoenzyme A2 and E5 were similar, but they were different from that of C1. The ionization with pK 5.29, which was detected only in isoenzyme C1, may be attributed to a group near the porphyrin ring as a stabilizer for the pi-cation radical.
Five components of neutral horseradish peroxidase were isolated and purified by means of column chromatography, and designated B1, B2, B3, C1, and C2, respectively. All the components contained 2 atoms of calcium and 16.8-to-21.0% as much carbohydrate as in the enzyme molecule. They were very similar to one another with respect to physicochemical and chemical properties such as molecular weight, molar absorption coefficient, rate constants of the catalytic reaction and dissociation of cyanide compound, but were dissimilar with respect to isoelectric point. Values of the isoelectric points determined from column isoelectric focusing at 20 degrees C were 5.75 (B1), 7.15 (B2), 7.10 (B3), 9.40 (C1), and 9.63 (C2). However, these values varied significantly depending upon the method and conditions of the focusing. The acid-alkaline titration curves of components B2 and C1 were flat in the pH region of 6 to 9. The facts suggest that a slight difference in the number of ionized groups of the components causes a large difference in the isoelectric points.
Six basic isoenzymes, E1 to E6, of horseradish peroxidase were isolated and purified by CM-Sephadex column chromatography, and were then crystallized. The crystalline basic isoenzymes had a lower molecular weights than the neutral isoenzyme C, but the molecular weights of their protein moieties were similar to that of the latter. The lower molecular weights of the basic isoenzymes were thus essentially due to smaller contents of carbohydrate. Of the six isoenzymes, four isoenzymes, E3 to E6, had extremely high pI values of over 12 and appreciably low contents of carbohydrate, 0.8 to 4.2%, whereas the isoenzymes E1 and E2 contained relatively large amounts of carbohydrates, 12.8 and 14.1%, respectively, and had lower pI values, 10.6. The amino acid compositions of the basic isoenzymes were different from one another, and the differences suggested that the isoenzymes were allelic. The relatedness of the amino acid compositions of these isoenzymes is discussed and the compositions are compared with those of other peroxidases. Finally, one of the six isoenzymes, E4, was obtained as large single crystals suitable for X-ray structural analysis, and the preliminary crystallographic data are presented.
General pharmacological properties of 4'-fluoro-4-[4-(2-thioxo-1-benzimidazolinyl) piperidino] butyrophenone (timiperone), a new neuroleptic drug, were compared with those of haloperidol. 1. Central nervous system: In behavioral observation, timiperone showed a typical neuroleptic profile at doses of 0.1 mg/kg p.o. and more (mice). The drug produced a moderate hypothermia at 10 mg/kg p.o. (rabbits), a mild increase in pain threshold at 3 mg/kg p.o. (mice and rats) and a slowing of cortical EEG at 1 mg/kg i.v. (cats). ED50 values of drug for the potentiation of ether and alcohol anesthesia were 0.34 and 0.22 mg/kg p.o., respectively (mice). Timiperone ahd neither an anticonvulsant activity at 30 mg/kg p.o. (mice) nor an effect on the spinal reflex at 1 mg/kg i.v. (cats). These effects of timiperone on the central nervous system were almost similar to those of haloperidol. 2. Respiratory and cardiovascular system: At dose of 0.03 mg/kg i.v. and more, timiperone produced transient increases in respiratory rate and regional arterial blood flow which were accompanied by a fall in blood pressure (dogs). Haloperidol had qualitatively similar effect, but was weaker than timiperone. Both drugs at high concentration (3X10-6 g/ml) exerted negative inotropic and chronotropic effect in isolated atrial preparations (guinea-pigs), and non-competitively antagonized the positive chronotropic action of isoprenaline. Atropine (2.5X10-7 g/ml) failed to modify the chronotropic action of timiperone (3X10-6 g/ml). 3. Autonomic nervous system: Timiperone at 0.1 mg/kg p.o. and haloperidol at 0.3 mg/kg p.o. induced a moderate miosis (rabbits) and antagonized blood responses to noradrenaline and acetylcholine (dogs). Both drugs at 1 mg/kg i.v. had no ganglion-blocking activity (cats). 4. Smooth muscle: In isolated guinea-pig ileum and vas deferens, timiperone and haloperidol (10-5 g/ml) antagonized the contractile responses of the muscles to various spasmogens, Both drugs at approximately 10-6 g/ml decreased spontaneous motility of the isolated rat uterus and inhibited the gastric secretion at 1 mg/kg i.p. (rats). At high doses, both drugs inhibited the gastrointestinal propulsion (mice), motility (dogs) and gastric emptying rate (rats), and had no damaging effect on the gastric mucosa (rats). 5. Skeletal muscle: At 0.1 mg/kg i.v., timiperone and haloperidol slightly enhanced twitch response of the anterior tibial muscle to electrical stimulation (rabbits). 6. Urine volume and urinary electrolytes: Timiperone and haloperidol showed a diuretic effect at 3 mg/kg p.o. whereas they inhibited urine output and electrolytes excretion at 30 mg/kg p.o. (rats).
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Cetraxate increased gastric mucosal blood flow and inhibited the decrease in blood content of the rat gastric preparation, as induced by serotonin. In addition, this compound decreased the inside pressure of the rat stomach and the tone of the rat fundus strip and inhibited the increase in gastric emptying rate induced by serotonin. Further effects of the drug were decrease in the tone of isolated rabbit aorta and inhibition of platelet aggregation as induced by thrombin. Decrease in the vascular resistance was apparently related to increase in the gastric blood flow. These experimental results suggest that the antiulcer effect of cetraxate is due to the improvement of blood circulation in the gastric mucosa.
Serotonin in doses of 5 to 80 mg/kg injected subcutaneously to rats injured the gastric mucosa dose-dependently and the lesion was histopathologically regarded as erosion. Aggregated platelets were observed in small blood vessels of the gastric mucosa prior to the development of erosion when serotonin was injected in a subcutaneous dose of 20 mg/kg effective in producing the lesion. Subsequently fibrinous thrombi could be found in the lesional mucosal area at the stage of erosive change. Serotonin in the same dose decreased gastric mucosal blood flow and enhanced gastric motility in the peroid of time when pre-erosive or erosive change occurred. Circulatory disturbance of the gastric mucosa induced by these histological and functional changes seemed to play an important role in development of the gastric mucosal lesion by serotonin. It is unlikely that gastric acid takes part as a trigger in producing the lesion.
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The molecular weight of soybean beta-amylase [EC 3.2.1.2] was determined to be 57,000 daltons by the sedimentation equilibrium method, and the enzyme was found to consist of 494 amino acid residues. No difference was found in molecular weight or composition between two components of beta-amylase separated by ion-exchange chromatography. The N-terminus of the enzyme was not detectable by the fluorodinitrobenzene method or phenylisothiocyanate method, and the C-terminus was determined to be alanine by the carboxypeptidase [EC 3.4.12.2] method. Five half-cystine residues were found in the form of cysteine; all the sulfhydryl groups could be titrated by p-chloromercuribenzoate after denaturation of the enzyme with guanidine hydrochloride, but only some in the native enzyme. The rates of mercaptide formation of these groups were dependent on pH and were different from each other, all being much lower than the rate for the free sulfhydryl group in mercaptoethanol. Differential titration experiments at different pH's and in the presence of maltose showed that mercaptide formation by only one sulfhydryl group caused loss of activity, and the reaction was accompanied by changes in the environment around aromatic side chains in the enzyme, which were detected by difference spectra and fluorescence emission spectra. These facts suggest that modification of the sulfhydryl groups causes a conformational change of the enzyme. Some preliminary crystallographic data for crystals formed at pH 4.0 were obtained, and inactivation by heavy metal salts was examined in relation to the preparation of isomorphous heavy atom derivatives.
Beta-Amylase [1, 4-alpha-D-glucan maltohydrolase, EC 3.2.1.2] has been purified from defatted soybean meal by fractional precipation with ammonium sulfate, ion-exchange chromatography on CM- and DEAE-Sephadex and gel filtration chromatography on Sephadex G-100. Two different components of beta-amylase were crystallized from ammonium sulfate solutions, and the homogeneity of each preparation was confirmed by sedimentation and disc electrophoretic analyses. Both components of soybean beta-amylase formed large single crystals (trigonal crystal system) from 40--50 per cent saturated ammonium sulfate solution buffered at pH 5.4 on dialyzing concentrated protein solution in the apparatus of Zeppezauer et al. Preliminary X-ray diffraction data gave a hexagonal lattice with unit cell dimensions a=86.1 A and c=144.4 A. The space group corresponds to P3121 or P3221, and one asymmetric unit contains one molecule of beta-amylase, assuming a crystal density of 1.25 g/ml and a molecular weight of the enzyme of 60,000 daltons. In this case, the crystal has a volume of 2.53 A-3 per atomic mass unit, and the percentage of protein in the crystal is about 52.
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