Biomedical subjects
Publications and source records attributed to M Ohno.
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The interaction between BPN' or Carlsberg subtilisins and peptides of the type Ac-Glyn-X-OMe (n = 0, 1, 2, 3), where X denotes one of five different aromatic amino acids, was investigated to elucidate the effect of the secondary interaction on catalysis in relation to the nature of the X residue. The increase in interaction upon elongation of the chain was accompanied by a large increase in kcat but with no marked change in Km in all the series of sensitive substrates. The peptides containing 2-(2-nitro-4-carboxyphenylsulfenyl)-tryptophan, however, acted as competitive inhibitors and exhibited an invariant dissociation constant in spite of the different chain lengths. These observations suggest that the secondary enzyme-substrate interaction induces a conformational change in the active site of the enzyme or in the substrate in such a way as to lower the activation energy and to form a stabilized transient complex. In this respect, BPN' and Carlsberg subtilisins are similar to porcine pepsin and Streptomyces griseus protease 1 rather than to alpha-chymotrypsin.
Presteady state and steady state analyses of the alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis of three specific ester substrates and three ring-substituted derivatives were carried out to elucidate the effect of hydrophobic interactions due to the different side chains of the substrates on the individual steps of the reaction. Hydrolysis of all the substrates except for N alpha-acetyl-Nin-formyltryptophan methyl ester (Ac-Trp(CHO)-OMe) was controlled by the deacylation rate. In spite of their comparable Ks values, the substrates with small kcat, such as N alpha-acetyltryptophan methyl ester and N alpha-acetyl-2-(2-nitro-4-carboxyphenylsufenyl)-tryptophan methyl ester, characteristically gave Km values one order of magnitude smaller than the others. For the reaction of Ac-Trp(CHO)-OMe, it was ascertained that the deacylation step was not rate-controlling. It is suggested that the acylation step controls the rate in this case.
A 31-year-old woman of short stature with severe oligomenorrhea was found to carry a duplication-deficiency X chromosome, 46,X,rec(X)dup q,inv(X)(p22q11), inherited from her mother who carried a pericentric inversion X chromosome, 46,X,inv(X)(p22q11). By a combination of autoradiography and BUdR incorporation, the duplication-deficiency X chromosome was always found to be the inactive and late replicating one. In the cultured fibroblasts with the recombinant X chromosome, some of the cells were seen to have bipartite X chromatin bodies. In the mother with inv(X), the normal and the inverted X chromosome were inactivated at random.
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The effects of l-penbutolol and dl-propranolol on A-V conduction, on excitability of ventricular muscle, and on ventricular tachycardia threshold in acute ischemia were compared, using anesthetized dogs. 1. L-penbutolol (100 microgram/Kg) and dl-propranolol (100 microgram/Kg) prolonged A-V conduction and reduced excitability of ventricular muscle significantly. L-penbutolol less prolonged A-V conduction than dl-propranolol. 2. Both l-penbutolol (50 microgram/Kg) and dl-propranolol (50 microgram/Kg) significantly prevented the lowering of ventricular tachycardia threshold in acute ischemia. These beta-blocking drugs are effective in the treatment of arrhythmia and l-penbutolol could be used safely, because of its weaker inhibition on A-V conduction.
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A method is described for the isolation of large quantities of basement membrane from bovine renal glomeruli under conditions which minimize or prevent degradation by tissue-associated proteases. The method incorporates the use of moderately-high concentrations of the protease inhibitors: ethylenediamine tetraacetic acid, epsilon-amino caproic acid, N-ethymaleimide, and diisopropylfluorophosphate; and the maintenance of a temperature of 0-4 degrees C throughout the procedure. Glomeruli preparations are isolated by a modified sieving technique and are routinely of purity greater than 97%. Under optimum conditions, three to four grams of basement membrane can be isolated under safe and rapid conditions in one week.
The kinetic specificities of BPN' and Carlsberg subtilisins [EC 3.4.21.14] were examined with various nucleus-substituted derivatives of Nalpha-acetylated aromatic amino acid methyl esters for mapping their hydrophobic binding sites in comparison with that of alpha-chymotrypsin. The Carlsberg enzyme was generally much more reactive than the BPN' enzyme due to the larger kcat value. The fact that the two sutilisins hydrolyzed Ac-Tyr(PABz)-OMe, which is a derivative of tyrosine bearing a planar trans-p-phenylazobenzoyl group at the OH-function, with the smallest Km value showed that these enzymes possess a more extended aromatic binding site than has so far been demonstrated. Ac-Phe(4-NO2)-OMe was remarkable in being hydrolyzed with a particularly large kcat value (5,500 +/- 700 s-1 at pH 7.8 for Carlsberg subtilisin). Ac-Phe(4-NO2)-OMe and Ac-Tyr-OMe were distinguished by Carlsberg subtilisin in terms of kcat but not by BPN' subtilisin, suggesting that the specificity site of the former is more sensitive to a small change in size of substituent than that of the latter. Ac-Trp(NCps)-OMe and Ac-Trp(NCps)-OH were bound to the enzyme's active site but in a competitive manner. A difference in the standard free energies of binding between the two enzymes may indicate that the hydrophobic cleft of Carlsberg subtilisin is somewhat deeper and/or narrower than that of BPN' subtilisin.
Papain [EC 3.4.22.2] polymerizes readily upon treatment with tetranitromethane (TNM) by forming intermolecular covalent linkages through its tyrosine residues (Tsukamoto, S. & Ohno, M. (1974) J. Biochem. 75, 1377-1380). Polymerization occurred optimally at pH 9.0 with S-sulfenylsulfonate papain. Circular dichroic spectra of polymerized papains showed a small change in ellipticity when compared with that of unmodified papain. Esterolytic activity of the modified enzyme toward benzoyl-L-arginine ethyl ester (BAEE) was almost fully retained, at least up to the formation of hexamer, with an unchanged Km value. Spectrophotometric and amino acid analyses indicated that two or three tyrosine residues are involved in intermolecular crosslinks depending on the amount of TNM used. The tyrosine residues nitrated were identified as those at positions 61, 116, 103, and 4, the extent of nitration decreasing in this order. When activated papain was treated with increasing molar ratios of TNM, an essential sulfhydryl function was first oxidized and, at a 2-fold molar excess of the reagent, restoration of activity was no longer observed even after addition of dithiothreitol (DTT). The evidence obtained in the present study eliminates the possibility of inactivation due to nitration of a tryptophan residue, which had been suggested previously.
The decapeptide corresponding to the amino acid sequence of porcine luteinizing hormone-releasing hormone (LH-RH) which involves 1 mol of tryptophan was synthesized via solid phase synthesis with two different deblocking procedures which used hydrogen chloride in formic acid and hydrogen chloride in acetic acid containing 1% 2-mercaptoethanol. After some fundamental studies on the former reagent with respect to deblocking efficiency toward the Boc group, 0.5 M hydrogen chloride (a 10-fold molar excess with respect to the N-terminal Boc group) in formic acid was used in the present synthesis. The two synthetic products exhibited the same chemical and biological properties as an authentic LH-RH. Hydrogen chloride in formic acid has proved effective without a scavenger although loss of peptide from the resin occurred to a somewhat greater extent than that with hydrogen chloride in acetic acid. A derivative of the synthetic LH-RH formylated at the indole nitrogen had a greatly diminished biological activity, indicating that the intact indole side chain is essential for the activity.
Decarboxylation of citrulline by Escherichia coli enzymes was presented. The N-carbamyl putrescine produced showed the same properties as those of synthesized authentic samples in column chromatography, paper chromatography, and paper electrophoresis.
The correlations between serum triidothyronine (T3), thyroxine (T4), 131I-triiodothyronine resin sponge uptake (RT3U) or free thyroxine index (T7) and the basal metabolic rate (BMR) during antithyroid drug treatment in 86 patients with Graves' disease were investigated. Although serum T3, T4, RT3U and T7 during therapy with MMI showed significant positive correlations with BMR, the coefficient of correlation (r = 0.6088, P less than 0.001) between T3 and BMR was the highest of all. While the normal range of BMR in control subjects was between -1.9 and +14.9 (the range of mean +/- SD), the corresponding values of T3, T4, RT3U and T7 calculated from the regression lines, ranged from 94.2 to 184.0 ng/dl, from 5.32 to 8.75 microgram/dl, from 26.5 to 28.9% and from 1.57 to 2.47 respectively. On the other hand, when the corresponding values of BMR to normal values of T3 (100-170 ng/dl), T4 (7.6-12.2 microgram/dl), RT3U (26.7-36.5% and T7 (2.29-3.49) in control subjects were calculated from the regression lines, the range of value obtained from the regression line of T3 coincided better with normal value of BMR in control subjects that those calculated from other regression lines (T4, RT3U and T7). These results suggest that serum T3 level would be a better index of evaluation of the thyroid function that T4 or RT3U in patients with Graves' disease under antithyroid drug treatment.
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