Biomedical subjects
Publications and source records attributed to T Takuma.
Butylated hydroxytoluene (BHT) which is widely used as an anti-oxidant in food has been found to induce the differentiation of murine erythroleukemia cells. BHT also amplifies the differentiation inducing activity of DMSO.
Epinephrine, a mixed alpha- and beta-adrenergic agonist, modulated new protein synthesis in dispersed rat submandibular cells. Both adrenoreceptors separately could increase protein synthesis after cells were exposed to [14C]-leucine for 10 min and then with 1 mM unlabelled leucine for 10-30 min. The extent of response was dependent on the concentration of epinephrine to which cells were exposed. Alteration in the sequence of exposure of cells to tracer and agonist changed the direction of agonist response.
An acidic isozyme of a chymotrypsin-like esteroprotease from the mouse submandibular gland was purified and its properties were compared with those of the basic isozymes purified previously (Takuma, T., et al. (1983) Biochim. Biophys. Acta 755, 70-75), bovine pancreatic alpha-chymotrypsin, and other chymotrypsin-like enzymes of mice. The isoelectric point of the purified enzyme was pH 4.7, and the molecular weight was estimated to be 25,000 by gel filtration on Sephadex G-100. The enzyme hydrolyzed benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) 7 times more slowly than basic isozymes did, but hydrolyzed casein as slowly as the basic isozymes did. The acidic isozyme was 40 times more sensitive to chymostatin than basic isozymes were, but 10 times less sensitive than alpha-chymotrypsin was. Moreover, acidic and basic isozymes were immunologically distinct. Chymotrypsin-like esteroproteases in the submandibular gland were antigenically unique among chymotrypsin-like enzymes in various tissues of mice.
The incorporation of [14C]leucine into protein in dispersed cells from rat submandibular gland was inhibited by epinephrine. The dose required for a half-maximal effect was approximately 10(-6) M. This sensitivity is similar to that of mucin and potassium secretion induced by epinephrine from these cells. The inhibitory effect of epinephrine was mediated by the activation of alpha 1-adrenergic receptors and was partially independent of extracellular calcium. The calcium ionophore A23187 also decreased protein synthesis in the presence and absence of extracellular calcium. Epinephrine diminished alpha-[14C]aminoisobutyric acid (AIB) influx, but the extent of altered AIB uptake was much less than the extent of decrease in protein synthesis. Analysis of newly synthesized protein by gel electrophoresis and autofluorography showed that epinephrine selectively inhibited the synthesis of specific submandibular proteins. In aggregate these results suggest that activation of alpha 1-adrenergic receptors in rat submandibular cells influences the regulation of protein synthesis by a mechanism that uses intracellular calcium.
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Basic isozymes of chymotrypsin-like esteroprotease from mouse submandibular glands were purified 60-80-fold by a rather simple procedure consisting of CM-Sepharose CL6B chromatography and gel filtration on Sephadex G-100. The purified sample contained three major isozymes (A, B, C) and some minor ones. Their isoelectric points were between pH 10 and 11. The molecular weights of the main isozymes were estimated at 28000 by SDS-polyacrylamide gel electrophoresis. The acidic isozyme (A) separated into two polypeptide chains whose molecular weights were 21500 and 6500. Specific activities of these isozymes using Bz-Tyr-OEt as substrate were comparable to that of bovine pancreatic alpha-chymotrypsin, but they hydrolyzed casein 10 times slower than did alpha-chymotrypsin. The hydrolytic activities of these isozymes on Bz-Tyr-OEt were inhibited by diisopropylfluorophosphate, tosyl-L-phenylalanine chloromethyl ketone and chymostatin, but they were 400 times less sensitive to chymostatin than was alpha-chymotrypsin.
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The activities of chymotrypsin-like esteroproteases in the mouse submandibular gland were additively induced by 5 alpha-dihydrotestosterone (DHT) and triiodothyronine (T3). Zymograms showed that there are many isozymes whose activities are regulated by DHT and/or T3. Some isozymes seemed to be hormone-independent. Histochemical studies revealed that all these isozymes are localized in the granular convoluted tubules.
The development of trypsin-like esteroproteases in the submandibular gland of mice was studied, using a newly synthesized naphthyl ester (tosyl-L-lysine alpha-naphthyl ester) for the preparation of zymograms and for histochemical demonstration of the enzyme. Esteroprotease activities were first detected spectrophotometrically on day 15 after birth; then increased markedly after day 20. A sex difference in esteroprotease activity appeared on day 25. Zymograms prepared after isoelectric focusing in polyacrylamide slab gels showed that the glands of neonatal mice contained esteroproteases with a rather different composition from that of adult mice. The adult type isozymes appeared first on day 15, and their activities increased markedly after day 20. Histochemical studies revealed that the isozymes of neonatal mice were derived from mast cells. A few striated ducts were first stained on day 15, and the sex difference of the granular tubules became obvious on day 25. These results indicate that the development of trypsin-like esteroproteases faithfully reflects the differentiation of granular tubules in the mouse submandibular gland, except in the neonatal period.
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The relationship between the actions of androgen and thyroid hormone in induction of trypsin-like esteroproteases in mouse submandibular gland was studied. Zymograms prepared using tosyl-L-lysine alpha-naphthyl ester as substrate after isoelectric focusing in polyacrylamide slab gel showed that the isozymes of trypsin-like esteroprotease induced by 5 alpha-dihydrotestosterone and triiodothyronine were identical. The time courses of esteroprotease induction by these hormones were very similar, and the lag time was not shortened by treatment with both hormones. The doses of 5 alpha-dihydrotestosterone and triiodothyronine for half-maximal induction were 0.5 mg and 2.5 micrograms per 100 g body weight, respectively, and these values were not altered by simultaneous injection of other hormones. The binding capacity and affinity of androgen receptor for methyltrienolone, a synthetic androgen, were not affected by daily injections of triiodothyronine for 5 days. These results suggest that androgen and thyroid hormone act independently, not competitively, and so the two hormones induce trypsin-like esteroproteases additively.
The effects of thyroxine and 5 alpha-dihydrotestosterone on the activities of various marker enzymes in the submandibular glands of immature mice were studied. The responsiveness to thyroid hormone appeared at latest by day five after birth, which was ten d earlier than the appearance of androgen responsiveness. The activity of trypsin-like esteroprotease, however, was not increased by these hormones until day 15 after birth.
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The effect of L-thyroxine (T4) on amylase activity in the developing rat pancreas has been investigated. Administration of T4 (0.2 microgram/g body wt) alone to intact rats on days 5-10 after birth did not induce pancreatic amylase but the enzyme was induced significantly by daily injection of cortisol (10 microgram/g body wt) alone into intact rats over the same period. In thyroidectomized, adrenalectomized rats pancreatic amylase was not induced by the injection of cortisol alone but it was induced by the administration of cortisol plus T4. Increase in enzyme activity was much less in thyroidectomized animals than in intact animals. These results suggested that T4 does not have a direct effect in increasing pancreatic amylase activity but plays a permissive role in increasing enzyme activity.
The effects of cortisol (10 microgram/g body weight) and L-thyroxine (T4; 0.2 microgram/g body weight) on the activity of parotid gland amylase in young rats were investigated. Administration of cortisol or T4 for 5 consecutive days from day 5 after birth caused the precocious appearance of amylase, T4 having almost twice the effect of cortisol. Cortisol and T4 did not have synergistic effects. In thyroidectomized-adrenalectomized rats, T4 increased amylase activity but cortisol did not. The increase in enzyme activity after day 20 was much less in rats thyroidectomized on day 10 than in rats adrenalectomized on day 10. These results suggest that T4 has a direct effect on the early increase of amylase activity (days 15-25) and that the action of glucocorticoid requires the presence of endogenous thyroid hormones. The hormone-induced level of amylase in intact rats was less than that of normal adult rats. Forced weaning of intact rats resulted in a further increase in amylase activity, suggesting that further amylase accumulation (after day 25) may be due to dietary factors.
Subcutaneous injection of cortisol (10 micrograms/g body wt daily for 5 days) into normal neonatal rats increased the activity of stomach pepsinogen after day 5. L-Thyroxine (T4; 0.2 microgram/g body wt daily for 5 days) alone did not affect the activity but it somewhat enhanced the effect of cortisol. Even before day 5, when no effect of cortisol alone was observed, T4 plus cortisol increased pepsinogen activity. In adrenalectomized, thyroidectomized neonatal rats, injection of cortisol alone did not induce enzyme activity but cortisol together with T4 did induce it. Moreover, the increase in pepsinogen activity was depressed in rats thyroidectomized on day 10 but not in those thyroidectomized on day 15. These results suggest that T4 does not have a direct effect on the stomach but plays an important role in making the stomach responsive to glucocorticoids, resulting in increased pepsinogen activity.