Biomedical subjects
Publications and source records attributed to S Tamura.
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The characteristics of the in-beam electron impact mass spectra of 13 N-carbobenzoxyoligopeptides (up to pentapeptides) composed mainly of leucine and isoleucine, and their alkyl and N-hydroxysuccinimide esters are described. In the cases of di- and tripeptide derivatives, each in-beam electron impact spectrum exhibited an abundant [M + 1]+ peak as well as amino-acyl fragments. However, in the case of pentapeptides the in-beam spectra measured under the routine conditions corresponded to the conventional electron impact spectra of the cyclopeptide derivatives formed by thermal cyclization in the mass spectrometer and few [M + 1]+ ions of the original peptides were observed. From the studies on temperature spectral change of in-beam electron impact spectra it became clear that rapid heating of the sample over the temperature range 200-300 degrees C gave abundant [M + 1]+ ions as well as fragment ions originating from the thermally intact peptide as the major peaks in the spectrum.
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Age related changes of the thyroid gland of CF1 mice ranging in age from 15 days to 24 months were studied morphologically and biochemically. The mean diameter of follicles becomes larger with advancing age due to fusion of adjacent follicles, and there is also an increasing variability of follicle diameter. The volumetric density of lysosomes in the cytoplasm of epithelial cells lining follicles increases with age. In the younger (seven days to 1.5-month-old) mice, most lysosomes are small and homogeneously dense; these are considered to be the primary lysosomes necessary for the hydrolysis of reabsorbed colloid droplets. The heterogeneously dense, large lysosomes containing lipid-like droplets increase in number with age, especially in mice over 15 months. The specific activities of acid phosphatase, beta-glucuronidase and beta-galactosidase show a relative decrease with age after six months; although there may be an absolute increase. Some of the lysosomes observed in old animals may be residual bodies.
1. Phosphatase II is a form of phosphoprotein phosphatase originally found in rat liver extract; it has a molecular weight of 160 000 by gel filtration and is highly active towards phosphorylase alpha. This phosphatase has been purified 1800-fold by using DEAE-cellulos (DE-52), aminohexyl--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200 chromatography. Throughout the purification steps, the original molecular weight and substrate specificity of phosphatase II were almost perfectly preserved. 2. The product of the final purification step migrated predominantly as a single protein band on non-denaturing gel electrophoresis. Sodium dodecyl sulfate gel electorphoresis revealed that the enzyme contains two types of subunit, alpha and beta, with molecular weights of 35 000 and 69 000, respectively. When treated with 0.2 M 2-mercaptoethanol at -20 degrees C, phosphatase II was dissociated to release the catalytically active alpha subunit. The beta subunit may be catalytically inactive but interacts with the alpha subunit so that phosphatase II becomes much less susceptible than the alpha subunit to inactivation by ATP or pyrophosphate.
1. Phosphoprotein phosphatase IB is a form of rat liver phosphoprotein phosphatase, distinguished from the previously studied phosphoprotein phosphatase II [Tamura et al. (1980) Eur. J. Biochem. 104, 347-355] by earlier elution from DEAE-cellulose, by higher molecular weight on gel filtration (260000) and by lower activity toward phosphorylase alpha. This enzyme was purified to apparent homogeneity by chromatography on DEAE-cellulose, aminohexyl--Sepharose-4B, histone--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200. 2. The molecular weight of purified phosphatase IB was 260000 by gel filtration and 185000 from S20,W and Stokes' radius. Using histone phosphatase activity as the reference for comparison, the phosphorylase phosphatase activity of purified phosphatase IB was only one-fifth that of phosphatase II. 3. Sodium dodecyl sulfate gel electrophoresis revealed that phosphatase IB contains three types of subunit, namely alpha, beta and gamma, whose molecular weights are 35000, 69000 and 58000, respectively. The alpha subunit is identical to the alpha subunit of phosphatase II. While the beta subunit is also identical or similar to the beta subunit of phoshatase II, the gamma subunit appears to be unique to phosphatase IB. 4. When purified phosphatase IB was treated with 2-mercaptoethanol at -20 degrees C, the enzyme was dissociated to release the catalytically active alpha subunit. Along with this dissociation, there was a 7.4-fold increase in phosphorylase phosphatase activity; but histone phosphatase activity increased only 1.6-fold. The possible functions of the gamma subunit are discussed in relation to this activation of enzyme.
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When the crude phosphoprotein phosphatase fraction of rat liver cytosol was treated with 80% aqueous ethanol at room temperature, the activity with phosphorylase alpha as substrate was increased by 110%, but those with glycogen synthase D and phosphohistone were decreased by 53 and 34%, respectively. Chromatography of the ethanol-treated fraction on DE-52 revealed that while phosphoprotein phosphatase IA (Mr=69,000) remained to exist even though it was reduced, phosphatases IB (Mr=-300,00) and II (Mr=160,000) were totally replaced by a new phosphatase form with an approximate molecular weight of 35,000. This low molecular weight form has been designated phosphatase III. When partially purified phosphatases IB and II were separately treated with ethanol, they were converted to phosphatase III. These results suggest that phosphoprotein phosphatases IB and II, but IA, contain phosphatase III as a subunit. Phosphatases IB and II, however, must differ in structure since "IB to III" is accompanied by an increase in phosphorylase phosphatase activity much greater than that for "II to III"
1. The changes of beta-glucuronidase activity in Wistar and Gunn strain rats from infants to adults, were determined. Consequently, the enzyme activity in liver was found to increase remarkably during the time of growth and to keep a certain level of activity at maturity. The beta-glucuronidase activity in Gunn strain rats was detected to be low in comparison with that of Wistar strain rats. 2. The beta-glucuronidase activity in liver of rats in over 50 days after birth is quite stable. It is concluded, therefore that the rats of 50-80 days old are most stable to examine the influence of drugs on beta-glucuronidase activity. 3. In the experiments using the above rats, the beta-glucuronidase activity in liver showed an increase by the administration of borneol, chloretone and barbital-Na. However, Gunn strain rats indicated rather low enzyme activity as compared to Wistar strain rats. 4. The beta-glucuronidase activity in kidney and intestine did not show an extensive alteration and was hardly affected by the drugs used.
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Prothoracicotropic hormone has been successfully extracted from as many as 2,800,000 silkworm male adult heads and purified partially through an 8-step procedure. Approximately 7 microgram of the crude preparation ("crude PTTH") shows activity in the debrained Samia pupa test. A further 4-step chromatographic purification of the "crude PTTH" derived from 100,000 Bombyx heads yields 3.4 mg of purified preparation ("highly purified PTTH"), of which 7 ng causes adult development in a debrained Samia pupa.
The alkaline nuclease (pH optimum 9.0) has been purified about 500-fold in 25% yield from the extract of rat liver mitochondria. The enzyme cleaves yeast RNA, poly(U), poly(U), poly(C) and denatured DNA to yield oligonucleotides with 5'-phosphoryl and 3'-hydroxyl ends. The enzyme has a molecular weight of about 60 000, a sedimentation coefficient of 4 S and an isoelectric point of 9.0. The behaviors of RNAase activity of the nuclease are identical with those of DNAase activity in column chromatography as well as in catalytic nature. The affinities of RNAase activity for substrate, Mg2+, spermidine and polyvinyl sulfate are lower than those of DNAase activity. The alkaline nuclease activity measured in the homogenate of regenerating rat liver is not significantly changed.
DNA ligase activity was distributed mainly in the extract of nuclei rapidly isolated from rat liver and about 20% of the nuclear activity in the cytoplasmic fraction. The DNA ligases of nuclear extract and cytoplasmic fraction showed similar gel-filtration patterns and the same sedimentation coefficient of 5.5 S. The appearance of 4-S enzyme from nuclei or nuclear extract was partly inhibited by phenylmethylsulfonyl fluoride. These results suggest that the major activity of DNA ligase in rat liver is due to a single species of the enzyme localized in nuclei and that 4-S nuclear DNA ligase reported previously is an artifact arising from 5.5-S enzyme during the isolation procedure.
The Weibull distribution function was applied to solid-state enzyme inactivation. On Weibull probability paper (within a narrow range), the plots of the accumulated inactivation ratio of each enzyme versus time regressed to a straight line. The parameters m and k, which correspond to the type and rate of the type and rate of the inactivation, were characteristic of each enzyme. The effect of temperature and parameter reproducibility are discussed.