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Biomedical subjects

K Tsukada

Publications and source records attributed to K Tsukada.

At least 307 records · Page 17Linked to original sources
PubMed1982

Eukaryotic DNA ligase. Purification and properties of the enzyme from bovine thymus, and immunochemical studies of the enzyme from animal tissues.

DNA ligase has been purified to near-homogeneity from the extract of bovine thymus with a yield of 5%. The purified enzyme catalyzed the joining of single-stranded breaks in duplex DNA at a rate of 33 nmol of phosphodiester bonds/min/mg of protein. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis and Ouchterlony double diffusion analysis. The enzyme is composed of a single polypeptide with a molecular weight of about 130,000. The enzyme has a Stokes radius of 52 A, a sedimentation coefficient of about 5 S, and a frictional ratio of 1.6. Apparent Km values for ATP and Mg2+ are 2 microM and 0.9 mM, respectively. Antibody against bovine thymus DNA ligase was prepared by injecting a rabbit with the purified enzyme. Immunochemical titrations revealed that the increased activity of DNA ligase observed after partial hepatectomy of rat and 16-fold higher activity level of mouse Ehrlich tumor cells compared with the host liver are due to a change in the enzyme quantity but not to a change in the catalytic efficiency of the enzyme molecule. Wide variations in the level of DNA ligase activity in extracts from various tissues of rat and mouse were accompanied by proportionate changes in the quantity of immunochemically reactive protein. The antibody inhibited DNA ligase activity from bovine tissues with 20-fold higher efficiency, compared with the enzyme from the rodent tissues. The enzyme activity from chick embryo was unaffected by the antibody.

Animals↗
PubMed1982

Immunological distinction of S-adenosylmethionine synthetase isozymes from rat liver and kidney.

The beta-form of S-adenosylmethionine synthetase among three isozymes has been purified from rat liver, and proven to be homogeneous. The molecular weight of this enzyme was estimated to be 100,000 by gel filtration on Sephacryl S-200, and the enzyme was shown to be composed of two subunits of 48,000 daltons. A rabbit antiserum against the normal rat liver beta-form of S-adenosylmethionine synthetase was used for immunochemical characterization. The alpha- and beta-forms of isozyme are immunochemically identical, but the antiserum did not react with the gamma-form from rat kidney.

Animals↗
PubMed1982

Purification and properties of magnesium- and manganese-dependent ribonucleases H from chick embryo.

Two RNases H, Mg2+- and Mn2+-dependent RNases H, are present in extracts of chick embryo. These RNases H can be separated by phosphocellulose column chromatography. Mg2+-dependent RNase H was purified over 900-fold and Mn2+-dependent RNase H over 1,700-fold from chick embryo extracts. The molecular weight of the purified Mg2+-dependent RNase H was about 40,000 and of the Mn2+-dependent RNase H about 120,000, when estimated by gel filtration. Mg2+-dependent RNase H exhibits maximal activity at pH 9.5, and requires 15 to 20 mM Mg2+ for maximal activity, whereas Mn2+-dependent RNase H is most active at pH 8.5, and is maximally active at the concentration of 0.4 mM Mn2+, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. Mn2+-dependent RNase H was inhibited by o-phenanthroline, pyrophosphate, and those polyamines tested, whereas Mg2+-dependent enzyme was not, although it was inhibited by NaF. Both RNases H liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini endonucleolytically.

Ammonium Sulfate↗
PubMed1982

Studies on liposome-encapsulated carboquone II. Effect of liposomal encapsulation of carboquone on distribution and clearance in animals.

The effects of encapsulation of carboquone (CQ) in the liposomes were investigated on the plasma clearance and tissue distribution of CQ in rabbit and rat. CQ-Liposome, the liposomes in which CQ was encapsulated, was prepared by the method described in a previous report. By intravenous administration at the same dose. CQ-liposome showed approximately three times higher plasma concentration than free CQ in rabbit and rat. However, prolonged release from liposomes or stabilization of CQ in plasma was not observed by encapsulation. The distribution to lung and reticuloendothelial-rich organs such as liver and spleen was enhanced, but on the contrary the distribution to the peripheral tissues such as muscle and skin was decreased. In rats pretreated with drug free liposomes, high plasma CQ concentration was obtained after intravenous administration of CQ-liposome. From these results, it is considered that the large parts of CQ-liposome is distributed into reticuloendothelial system (RES) and RES is saturable with liposomes. And it is also presumed that the high plasma concentration obtained after intravenous administration of CQ-liposome, would be caused by altering the tissue distribution of CQ by encapsulation.

Animals↗
PubMed1982

Studies on liposome-encapsulated carboquone. III. Enhancement of lymphatic transport of carboquone by encapsulation.

To examine the possibility of utilizing liposomes as drug carriers of carboquone (CQ), an antitumor agent, effects of encapsulation on transport into lymphatics have been studied. Enhancement of lymphatic transport of CQ was obtained by the administration of liposome encapsulated CQ (CQ-liposome) into abdominal cavity in thoracic duct cannulated rats. And also, the enhancement of CQ transport into regional lymph nodes belong to gastrointestinal tract was also demonstrated in normal rats. When CQ-liposome was administered intravenously or intramuscularly, however, excellent transport into lymphatics was not recognized. Various pharmaceutical formation such as emulsions and oily solution failed to produce as high a lymph level as CQ-liposome. These results show that liposome may represent an effective system to deliver CQ into lymphatics by intraperitoneal administration.

Animals↗
PubMed1982

Studies on liposome-encapsulated carboquone. IV. Enhancement of antitumor activity of carboquone against Ehrlich ascites carcinoma by encapsulation.

The antitumor effects of liposome-encapsulated carboquone (CQ-liposome) were studied following intraperitoneal administration into mice bearing Ehrlich ascites carcinoma (EAC). CQ-liposome showed nearly 3 times superior efficiencies to parent CQ (free CQ) in the growth inhibitory effect on EAC cells inoculated in mice, and also showed almost the same toxicity as free CQ in normal mice. CQ-liposome prolonged the life span of EAC bearing mice about 2 times that of free CQ. In the results of electrophoresis, it was found that CQ-liposome and EAC cells had positive and negative surface charges, respectively. These observations suggest that CQ-liposome is a favorite delivery system to enhance the efficiency of CQ to EAC in mice. One of the reasons for its excellent effect may be explained by an easy contact of CQ-liposome with EAC cells because of its opposite surface charges.

Animals↗
PubMed1981

On the mechanism by which insulin stimulates protein synthesis in chick embryo fibroblasts.

It has been known that insulin raises the rate of incorporation of [3H]leucine into the total protein of hormone-deficient chick embryo fibroblasts by approximately 1.5-fold. The elevation is not dependent upon the production of new messenger ribonucleic acids (mRNAs). Evidence is now presented in support of the following points: the greater labelling is due to more rapid polypeptide synthesis, not to an increase in the specific activity of leucyl-tRNA; the enhanced synthesis derives largely or entirely from a speeding up of the process of initiation, rather than that of elongation or termination; and the 1.5-fold stimulation is due to the elevated rates of formation of at least many of the fibroblast proteins. The hormone was shown before to stimulate posttranscriptionally and highly preferentially for formation of ribosomal proteins in the resting chick embryo cells. The question has been asked here whether insulin increases the production of total cell ribosomal protein by chemically altering preformed mRNAs. Results obtained by translating messages from deprived and hormone-treated cells in wheat germ and reticulocyte preparations do not support a mechanism involving covalent modification of preformed mRNAs. The observations, coupled with those previously made with inhibitors of translation, lead us to suggest that insulin stimulates protein synthesis in the resting chick embryo cells by activating limiting components of the initiation system. The effects of the hormone are greatest with messages, such as those for the ribosomal proteins, that have low affinities for the limiting initiation components.

Animals↗
PubMed1981

Induction of DNA ligase during stimulation of DNA synthesis in intact rat liver by a dietary manipulation.

After a nutritional shift from a protein-free to a diet containing 50% casein, the activity of DNA ligase increases in intact rat liver in correlation with the induction of hepatic DNA replication. The treated rat liver as well as control rat liver contains a single species of DNA ligase having a sedimentation coefficient of about 5.5 S. The administration of cycloheximide in vivo completely inhibits the increase in DNA ligase activity and in DNA synthesis, indicating that DNA ligase is induced in the hepatic cells replicating DNA. In contrast to DNA ligase, DNA kinase is unchanged in the activity level by the dietary manipulation.

Animal Nutritional Physiological Phenomena↗
PubMed1981

Lodish model and regulation of ribosomal protein synthesis by insulin-deficient chick embryo fibroblasts.

The production of ribosomal proteins in chick embryo fibroblasts that have been deprived of insulin is depressed in a much greater degree than that of most or all other cell proteins. Previous observations ruled out explanations for the preferential decrease in ribosomal protein formation that depend upon a selective destruction of ribosomal protein messages or a regulatory role for nascent ribosomal ribonucleic acid. The proposition has now been examined that ribosomal protein messenger ribonucleic acids (mRNAs) in the hormone-deficient chick embryo cells have a lower affinity for a limiting, early acting component of the initiating machinery than do most other cell messages and, in consequence, suffer from a translational disadvantage. The approach that was used depends upon the findings of Lodish and others that all mRNAs are not initiated with equal ease, that inhibitors of elongation favor the initiation of low-affinity mRNAs, and that agents that dampen an early step in initiation discriminate against the low-affinity messages. The idea was tested by comparing the effects of various inhibitors on the rates of synthesis of total cell protein and individual nonribosomal proteins, on the one hand, with those of individual ribosomal proteins, on the other. The results fit the Lodish model and are consistent with the conclusions that ribosomal protein mRNAs are more poorly initiated in the resulting fibroblasts than are most or all other cell messages and that this condition is largely or entirely responsible for the low rate of ribosomal protein formation.

Animals↗
PubMed1981

Multiple species of mammalian S-adenosylmethionine synthetase. Partial purification and characterization.

Two species of S-adenosylmethionine (S-Ado-Met) synthetase (EC 2.5.1.6) exist in rat liver cytosol and a distinct species of the enzyme exists in kidney cytosol. S-Ado-Met synthetases alpha and beta in rat liver cytosol have been partially purified about 200- and 80-fold, respectively. The apparent molecular weight estimated by gel filtration and the sedimentation coefficient are 210 000 and 9 S for S-Ado-Met synthetase alpha and 160 000 and 5.5 S for S-Ado Met synthetase beta. Both enzymes absolutely require Mg2+ and K+ for the activity and are completely inhibited by p-(chloromercuri)-benzoate. Kinetic studies indicate that S-Ado-Met synthetases alpha and beta exhibit negative cooperativity with low S0.5 (ligand concentration required for half-maximal velocity) for L-methionine (17 microM) and ATP (0.5 mM) and positive cooperativity with much higher S0.5 values (S0.5 (L-methionine) = 0.5 mM, S0.5 (ATP) = 2 mM), respectively. The cryoprotectants dimethyl sulfoxide and glycerol markedly lower the S0.5 values of S-Ado-Met synthetase beta without significant effect on Vmax. A single species of S-Ado-Met synthetase has been purified about 1000-fold from rat kidney cytosol. The kidney enzyme, termed S-Ado-Met synthetase gamma, has an apparent molecular weight of 190 000 and a sedimentation coefficient of 7.5 S and is resistant to the inhibition by p-(chloromercuri)benzoate. S-Ado-Met synthetase gamma exhibits slightly negative cooperativity with an apparent S0.5 value for L-methionine of 6 microM and for ATP of 70 microM.

Adenosine Triphosphate↗
PubMed1981

Nuclear location of ribonuclease H and increased level of magnesium-dependent ribonuclease H in rat liver on thioacetamide treatment.

Rat liver nuclei were isolated in aqueous solutions of low ionic strength or anhydrous glycerol. The presence of ribonuclease H (RNase H) [EC 3.1.4.34] activity in the cytoplasm is due to extraction of the nuclear enzyme by buffer and inorganic salts. Two forms of RNase H were separated from rat liver nuclei by affinity chromatography using a DNA-cellulose column. When the RNase H in the wash solution of nuclei with 0.3 M sucrose and in nuclear solution extracted with 0.15 M NaCl were fractionated on a single-stranded DNA-cellulose column, two peaks corresponding to Mn2+- and Mg2+-dependent RNases H were eluted at 0.1 M and 0.2 M NaCl, respectively, and a peak having both RNase H activities was recovered in the wash-through fraction from the column. Among the enzymes from these two fractions in the nuclei, the activity of the Mg2+-dependent RNase H which binds to DNA-cellulose increased several-fold within 24 h of a single injection of thioacetamide. The activities of Mg2+-dependent RNase H extracted with higher-salt solution from the nuclei and recovered in the flow-through fraction from the DNA-cellulose column and the Mn2+-dependent RNase H activities were relatively unaffected by an injection of thioacetamide.

Acetamides↗
PubMed1981

S-adenosylmethionine synthetase isozymes in the liver of tumor-bearing mice.

Isozyme patterns of S-adenosylmethionine synthetase have been determined with and without dimethylsulfoxide in liver of tumor-bearing mice. The activity of the beta-form markedly increased following intraperitoneal transplantation of Ehrlich ascites tumor cells, whereas the activity of the alpha-form did not change so strikingly. Tumor cells were shown to have only the gamma-form of the enzyme. When the ascites fluid, after removing tumor cells, was injected into a normal mouse, the activity of the beta-form increased over that of control animals.

Animals↗