Masamitsu Miyoshi

α-Adrenergic regulation of the activity of thymidylate synthetase and thymidine kinase during liver regeneration after partial hepatectomy

The increases in activity of hepatic thymidylate synthetase and of thymidine kinase, which catalyze the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed during liver regeneration in rats which had been given alpha-adrenoceptor antagonists (phenoxybenzamine and phentolamine) or adrenergic neuron blockers (guanethidine and reserpine). These suppressions were not observed with a beta-adrenoceptor antagonist (propranolol), or an anticholinergic agent (atropine methyl nitrate). The rise in the activity of the thymidylate-synthesizing enzymes was closely correlated with the increase in the DNA content of the liver. It is concluded that catecholamine regulates the increase in the activity of thymidylate synthetase and thymidine kinase, which are key enzymes in DNA synthesis in regenerating liver. It is also suggested that sympathetic nerves play an important role in liver regeneration.

A new immunoblotting assay for thymidylate synthetase and its application to the regulation of enzyme activity in regenerating rat liver.

A highly sensitive and specific immunoblot assay has been developed to quantitate the content of rat liver thymidylate synthetase (EC 2.1.1.45). Applying the method, it is demonstrated that the increase of the activity of thymidylate synthetase in liver regeneration after partial hepatectomy is due to the de novo synthesis of the enzyme protein. Administration of cycloheximide, phenoxybenzamine, phorbol 12-myristate 13-acetate, nifedipine, dexamethasone or indomethacin to partially hepatectomized rats prevented the synthesis of thymidylate synthetase in regenerating liver. Thyroparathyroidectomy also inhibited the increase of the enzyme in liver regeneration. These observations are discussed in relation to the signal transduction concerning the alpha 1-receptor, which was shown to regulate liver regeneration in our previous papers.

Purification and characterization of thymidylate synthetase from rat regenerating liver

Thymidylate synthetase (EC 2.1.1.45) from rat regenerating liver has been purified over 5000-fold to apparent homogeneity by a procedure involving two affinity methods. Molecular weight of the native enzyme was found to be about 68,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band of molecular weight of 35,000, suggesting that thymidylate synthetase is a dimer of very similar or identical subunits. The Michaelis constants for deoxyuridylate (dUMP) and (+/-)L-5,10-methylenetetrahydrofolate are 6.8 microM and 65 microM, respectively. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be ordered sequential. 5-Fluoro-dUMP, halogenated analog of the nucleotide substrate is a competitive inhibitor of the enzyme, with an apparent Ki value of 5 nM. Amethopterin, analog of the cofactor is also a competitive inhibitor with an apparent Ki value of 23 microM.

Chemical Modification of κ-Casein

この実験では, κ-カゼインのアミノ基, カルボキシル基, SH基, チロシン, トリプトファン, リジン, セリン, ヒスチジン, アルギニン, およびメチオニンを種々の方法で化学修飾し, それらがαs-カゼイン安定化作用に及ぼす影響について調べた。 κ-ヵゼインは分子間会合を通して複合体を形成しS20, wは14程度であり, アルカリとか尿素処理で始めてS20, w 2-3に分散する。 このため, 今回の修飾反応条件であるpH 7-9においてはκ-カゼイン分子はじゅうぶんに解離していないため, 反応基が複合体の表面に位置しているかどうかによって反応速度, ひいては修飾率が大きく左右されたと思われる。 アミノ基とカルボキシル基を修飾するとほぼ完全に安定化作用が消失し, ヒスチジンとチロシンの修飾も顕著な安定化力低下をもたらした。 κ-カゼインを還元してより低分子化すると未修飾κ-カゼインより安定化力が高まった。 またその他のアミノ酸残基の修飾は, αs-カゼインの安定化にほとんど関係がなかった。焦点電気泳動法による分析により, 6M尿素中においてκ-カゼインの等電点がpH 5より酸性側へ移ると急にαs-カゼインに対する安定化作用が失われることがわかった。 還元κ-カゼインの焦点電気泳動により6成分が分離され, そのうち等電点の中性側の成分が最も早く修飾を受けるので, これらの成分がκ-カゼイン複合体の表面に位置しているものと推定された。 デンプンゲル電気泳動により, κ-カゼインの化学修飾は分子電荷のみならず分子の大きさも変化させることが判明し, 特にセリン, ヒスチジン, それにチロシンを修飾したものは, 分子会合が進み, 他方TFA化したκ-カゼインとSH基を修飾したκ-カゼインはより小さい分子へ解離した。