Tsutomu Yasui

Physicochemical studies on denaturation of myosin-adenosinetriphosphatase

Abstract Denaturation of myosin A- and myosin B-ATPase was investigated in 0.5–0.6 M KC1 and at 30 °C. and pH 7.0. The denaturation of myosin A proceeded in accordance with the first-order law until ATPase disappeared, while that of myosin B proceeded according to the first-order law to a constant value of ATPase. From the results on denaturation of the three parts of myosin B separated by ultracentrifugation and of synthetic actomyosin, it was shown that the rapid denaturation of the first-order myosin B was due to myosin A contaminant in the preparation and/or released from the “heavy” components of myosin B. Under present conditions, the denaturation of myosin A was not affected by the addition of cysteine and by passing nitrogen or oxygen through the solution. By salting-out analysis, it was found that the main peak of myosin A moved from 38.5 to 32% saturation of ammonium sulfate with denaturation, and that a subunit, occupying about 10% of the total, was revealed in the precipitation range of 50–60%. This subunit was water soluble and considerably heat stable and traveled in the ultra-centrifuge as one peak, its s20w being 2.1–2.8. The reduced viscosity of myosin A and the weight-average molecular weight of myosin A increased with decrease of ATPase. When PP was added and ATPase was assayed using Ca++ as an activator of ATPase, a lag phase appeared in the denaturation of ATPase. After the lag phase, the denaturation proceeded with a smaller velocity than in the absence of PP, while, when Mg++ was used as a modifier of ATPase, the effect of PP could not be observed. From these results a reaction scheme was proposed which satisfactorily explained the course of the denaturation under various conditions.

Physicochemical studies on denaturation of myosin—Adenosinetriphosphatase. II. Changes in chromatographic profile and optical rotation

Abstract The inactivation of myosin A-ATPase proceeds according to the first-order law. The rate constants of the inactivation were proportional to the 1.3, 0, and −3.3 powers of [H+], respectively, in the ranges of pH 5.2–6.0, 7.5–8.5, and 10.0–10.5. In these three ranges of pH, ΔH‡ values were 31.6, 52.6, and 42.1 kcal./mole, respectively. During the incubation at 36 °C. the α component in chromatographic profiles on diethylaminoethylcellulose decreased, the β component increased, and finally the β component decomposed. However, specific ATPase activities of both the α and the β components were not constant and decreased with time. Under three conditions (pH 7.0 and 30 °C., pH 5.7 and 20 °C., and pH 10.3 and 20 °C.), the change in optical rotatory dispersion with time was measured and compared with that of ATPase activity. The helical content decreased very slowly and only by a few per cent, even after ATPase activity disappeared.

Proteolytic separation of an enzymic active subfragment from the myosin-subfragment (S-1)

Abstract Direct visualization of myosin molecule by electron microscopy has shown it to be a long rod with a globular head at one end (Rice 1961 Huxley 1963). The use of proteolytic enzymes such as trypsin and chymotrypsin can cleave myosin into well defined and high molecular weight fragments, heavy and light meromyosins (Szent-Gyorgyi 1953 Gergely 1955). The globular head was found in the heavy meromyosin (HMM), and the light meromyosin was regarded as a main component of the rod. Both the ATPase activity and the actin binding ability, which are the characteristics of myosin, are found with HMM and also with the myosin-subfragment, so called S-1 (Mueller and Perry 1962), but not with the light meromyosin. It is then generally accepted that the two characteristics of myosin were confined to the globular head, and the S-1 has been assumed to be a primary component of the globular head. In this report, isolation of an active subfragment from the S-1 is presented. The most remarkable difference of the new subfragment from the S-1 has been shown in the effect of pH on the ATPase and ITPase activities.

Molecular Adaptability of Carp Myosin: a Study of Some Physico-Chemical Properties and Their Comparison with Those of Rabbit Myosin

During thermal inactivation, the addition of as low as M urea resulted in the reduction of delta G identical to barrier of the inactivation of carp myosin Ca2+-ATPase, whereas that of rabbit myosin remained unaffected. In the absence of urea, a four-hour incubation of carp myosin was accompanied by the release of light chains at 30 degrees C, a value 10 degrees C lower than that for rabbit myosin. Electron micrographs revealed that carp myosin forms artificial thick filaments, which were uniform in size and may differ in a few details from those of rabbit. Not only that helical content of carp myosin was about 4% less than those of rabbit myosin, but it showed more sensitivity to thermal and urea denaturation; and its reversibility upon subsequent cooling or removal of urea was rather poor. The loss in helicity of myosins by urea was a concentration- and temperature-dependent biphasic reaction, with the most obvious effect observed on carp myosin. That carp myosin has increased tendency of unfolding in urea solutions was confirmed by viscosity data and the exposure of thiols also. Even in the absence of urea more SH groups of carp myosin were incorporated by DTNB, and more epsilon-amino groups reacted with NQS. Carp myosin remained in solution till the modification of about 52 surface myosin remained in solution till the modification of about 52 surface amino groups, whereas no precipitation effect was noted in case of rabbit myosin. Neither amino-acid composition nor some parameters derived from it, such as average hydrophobicity polarity index and number of polar side chains, revealed any difference pertinent to the relative stability of the two myosins. On the contrary, the contractile efficiency of carp myosin in the near physiological range was high and thus inversely related with the thermostability. This relationship along with the above evidence has been regarded to demonstrate the adaptability of carp myosin through a loose molecular conformation, which has probably been achieved by the addition of weak interactions in the course of evolution.

Urea tolerance of myofibrillar proteins of two elasmobranchs: Squalus acanthias and Raja tengu

Some biochemical properties of actomyosin and myosin from elasmobranchs, Squalus acanthias and Raja tengu are compared with those of a freshwater (Cyprinus carpio) and a marine teleost (Seriola quinquiradiata). Whereas Ca2+-ATPase of teleost actomyosins are more stable in the absence of urea, the reverse is true for elasmobranchs up to 1.0 M urea. In contrast to that of teleosts, the Mg2+-ATPase of S. acanthias actomyosin shows an activation in the presence of urea, where as that of R. tengu persists. Below 1.0 M urea, there is low incorporation of DTNB into thiols of elasmobranch myosins, and losses in alpha-helicity are reversible up to 5.0 M urea. The results, thus, demonstrate that for a certain concentration of urea, elasmobranch myofibrillar proteins may exhibit a group specific tolerance to urea.

Preparation and some properties of carp heavy meromyosin

Trypsin fragments carp myosin heavy chain into components of lower molecular weight. These changes are concomitant with the loss of Ca2+-ATPase and a weakening in actin-myosin interaction. As heavy meromyosin (HMM) prepared from myofibrils is more homogeneous, the above changes are due to overfragmentation of heavy chain mass. Amino-acid composition of carp and rabbit HMMs are similar, but differences exist in response to DTNB and incorporation.

Inactivation of Myosin B and Myofibrillar ATPases as a Function of Water Activity and the Inhibitory Effect of Sucrose on the Dehydration-Induced Inactivation of Myosin-ATPase

水分活性の変動にともなうミオシンBと筋原線維の失活度合をATPase活性を目安にして調べた.ミオシンB及び筋原線維ATPaseは脱水により失活し,その度合は多分子層(水分活性=0.55)に到達するまで増大する.さらに水分活性が低下して単分子層に移行すると,失活度合は逆に減少する.ATPase活性の最大減少度合は,水分活性0.55におけ.約50%であった.種々の脱水水準にある筋原線維をSDS-ゲル電気泳動法により調べてみると,ミオシン重鎖の蛋白質分解反応による崩壊が明らかに認められた.このことは本標品が固有の筋肉蛋白質分解酵素により汚染されていることを示すものである.しかし,単離されたミオシンやミオシンBはミオシン重鎖の分解を示さなかった.これらのことから,脱水過程で認められた筋原線維のATPase活性の変化は,ミオシンATPase活性点近傍における構造変化を反映するものと考えられる.また,この脱水に由来する失活反応の完全な抑制に必要な蔗糖の最低濃度は0.05Mであることが判明した.

The Study on the Thermostability of Myosin Rod Isolated by a Bacterial Protease

ウサギ骨格筋のミオシンからBacidlus polymixaの中性タンパク質分解酵素(BPNP)処理によってミオシン分子のサブユニット,すなわちライトメロミオシン(LMM-BPNP)を調製しこれを試料として実験を行った.6Mグアニジン塩酸中の粘度測定から計算されたLMM-BPNP分子の分子量は135,000-150,000であった.このLMM-BPNPの熱安定性を,ミオシンをトリプシン処理して得られるミオシン尾部(LMM Fr1)や臭化シアン処理によって得られるミオシン尾部(LMM-C)のそれと溶解性やその他いくつかの物理化学的手法によって比較検討した.その結果LMM-BPNPはLMM-CとLMM Fr1の中間的性質をもっていることが明らかになった.

Studies on the forms of casein micelle. I. Research with phosphoprotein phosphatase.

カゼインミセルは,種々なる相互作用により球状を保持しているのであるが,その相互作用の1つに,有機結合性リン,すなわちセリンリン酸がある,筆者らは,REVELらの方法により分離精製を行なったPPaseをカゼインミセルに作用させた際の形態変化についてSephadex G-10, G-200, Polyacrylamide gel電気泳動,および電子顕微鏡観察により追究を行なった.その結果は次の通りである.1. PPaseの活性を,酸カゼインとカゼインミセルにより比較した所,酸カゼインでは,約95%,カゼインミセルでは,約86%の脱リン率を得た.2. 脱リン酸カゼインミセルのSephadex G-10ゲルロ過によると,PPaseによりカゼインミセルから遊離したリン,カルシウムの大部分は,それぞれ異なる位置に流出したが,両無機物が蛋白質区分にわずか認められた.3. カゼインと脱リン酸カゼインを電気泳動により観察した所,易動度に差異が見られた.また,有機リン量の多いαs-, β-カゼインほど顕著であった.4. native casein micelleにPPaseを作用させ経時的に電子顕微鏡観察を行なった所,反応5分経過後には,カゼインミセルの表面がくずれた様相を呈し,互いに結合したミセルも観察された.反応15分経過後には,カゼインミセルの凝集と集合が見られた.反応30分経過後には,カゼインミセルの集合も大形化し,くずれたカゼインミセルの集合が多く見られ,しかもback groundに60～500Aの微細なミセル状小粒子が観察された.反応60分経過後では,大部分は不定形の凝集であったが,不完全なミセルの集合も認められ反応30分経過時と同様なミセル状小粒子が観察された.5. 脱リン酸カゼインミセルのSephadex G-200ゲルロ過によると,native casein micelleと異なる2つのピークが得られた.その1つは,αs-, β-カゼイン,もう1つは,as-, κ-カゼインが多く,わずかβ-カゼインを含んでいた.終りに臨み懇篤なる指導と有益なる助言をいただいた北海道大学,農学部,高橋興威,同三河勝彦助手に深謝の意を表する.なお,本研究は,昭和43年度,文部省科学研究費の補助をえて遂行したものである.

Researches on the Color of Cured Meat

Cured-meat pigmentと一酸化窒素ミオグロビン (MbNO) について, その酸化機構を検討した結果, 次の点が明きらかとなつた。1. Cured-ment pigmentはヘム蛋白質の一酸化窒素結合物であり, その主体はMbNOである。2. Cured-meat pigmentは, 空気中の酸素により徐々に酸化され, これに光が加わると, 急激に酸化されてメトミオグロビン (met Mb) に変化する。3.温度20°～25℃, 照度3,000～4,000ルツクスの室内で, pH5.6～5.9のcured-meat pigmentおよびMbNOの光酸化速度恒数は1.26hr-1で, pH7付近ではかなり光酸化が抑制される。4.還元剤の使用によつて, 酸素による一次的酸化を抑制することにより, 光の存在の有無にかかわらず, MbNOを長時間安定に保つことが可能である。