K. Maruyama

The identification of randomly formed thin filaments in differentiating muscle cells of the chick embryo.

Abstract The appearance of thin filaments, 60–80 A in width, in the myoblasts of early chick embryos incubated for 3 days at 39°C was observed with the electron microscope. The filaments were distributed randomly throughout the cytoplasmic matrix of the myoblasts where myofibrils had not yet been formed. These filaments were isolated from 4-day embryos by differential centrifugation; they were found in the top layers of the so-called “microsomal fraction.” The fraction showed considerable flow birefringence and an ATPase activity. Mixing of the fraction with rabbit myosin caused marked increase in the flow birefringence, and the addition of ATP to the mixture in 0.6 M KCl solution resulted in a decrease in the birefringence. The ATPase activity of myosin was enhanced about 100% by the addition of the fraction at low ionic strength. No changes in birefringence or ATPase activity took place when actin was added to the fraction. The demonstration of the reaction of the “microsomal fraction” with added myosin strongly suggests that the thin filaments in question are F-actin filaments.

A new protein-factor hindering network formation of F-actin in solution

Abstract It is known that F-actin, one of the main structural proteins of myofibrils, forms a network structure in solution, resulting in complicated behavior under hydrodynamic force. The present study demonstrates that a factor of protein nature, isolated from myofibrils, inhibits the network formation of F-actin. Under a field of sonic vibration or high shearing force in the presence of this “actin-factor”, F-actin is dispersed with a dimension of about 1 μ in particle length, which is close to the unit length of F-actin in situ (1 filaments in sarcomeres).

Natural F-actin: I. Direct isolation of F-actin from myofibrils and its physico-chemical properties

Abstract 1. 1. F-actin was directly isolated from myofibrils without the use of any depolymerization process or acetone treatment. 2. 2. Natural F-actin was very similar to Straub F-actin with respect to its interaction with myosin. 3. 3. The particle length calculated for natural F-actin was shorter than that of Straub F-actin, and corresponded with the possible unit length of the I-filaments in a sarcomere (approx. 1–2 μ). 4. 4. Natural F-actin differed from Straub F-actin in its poor intermolecular interaction or lack of network formation. It had an intrinsic viscosity of about 4 (100 ml/g) and a sedimentation coefficient, s 20, w 0 , of about 40 S.

Some physico-chemical properties of β-actinin, “actin-factor”, isolated from striated muscle

Abstract Purification of β-actinin, the protein factor that hinders network formation of F-actin, has been carried out. β-Actinin, in a weight ratio of 1:10, dispersed F-actin into particles having a length of 1 μ. The molecular weight of β-actinin appeared to depend on ionic conditions. It was 6.5 · 10 4 in 0.1 N NaOH, 13 · 10 4 in 5 mM KHCO 3 , and more than 30 · 10 4 in 0.1 M KCl. The extrapolated value of the sedimentation coefficient was 8 S, when 5 mM KHCO 3 was present. The amino acid composition of β-actinin was similar, though not identical, to that of actin and of α-actinin.

Interaction of myosin B with adenosine triphosphate, a flow-birefringence study.

Abstract The interaction of myosin B, or natural actomyosin, with adenosine triphosphate (ATP) was investigated employing chiefly the flow birefringence technique. Unless the myosin B sample was clarified, anomalous behaviour of the extinction angle (χ) in the low velocity gradient (G) range was frequently observed on the addition of ATP. On addition of ATP, the rotary diffusion constant of myosin B extrapolated to G = 0 increased slightly from 0.7 to 1.2 sec −1 . The molecular size in the myosin B solution was polydisperse. The range of length distribution was from 1 to 3 μ, and in the presence of ATP from 0.7 to 2.4 μ. No disaggregation of myosin B was observed upon dilution to 0.005%. The birefringence greatly decreased on the addition of ATP throughout the G range tested. In the high G range between 500 and 1,000 sec −1 , χ increased from 8° to 14° on addition of ATP. The myosin B solution was centrifuged in the presence of ATP at 100,000 g, and the supernatant showed the same flow birefringence characteristics as myosin A solution. The rotary diffusion constant of myosin A is 1,800 sec −1 . The minimum effective concentration of ATP on flow birefringence was 5.10 −5 M . Pyrophosphate was also effective in the presence of Mg ++ . The influence of KCl at a high concentration was similar to that of ATP. A much more drastic effect was caused by KOH and KI. In discussing the effects of ATP, the most adequate explanation of the change in the flow birefringence properties of the clarified solution with ATP, seems to be the dissociation of myosin B into actin and myosin.

Effect of β-actinin on the particle length of F-actin

Abstract The binding of β-actinin, the actin-dispersing factor, to F-actin has been investigated. The stoichiometric ratios for the binding of β-actinin to F-actin were found to be 1:10 and 1:8, by weight, for intact and sonicated F-actin, respectively. When an increasing amount of β-actinin was added to F-actin and subjected to sonication, F-actin could be dispersed into particles as short as 3000–4000 A. Such short F-actin particles were also formed when G-actin was polymerized in the presence of a large amount of β-actinin. Experimental conditions were checked for the action of β-actinin on the apparent particle length of F-actin. β-Actinin was found to become slowly inactivated in 0.1 M KCl.

The effect of myosin and calcium on the solubilization of F-actin from muscle mince.

Abstract 1. 1. Muscle mince was extracted with Weber-Edsall solution to remove myosin. From the residue, fibrous protein of 1–2 μ particle length was solubilitzed by 0.6 M KCl solution containing myosin and/ot 1–10 mM CaCl 2 . 0.6 M KCl solution alone solubilized hardly anything from the mince residue. Actomysin was also extracted with myosin, and natural F-actin was extracted with CaCl 2 . 2. 2. Ordinary F-actin of the Straub type was obtained by the depolymerization and repolymerization procedure from the solution extracted in the presence of Ca 2+ . 3. 3. An unknown component, different from myosin, F-actin and actomysin, was detected by ultracentrifugation of the solution extracted in the presnce of Ca 2+ . 4. 4. The chabge in muscle fibrillar structure during the extraction of muscle mince with Weber-Edsall solution was followed with an electron microscope. Separation appeared between the thin filaments and the Z-membrane before the solubilization of the thin filament.

Formation of actomyosin during the extraction of muscle mince with Weber-Edsall solution

Abstract The process of transformation of myosin A to myosin B during the prolonged extraction of muscle mince was investigated in detail by various physico-chemical techniques, viz. , viscosity, turbidity, flow birefringence, ultracentrifugation and ATPase activity measurements. All the determinations have shown that the formation of actomyosin started at around 8–10 h after the start of extraction and was completed at about 20 h. Flow birefringence measurements suggested that actomyosin particles of fairly uniform size-1–2 μ in length-were formed from the beginning and the amount increased during further extraction of muscle mince.

Kinetic analysis of the polymerization process of actin.

The polymerization process of actin was examined by measuring the amount of flow birefringence and by analyzing release of labeled inorganic phosphate from the bound [gamma-32P]ATP upon polymerization of G-actin to F-actin. Comparison of the above experimental results with the electron microscopic data of Kawamura and Maruyama (J. Biochem., 67, 437-457, 1970) suggested that growth and redistribution steps occurred simultaneously during polymerization. Attempt was made to simulate the polymerization process of actin by calculating the kinetic equations numerically. The results of simulation suggested that it was necessary to take into consideration the association and dissociation between F-actin particles as well as the association and dissociation between F-actin and G-actin.

Some physico-chemical properties of KI-extracted F-actin

Abstract An F-actin preparation, extracted from myosin-removed myofibrils by KI, showed an apparent particle length of approx.1–2 μ, which is much shorter than that of the ordinary F-actin of Straub type. There was a lack of “network formation” of the KI-actin in solution. Its viscosity was also lower than that of Straub-actin. The extrapolated value of the sedimentation coefficient was approx. 40 S. Acetone treatment of KI-actin slightly altered the particle-length distribution. Upon repeated purification of KI-actin strong intermolecular interaction occurred and finally the material became F-actin of Straub type.