Disassembly from both ends of thick filaments in rabbit skeletal muscle fibers. An optical diffraction study.

We show in this paper that the change of the internal structure of a sarcomere in a rabbit glycerinated psoas muscle fiber can be examined by analyzing the intensity change of the first- and the second-order optical diffraction lines. A unit-cell (sarcomere)-structure model has been applied to the estimation of the length of thick filaments in a muscle fiber while they undergo dissociation. The optical factors, except for the unit-cell-structure factor, hardly changed during the dissociation of the filaments. Our results show that thick filaments dissociate from both ends on increasing the KCl concentration in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed to some extent the dissociation of thick filaments. The disassembly of thick filaments occurred at higher KCl concentrations in the absence of pyrophosphate. There was a correlation between the stability of the thick filament structure and cross-bridge formation, which was induced either by the addition of micromolar concentrations of Ca2+ in the presence of Mg-pyrophosphate or by removal of Mg-pyrophosphate.     

Dynamic light-scattering study on polymerization process of muscle actin

Globular actin (G-actin) polymerizes into a fibrous form (F-actin) under physiological salt conditions. The polymerization process of muscle actin was studied by a dynamic light-scattering method. The intensity correlation functions G2(tau) of scattered light from a G-actin solution containing 2 mM Tris-HCl (pH 8.0) and 0.1 mM ATP were analyzed by a cumulant expansion method, and the translational diffusion coefficient was determined to be D = (8.07 +/- 0.10) X 10(-7) cm2/s at 20 degrees C. This D value gave a diameter of 5.3 nm for spherical G-actin including a hydration layer. Polymerization of 1-3 mg/ml G-actin in a solution containing 10 mM Tris-HCl (pH 8.0), 0.2 mM ATP and 60 mM KCl was followed by successive measurements of G2(tau) for a data accumulation period of 60-300 s/run. The time evolution of G2(tau) was analyzed by a least-squares fitting to the field correlation function of a multiexponential form g1(tau) = sigma iAi exp(-gamma i tau) with gamma 1 greater than gamma 2 greater than 3 greater than ..., and the static scattering intensity I(t) = mean value of I as a function of time t after initiation of polymerization was decomposed as I(t) = mean value of I sigma iAi. At the early stage of polymerization, a two-exponential fit gave results indicating that component 1 came from G-actin and component 2 from F-actin growing linearly with t. At the middle stage of polymerization, a three-exponential fit gave the results that component 1 came from G-actin and possibly its small oligomers, component 2 from polymers with a number-average length Ln of about 900 nm which was independent of t, and component 3 from 'ghosts' in dynamic light scattering in a semidilute regime. Component 3 was concluded to arise from restricted motions of polymers with lengths much longer than Ln in cages formed by polymers giving component 2, and a fragmentation-elongation process of F-actin was suggested to start at the middle stage of polymerization, resulting in the size redistribution of F-actin.     

Establishment and characterization of a human malignant schwannoma cell line (HKMS)

The malignant schwannoma cell line (HKMS) was established from the subcutaneous tumor of Axilla region of a 48-year-old Japanese woman. The HKMS line has the following biological properties. 1. The HKMS cells were spindle in shape and showed neoplastic and pleomorphic features. The monolayer sheet of HKMS cells showed the resemble cell-arrangement with that of the original tumor tissue. 2. The cells showed a stable growth and the serial passages were successively carried out 150 times within 3 years. Their population doubling time is about 40 hours. 3. The chromosome number varied widely, and the modal number was stable at the 78-80. The marker chromosomes were present. 4. The cells were transplanted into the subcutis of nude mice and produced the malignant schwannoma.     

Establishment and characterization of human neuroblastoma cell line (HSNB)

We cultured an aspiration fluid of the sternal bone marrow of the patient having adrenal neuroblastoma and established a neuroblastoma cell line (HSNB). The HSNB line has the following biological properties. 1. They are small round in shape and proliferate in flotation while forming cell aggregate, and often they attach the bottom of plastic dish and process the nerve-like fibers. A rough-endoplasmic reticulum are poorly developed, however, a lot of free ribosomes are scattered in the cytoplasm. In the peripheral area of the cells, small spherical secretory granules (60-140 nm in diameter) are existed. One characteristic of this cell is existence of microtubules in the cell-projections. 2. They show a stable growth and the doubling time is about 50 hours. 3. Their chromosome number varied widely and the mode is 46. The double minute chromosomes were present in 50% of cells. 4. When they are transplanted in the cheek pouch of hamster, they produced the neuroblastoma. 5. They produce neuron specific enolase. 6. N-myc gene was amplified ca 250 folds.     

Thermostable tryptophan synthase ofBacillus stearothermophilus expressed inEscherichia coli

Summary The tryptophan synthase genes,trpA andtrpB, from a moderate thermophile,Bacillus stearothermophilus IFO13737, were expressed efficiently inEscherichia coli. The recombinant tryptophan synthase amounted to 22% of the soluble cellular protein, and was purified to homogeneity by three steps. The enzyme is more thermostable thanE.coli tryptophan synthase, especially the subunit. The enzyme is also more resistant to sodium dodecylsulfate and methanol thanE.coli enzyme.     

Melting from both ends of an A-band in a myofibril. Observation with a phase-contrast microscope

Observation with a phase-contrast microscope clearly shows that melting of an A-band, i.e., a bundle of thick (myosin) filaments, in a rabbit skeletal myofibril occurs from both ends in the pressure of high concentrations of KCl and PPi. Thick filaments partially melted from both ends can be obtained in a myofibril.     

Temperature change does not affect force between single actin filaments and HMM from rabbit muscles

The temperature dependence of sliding force, velocity, and unbinding force was studied on actin filaments when they were placed on heavy meromyosin (HMM) attached to a glass surface. A fluorescently labeled actin filament was attached to the gelsolin-coated surface of a 1-microm polystyrene bead. The bead was trapped by optical tweezers, and HMM-actin interaction was performed at 20-35 degrees C to examine whether force is altered by the temperature change. Our experiments demonstrate that sliding force increased moderately with temperature (Q(10) = 1.6 +/- 0.2, +/-SEM, n = 9), whereas the velocity increased significantly (Q(10) = 2.9 +/- 0.4, n = 10). The moderate increase in force is caused by the increased number of available cross-bridges for actin interaction, because the cross-bridge number similarly increased with temperature (Q(10) = 1. 5 +/- 0.2, n = 3) when measured during rigor induction. We further found that unbinding force measured during the rigor condition did not differ with temperature. These results indicate that the amount of force each cross-bridge generates is fixed, and it does not change with temperature. We found that the above generalization was not modified in the presence of 1 mM MgADP or 8 mM phosphate.