Relaxation phenomena in human erythrocyte suspensions

Temperature-jump studies on the long S-2 fragment (100,000 daltons) isolated from myosin show that this structure can undergo a-helix-random coil transitions in a time range approximating the cycle time of a crossbridge. Two relaxation times are observed after temperature jumps of 50C over the range 35-55°C, one in the submillisecond (Tf) and the other in the millisecond (r,) time ranges. Both processes exhibit maxima near the midpoint of the helix-coil transition (ttm = 45 + 2°C) as determined by optical rotation melt experiments. Similar results were observed for the low temperature transition (t. = 45°C) of the myosin rod. Viscosity studies reveal that the S-2 particle has significant flexibility at physiological temperature. Results are considered in terms of the Huxley-Simmons and helix-coil transition models for force generation in muscle. The subfragment 2 (S-2) region of the myosin molecule plays a major role in most current theories of muscle contraction. In the model of H. E. Huxley (1) for contraction, this segment, which forms the coiled-coil a-helical tail of heavy meromyosin, swings away from the thick filament surface during each contractile cycle of a crossbridge, thus allowing the myosin heads (S-1 subunits) to attach to neighboring thin filaments with the same stereospecific orientation over a wide range of interfilament spacing. S-2 is considered to be a rigid, inextensible rod which is fastened to the thick filament backbone by a hinge at the junction of light meromyosin (LMM) and heavy meromyosin (HMM) and is joined to the myosin head by a second hinge-the S-1/S-2 linkage. This arrangement provides the freedom required to accommodate the variable interfilament spacing as the sarcomere shortens. After attachment of the S-1 subunit to the thin filament, it rotates relative to this structure, generating a relative sliding force between the thick and thin filaments. During each cycle of a crossbridge, ATP is hydrolyzed. In the A. F. Huxley-Simmons model (2-4), S-2 serves as an elastic element which, by spring-like extension, acts to exert a part of the tensile force developed when a crossbridge interacts with the thin filament. The force-generating event is again considered to be rotation of the myosin head on the thin filament with the S-2 elastic element stretching instantaneously as the S-1 subunit alters its angle of attachment through a series of stable positions with progressively lower potential energy.In the Harrington model (5) for contraction, the force-generating event is considered to be an a helix-random coil transition within the S-2 element during each cycle of a crossbridge. In this model the myosin head remains fixed in orientation during its transient attachment to the thin filament and the melting of part of the double a helix near the LMM-HMM junction is effected by the ATP cleavage reaction occurring on a neighboring myosin head.Our crosslinking studies of synthetic myosin thick filaments and glycerinated rabbit muscle fibers in rigor indicate that the The publicatio...

“…The 14 UV Absorbance Changes Accompanying Melting of Myosin Rod. The temperature-jump experiment was also performed on solutions of the myosin rod in 0.5 M NaCl at pH 7.…”

Section: Uv Absorbance Changes Accompanying Melting Of S-2mentioning

confidence: 99%

Rapid helix--coil transitions in the S-2 region of myosin.

Tsong¹,

Karr²,

Harrington³

1979

“…In the past few years, numerous studies bearing on the effects of high voltage pulsation and rapid Joule-heating on suspensions of cells and phospholipid bilayers have appeared in the literature (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). These studies have focused on two different aspects of the voltage pulsation: one deals with the lysis of, and the release of molecules from, cells treated with high electric fields (1)(2)(3)(4)(5), and the other deals with the relaxation phenomena of cell suspensions accompanying rapid Joule-heating (6)(7)(8)(9)(10). Both types of experiment foster the idea that the molecular release and the relaxation phenomena observed may be related to basic processes of membrane and nerve functions.…”

mentioning

confidence: 99%

“…Because the voltage pulsation in these studies introduces electric field and temperature jump at the same time, and because each of them can perturb the system in several different ways, the interpretation of the experimental data has been ambiguous. Possible perturbations of a cell suspension by a voltage pulse have been discussed elsewhere (4,9,11), and are summarized in a more comprehensive way in Table 1.…”

mentioning

confidence: 99%

Hemolysis of human erythrocytes by transient electric field.

Kinosita

Tsong

1977

257

118

Exposure of human erythrocytes, under isotonic conditions, to a high voltage pulse of a few kV/cm leads to total hemolysis of the red cells. Experiments described herein demonstrate that the hemolysis is due to the effect of the electric field. Neither the effect of current nor the extent of the rapid Joule-heating to the suspending medium shows a direct correlation with the observed hemolysis. Voltage pulsation of the erythrocyte suspension can induce a transmembrane potential across the cell membrane and, at a critical point, it either opens up or creates pores in the red cells. In isotonic saline the pores are small. They allow passage of potassium and sodium ions but not sucrose and hemoglobin molecules. The pores are larger in low ionic conditions and permit permeation of sucrose molecules, but under no circumstances can hemoglobin leak out as the direct result of the voltage pulse. Kinetic measurements indicate that the hemolysis of the red cells follows a stepwise mechanism: leakage of ions leads to an osmotic imbalance which in turn causes a colloidal hemolysis of the red cells.

“…This article must therefore be hereby marked "ad- Temperature-jump kinetic measurements were done with a Messanlagen temperature-jump apparatus (8). Experimental details of the relaxation measurements are given elsewhere (9,10). Spectroscopic measurements were performed with a McPherson 707K double-beam spectrophotometer.…”

mentioning

confidence: 99%

Rapid conformational changes of cytochrome P -450: Effect of dimyristoyl lecithin

Tsong

Yang²

1978

Binding of benzphetamine to purified microsomal cytochrome P450 from rat liver causes a shift in the heme spin state of the protein to favor the high-spin form. This shift is strongly temperature dependent. A rapid temperature jump of a cytochrome P450/e benzphetamine mixture, monitored by changes in the Soret absorptions of the heme, reveals two relaxation processes: one in a 50-msec time range (7f) and the other in a 0.3-sec time range (T5). Both relaxations reflect conformational changes of the protein after the substrate binding. No bimolecular reaction of benzphetamine and the enzyme has been resolved. This indicates that there is no absorption change of the heme associated with the initial binding. In the presence of dimyristoyl lecithin, at 250C Tf decreases by nearly one order of magnitude whereas r, decreases to one-third. The enhancement of rates by added phospholipid is both temperature-and concentration-dependent: rates are accelerated only above the gel-liquid crystalline transition temperature, and this effect saturates near the enzyme/lipid ratio of 1:20. In contrast, the lipid does not have significant effect on the equilibrium binding curve of the substrate. These results suggest that the lipid may form an envelope around the enzyme and, depending on its crystalline state, regulates the rate of the substrate-induced conformational changes of cytochrome P450.Several factors are known to affect the equilibrium between the high-and low-spin states of cytochrome P-450 (P-450). The equilibrium shifts toward the high-spin state upon binding with certain substrates or when the temperature is increased. Equilibrium constants and other thermodynamic parameters have been calculated for P-450 from the soil bacterium Pseudomonas putida, whose high-and low-spin states are characterized by Soret bands around 391 and 417 nm, respectively (1,2). The assignment of the spin states and the point of equilibrium is less certain in liver microsomal P-450. Haugen and Coon (3) have purified, from rabbit liver microsomes, a low-spin form of P-450 with a Soret band at 418 nm and another species of P-450 that is predominantly in the high-spin state with a Soret band at 395 nm. Rein et al. (4) have shown that in the presence of benzephetamine the heme group of P-450 shifts toward the fiigh-spin state. Ebel et al. (5) have reported that, at room temperature, 40% of the P-450 in rat liver microsomes is in the high-spin state, and an additional 35% is converted to the high-spin form upon binding with substrates. They have also reported the temperature-induced difference spectrum to be a typical "type I" binding spectrum (6).We have obtained similar results with highly purified P-450 preparations. This provides an excellent system for investigating the molecular basis of the spectral and spin state changes by the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this f...