Rapid relaxation of single frog skeletal muscle fibres following laser flash photolysis of the caged calcium chelator, diazo‐2

Abstract

Diazo-2 is a calcium chelator based on BAPTA [(1989) J. Biol. Chem., in press], whose electron withdrawing diazoacetyl group may be rapidly (2000 s -1) converted photochemically to an electron donating carboxymethyl group by exposure to near ultraviolet light, producing an increase in its calcium affinity (Kd changes from 2.2/iM to 0.073/zM) without steric modification of the metal binding site. Photolysis of a 2 mM solution of this compound with a brief flash of light from a frequency-doubled ruby laser (347 nm) caused single skinned muscle fibres from the semitendinosus muscle of the frog Rana temporaria to relax with a mean half-time of 60.4 + 5 ms (range 30-100 ms, n = 15) at 12°C, which is faster than the relaxation observed in intact muscles (half-time 133 ms at 14°C [(1986) J. Mol. Biol. 188, 325-342]) and similar to the rate of the fast phase of tension decay in intact single fibres (20 s -1 at 10°C [(1982) J. Physiol. 329, 1-20]).

Ca2+and activation mechanisms in skeletal muscle

“…20 a) can be achieved with a caged chelator, which are at least as fast as those seen in vivo whilst the fall in free calcium is likely to be much faster (h < 1 ms) (Mulligan & Ashley, 1989;Ashley et al 1990;Mulligan et al 1990;Palmer et al 1990) than even the fast phase of the relaxation in vivo at 12 °C (h = 70-80 ms), while in myosin-regulated scallop striated muscle the rates achieved are far faster than intact muscle (Fig. 206).…”

Muscle cell function during prolonged activity: cellular mechanisms of fatigue

“…However, evidence is starting to accumulate which suggests that changes in cross-bridge function may be more important than realized so far and, in this context, the use of caged BAPTA in skinned muscle now offers an experimental (Modified from approach to the study of relaxation which is not limited by the time course of Ca2+ removal (Mulligan & Ashley, 1989).…”

A diazo-2 study of relaxation mechanisms in frog and barnacle muscle fibres: effects of pH, MgADP, and inorganic phosphate

“…1, arrow marked "L"). It was calculated that about 50% photolysis of the diazo-2 must be occurring to produce this amount of relaxation [34]. The fibres were air-flashed since absorbance ("inner filter" effect) and appreciable attenuation of the laser light occurs in diazo-2 solution, resulting in poor radial photolysis within the preparation.…”

“…In addition, barnacle muscle does not possess parvalbumins which may otherwise be the source of this elevated free Ca 2+ observed during relaxation. Certainly, the photolysis of the caged Ca 2+ chelator, diazo-2 [1], which reduces the free Ca 2+ to nanomolar levels within 2 ms, produces a faster relaxation than that observed in electrically stimulated fibres [34]. In intact electrically stimulated fibres, therefore the elevated levels of free Ca 2+ observed during relaxation may be regulated by uptake by the sarcoplasmic reticulum (SR), and this determines the rate of relaxation.…”

“…This will enable us to examine crossbridge transitions without the confounding influence of diffusional delays [34]. The effects of increased concentrations of the metabolites MgADP and P i , and H + on the rate of relaxation in two types of muscle were examined, under these conditions, where both the cell and SR membranes have been removed.…”