An investigation of myoplasmic magnesium adenosine triphosphate in barnacle muscle fibres with the firefly method.

Abstract

1. A study has been made of the feasibility of monitoring myoplasmic ATPMg in single barnacle muscle fibres by microinjections DuPont luciferase-luciferin. 2. Injection of DuPont luciferase-luciferin into a cannulated fibre results almost immediately in light emission the intensity of which reaches a maximum within 2 sec and then decays in two phases, the first rapidly owing to product inhibition, and the second less rapidly (the resting phase). The greater the peak flash is, the more rapid the rate of decline of the first phase. The entire kinetic picture bears a close resemblance to that observed with a light reaction mixture. 3. (i) Injection of dehydroluciferin before luciferase-luciferin reduces peak flash in a dose-dependent manner. (ii) Injection of 0.5 m-MgSO4 shortly after the onset of peak flash fails to raise the resting level of luminescence. By contrast, injection of 10 mM-EDTA promptly reduces resting luminescence. Injection of 10 mM-EGTA increases resting luminescence transitorily. (iii) Injection of NaCl and KCl in graded amounts reduces resting luminescence in a dose-dependent manner. 4. Reducing (or increasing) the pH of the external medium (10 mM-HCO3-ASW) shortly before injecting luciferase-luciferin results in a reduction in peak flash. The pHe optimum for light emission lies in the region of 7.8. 5. Injection of pyrophosphate (PPi) shortly after luciferase-luciferin causes a dual effect: PPi in low concentration stimulates light output, whereas PPi in high concentration reduces light output. Injection of PPi shortly before luciferase-luciferin results in a reduced peak flash. 6. Injection of CoA shortly after luciferase-luciferin increases resting luminescence in a dose-dependent manner. Both the growth and decay phases of light emission following the injection of CoA differ from the characteristic phases observed following the injection of luciferase-luciferin. Additionally, CoA causes a sustained increase in resting luminescence, but fails to modify the kinetic picture if injected prior to luciferase-luciferin. 7. Injection of ATPMg shortly after old DuPont luciferase-luciferin leads to a characteristic growth and decay curve which is indistinguishable from that caused by luciferase-luciferin. However, this is not true if ATPMg is injected after new DuPont luciferase-luciferin. The addition of 10(-4) M-ATPMg to the myoplasm produces a peak flash comparable to that produced by injecting old DuPont luciferase-luciferin. Application of the non-competitive inhibition equation fails to provide a reasonable estimate of myoplasmic ATPMg, presumably because it is difficult to arrive at a reliable Km value for ATPMg. Injection of new DuPont luciferase-luciferin twice in succession leads to identical flash heights. 8. Calibration performed with a solution containing 120 mM-K glutamate permits an estimate to be made of myoplasmic ATPMg if fibres are injected with luciferase-luciferin so that flash height is known beforehand. This approach leads to a myoplasmic ATPMg value of 1.2 +/- 0.1 mM (S.E...