A method is described for making continuous measurements of the pH of anaerobic muscle. The technique is shown to be applicable to measurement in an intact beef carcase post-mortem. This technique is compared with conventional measurements upon homogenates prepared from samples of metabolically inhibited muscle and the results are seen to be closely comparable. The continuous measurement of tissue pH has the advantage that relatively rapid events may be followed with ease.
Measurements have been made of the rate of pH fall post-mortem in excised beef sternomandibularis muscles held in a linear temperature gradient. A minimum rate of pH fall was observed between 10 and 12°C and the rate increased as the temperature was further reduced towards O'C, a phenomenon known to be accompanied by coldshortening and attributable to increased activity of the contractile actomyosin ATPase which stimulates glycolysis. The increase in rate observed as the temperature was raised above the 10-12°C minimum, had an activation energy of 40kJ/mol, similar to that of the calcium-independent myosin ATPase.
We have demonstrated the release from respiratory control in membrane particles from Micrococcus denitrificans by carbonyl cyanide m-chlorophenylhydrazone and combinations of gramicidin, valinomycin, nigericin, monensin and dianemycin (John & Hamilton, 1970, 1972. From these findings it has been concluded that the increases in membrane permeability show ion specificity identical with that demonstrated in other systems (Henderson et al., 1969). Tetrachlorosalicylanilide and trichlorocarbanilide have also been found to be proton-translocating uncouplers. Studies of protoplast and spheroplast stability in hyperosmotic media (Hamilton, 1970) and the decay of an acid pulse (W. A. Hamilton, unpublished work) have allowed us -to extend these findings to the organisms Bacillus megaterium, Micrococcus lysodeikticus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.The behaviour of trichlorocarbanilide is somewhat anomalous, in as much as it apparently increases both HI and Cl-permeability. Accordingly this compound has been further studied with black lipid membranes formed from oxidizedcholesterol or phosphatidylcholine (lecithin) +cholesterol.In KCI media, trichlorocarbanilide caused a timedependent increase in membrane conductance. This effect was decreased by the replacement of chloride by sulphate or acetate, but was unaffected by the replacement of KCI by NaCl. In the presence of trichlorocarbanilide, anionic diffusion potentials were observed across these membranes in concentration gradients of NaCl or K2SO4. This confirms the increase in anion permeability, since untreated membranes demonstrated cation diffusion potentials under these conditions. Where, however, the concentration gradient across the membrane was in the form of a pH gradient in an acetate-buffered medium with a low (10mM) chloride concentration, the diffusion potential measured in the presence of trichlorocarbanilide was acid-sidenegative. This implies an enhanced proton permeability of the same order as that for chloride. In these artificial membrane systems, therefore, we have been able to verify the action of trichlorocarbanilide in increasing the permeability to protons and to anions, in particular to those of small hydrated radius, such as chloride. We have found no evidence for significant effects with the alkali-metal cations.
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