Abstract
N,N′-Dicyclohexylcarbodiimide (DCCD) was found to be a potent inhibitor of the membrane-bound ATPase of Streptococcus faecalis but did not inhibit the solubilized form of the enzyme. Inhibited membrane-bound ATPase was reactivated by releasing the enzyme from the membrane. Conversely, sensitivity to DCCD was restored by reconstituting the ATPase-membrane complex from solubilized ATPase and depleted membranes. The results indicate that DCCD reacts covalently with a membrane component and inhibits the enzyme indirectly, perhaps by means of a transmitted effect on its conformation. When added to intact cells, DCCD reduced the rate of glycolysis yet the ATP content of the cells was as high in the presence of the inhibitor as in its absence. DCCD strongly inhibited the degradation of ATP which ensued when glycolysis was stopped, either by removal of glucose or by addition of iodoacetate. The results suggest that DCCD inhibits ATPase in vivo, just as it does in isolated membrane preparations. The inhibition of over-all glycolysis may be secondary to the inhibition of ATP degradation. DCCD also inhibited a number of energy-dependent transport processes, including the accumulation of K+ by exchange for H+ and Na+ and the uptake of phosphate and alanine. Inhibition was observed whether glucose or arginine served as energy source. The findings are consistent with the hypothesis that the membrane-bound ATPase of S. faecalis mediates energy transductions involved in membrane transport; inhibition of transport by DCCD would be a consequence of inhibition of the ATPase. Alternatively, DCCD may bind to sites on the membrane to inhibit indirectly both transport and ATP hydrolysis.
Highlights
MethodsIsolation of Native ATPase-Membrane Complex-The preparation of membrane-bound ATPase from X. faecalis has been described [15,17]
Sensitivity to DCCD was restored by reconstituting the ATPase-membrane complex from solubilized ATPase and depleted membranes
When added to intact cells, DCCD reduced the rate of glycolysis yet the ATP content of the cells was as high in the presence of the inhibitor as in its absence
Summary
Isolation of Native ATPase-Membrane Complex-The preparation of membrane-bound ATPase from X. faecalis has been described [15,17]. The resulting membrane ghosts were separated from the cytoplasmic fraction and washed twice with 1 mM MgS04. This twice-washed membrane preparation is referred to as (native) membrane-bound ATPase. Preparation of Solubilized ATPase and Depleted MembranesThe procedure for solubilizing the ATPase has been described previously [15, 17]. The membrane-bound ATPase preparation described above was washed further, as follows: once with 2 M LiCl - 0.25 M Tris Cl, pH 7.5; once with 0.033 M Tris Cl, pH 7.5,l mMMgC12; and three times with 1 mM Tris Cl, pH 7.5. About 70 to 80% of the membrane-bound enzyme is released abruptly in a soluble form in these final three washes
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