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Complex III of the mitochondrial electron transfer chain is an asymmetric unit with a diameter of 80-100 Al and a molecular weight of about 300,000.2 Preparations of the complex contain 20-30 per cent by weight of lipid and (in the presence of bile salts) are water soluble.3 Under appropriate conditions (involving dilution of the bile salts), the units aggregate into vesicular membranes.4 The complex exhibits reduced coenzyme Q-cytochrome c reductase activity with high specific activity.3 The following accumulated evidence strongly supports the contention that the complex represents the subunit of the inner mitochondrial membrane which catalyzes the corresponding span (reduced coenzyme Q-cytochrome c reductase) of the electron transfer chain: (i) The complex has been prepared from mitochondria by three different procedures. All preparations have the identical composition in terms of their content of cytochromes and nonheme-iron protein.3 5 6 (ii) At all stages in the purification of the complex, the ratios cytochrome b: cytochrome cl (ref. 7) and nonheme-iron protein: cytochrome c1 (ref. 8) remain constant.1' 6 (iii) All preparations of the complex exhibit, on ultracentrifugal analysis, a single peak with constant sedimentation characteristics,2 consistent with the minimum particle weight as assessed by cytochrome cl content. (iv) The dimensions of the particle as seen by electron microscopy are consistent with this molecular weight, and are similar to the dimensions of the "base-piece" repeating units of the mitochondrial inner membrane. (v) The complex represents the purest form in which the component species have been isolated without loss of native characteristics relating to spectra, oxidation-reduction potential, and solubility. (vt) Specific inhibitors of reduced coenzyme Q-cytochrome c reductase (such as antimycin A) exhibit much the same inhibitory behavior with the isolated complex as with intact mitochondria.3 Cleavage of the complex results in loss of both enzymic activity and the capacity to bind antimycin A.9-1' By all the above criteria the complex must be considered as a single, integrated enzymic unit. Indeed it probably represents one of the best-documented examples of such a multiprotein enzyme. This is not to say that it might not be possible to demonstrate partial reactions both with the intact complex and with isolated fragments. All available evidence, however, suggests that such reactions would be, to some extent, artifactual. An intensive study has recently been made to extend the available knowledge of the composition and structural organization of the complex.1 10-12 In addition, certain significant observations relating to spectral changes in the active complex have also been reported.13 It is the purpose of this communication to integrate

A stable papain membrane has been prepared on a collodion matrix by absorbing papain in a collodion membrane and then cross-linking the papain with bisdiazobenzidine 3,3'-disulfonic acid. The pH-dependence of the activity of the enzyme membrane on the low-molecular-weight substrate, benzoylarginine ethyl ester, was found to differ from that of crystalline papain; the activity was low in the neutral pH range where the native enzyme has its optimum and high at alkaline pH. This anomalous behavior is due to a lowering of the local pH within the membrane as a result of the release of acid by the enzymic hydrolysis of the ester substrate.

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