R E S E A R C H

CCP Uncouples Oxidative Phosphorylation Du Pont scientists describe new class of uncouplers many times more effective than substituted phenols

A new class of compounds that are remarkably effective uncouplers for oxidative phosphorylation has been de­veloped by Dr. Peter G. Heytler, Dr. William W. Prichard, and Dr. Richard A. Goldsby of Du Pont's central re­search department. Carbonyl cyanide phenylhydrazone (CCP) and its ring-substituted derivatives are more effec­tive than antibiotics, substituted phenols, or inorganic ions, Dr. Prichard told the 46th Annual Meeting of the Federation of American So­cieties for Experimental Biology, held in Atlantic City, N.J. Particularly effective uncouplers of oxidative phos­phorylation in mitochondrial systems are the m-chloro- and p-trifluoro-methoxy- derivatives of CCP. The parent compound is about 10 times more effective in uncoupling than 2,4-dinitrophenol ( DNP ) ; m-chloro-CCP is about 100 times as active as

DNP; and p-trifluoromethoxy-CCP is about 1000 times as active.

The CCP compounds may be con­sidered DNP-like in that both DNP and CCP uncouple oxidative phos­phorylation. But the similarity ends here. The CCP derivatives block photosynthetic phosphorylation. DNP does not. In fact, at a level of 10~4M, DNP actually stimulates photosyn­thetic phosphorylation. And while DNP uncouples the oxidative reaction at about 10~4M, the p-trifluoro­methoxy-CCP derivative uncouples even at 10~8M and causes complete loss of phosphorylative activity at 10"7M. Further, the CCP derivatives do not affect adenosine triphosphatase activity in purified ATP systems ob­tained from mitochondria, while DNP stimulates ATPase. As do most un­couplers, DNP inhibits the exchange of* inorganic phosphate with the ter-

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Carbonyl cyanide phenylhydrazone (CCP)

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m-Chloro-CCP m-Trifluoromethoxy-CCP

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m-Chloro-CCP

Minutes

UNCOUPLERS. Carbonyl cyanide phenylhydrazone and its derivatives (top) prove to be effective uncouplers for oxidative phosphorylation. The chart (representing a mouse liver mitochondria plus £-OH butyrate sys­tem) shows that the uncoupling occurs early in the phosphorylation cycle» before the oligomycin-sensitive site

minal phosphate of ATP. In this respect, the CCP compounds are simi­lar to DNP, Dr. Heytler says.

Mechanism. Thus the CCP ma­terials do not uncouple by competing with the inorganic phosphates for ac­tive sites. There is evidence that the uncoupling occurs very early in the phosphorylation cycle, even before the so-called oligomycin block of oxi­dative phosphorylation. The CCP materials probably act just before or at the time of phosphate incorporation, Dr. Heytler suggests. Although he has no data on the effect CCP has on ADP-ATP exchange, theoretically, at least, the CCP compounds should have none. This exchange, he adds, will be studied soon.

Because the compounds block both oxidative and photosynthetic phos­phorylation, it is likely that they attack components common to both systems, Dr. Heytler says. The compounds are active at a level of 0.2 millimicromole per mg. of mitochondrial protein, about the same range in which the electron transport components of mito­chondria occur. The compounds ap­pear to affect a poorly characterized site, may thus prove useful in study­ing the mechanism of phosphorylation.

Mostly, the group of CCP com­pounds has importance in funda­mental research as a tool with which to study biological energy transfers. But the Du Pont scientists say that the compounds are also being studied as fungicides, insecticides, and plant growth control agents. The materials reduce the permeability of plant roots to water and to certain ions, but in­crease the respiration rate of root cells. At a concentration of about 1 p.p.m. in a nutrient solution, m-chloro-CCP cuts water uptake almost completely, but does not result in the death of the plant. Amino acid uptake by algae is blocked by the compounds.

Ring substitution, as might be sus­pected, has a major effect on un­coupling activity, which is also modi­fied by both the ionization constant and the lipoid solubility of the CCP derivatives. Although the specific de­tails here have yet to be resolved, an increase in lipoid solubility or in the ionization constant, or both, causes a rise in uncoupling activity. And the activity of CCP compounds is pre­vented by cysteine, cysteamine, and homocysteine because of specific bind­ing even at low concentrations. The amino and thiol groups are both re­quired for this protective effect.

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