194 A. L. LEHNINGER, G. D. GREVILLE VOL. 12 (1953) 



Since the already described experiments with intact mitochondria showed that the 

 "sparking" or "priming" phenomenon was necessary to initiate oxidation of the d- 

 isomer, but not the /-isomer, it appeared that this "sparking" process might involve 

 ATP and Coenzyme A. Recently it has been demonstrated that the enzymic oxidation 



TABLE V 



REDUCTION OF DPN+ BY I- AND d-BOH IN EXTRACTS OF MITOCHONDRIA 



Basic test system contained o.io ml of dialyzed extract of acetone-dried mitochondria (prepared 

 by sucrose method; see "Experimental Details") o.i M KCl, 0.05 M tris (hydroxymethyl)amino- 

 methane-HCl ("tris") buffer pH 8.0, o.ooi M DPN+, o.oi M cysteine, and 0.025 M rf-BOH or /-BOH 

 as shown. Where added, ATP was 0.002 M , CoA was 0.0005 M, and MgClj was 0.005 ^- The volume 

 of all systems was i.o ml, made up with HgO. Temp. 20°, time 20 minutes. Optical path, i.o cm. 

 The test systems were exposed to air during the reaction periods. 



of butyrate, a process which also requires "sparking", only occurs after the Coenzyme 

 A derivative of butyric acid has been formed^^- ^^. The interpretation has been made that 

 the "sparking" phenomenon actually represents the enzymic formation of the CoA 

 derivative. By analogy it was reasoned that the "sparking" of (7-BOH oxidation likewise 

 involves CoA and ATP. Experiments soon revealed that the addition of ATP, CoA, and 

 Mg++ to the extracts described above enabled them to cause the reduction of DPN+ by 

 t/-BOH at a high rate but such additions did not stimulate the reduction of DPN^- 

 by /-BOH. It is seen from Table V that all three components (ATP, CoA, and Mg++) 

 were necessary to demonstrate maximal reduction of DPN+ by ^-BOH. There was very 

 little blank reduction of DPN+ in the presence of the three supplements. In the presence 

 of the additions the reduction of DPN+ by (/-BOH occurs at a linear rate for a period of 

 at least 30-40 minutes under the conditions studied. These clear extracts of mitochondria 

 therefore contain the enzymes catalyzing the reactions by which the d- and /-forms of 

 ^-hydroxybutyrate undergo at least the first stages of biological oxidation and it is 

 clearly evident that the oxidation of ^-BOH proceeds by a mechanism somewhat different 

 from that involved in the oxidation of /-BOH. 



By varying the manner of preparation of the extracts described above (see Experi- 

 mental Details) or by fractionation procedures still under study, it was found possible 

 to obtain extracts having very little or no demonstrable activity in the reduction of 

 DPN+ by /-BOH, in the presence or absence of ATP, CoA, and Mg++, but which showed 

 high activity toward (/-BOH in the presence of ATP, CoA, and Mg. Typical data obtained 

 from such extracts are shown in Table VI. It is seen that the rate of reduction of DPN 



References p. 202. 



