ANTIBODIES AGAINST RESPIRATORY ENZYMES 381 



2. Inhibition of Pyruvic Acid Reductase by Specific 

 Immune Serum 



In Warburg's Laboratory, Kubowitz and Ott (1943) isolated in crys- 

 talline form from rat Jensen-sarcoma and rat muscle the specific 

 protein of pyruvic acid reductase. From 3000 g. of tumors (ca. 600 g. 

 dry weight) which were harvested from about 3000 rats about 50 mg., 

 and from 1000 g. of muscle (ca. 200 g. dry weight), which were har- 

 vested from 10 to 12 rats, about 200 mg. of pure enzyme protein 

 were obtained. The crystalline proteins were indistinguishable with 

 respect to crystalline form and catalytic activity; furthermore, anti- 

 body against the tumor enzyme protein inhibited the catalytic activity 

 of the enzyme protein obtained from rat muscle, and vice versa. 



These enzyme proteins catalyzed the following reaction: 



Pyruvic acid-fdihydro-coenzyme I?=^lactic acid-j-coenzyme I. Under 

 the conditions of the experiments the reaction in the above equation, 

 measured spectroscopically, proceeded completely from left to right. 

 As a result of the completeness of the reaction from left to right, the 

 absorption band at A3 34 mju. shown by dihydro-coenzyme I disappeared 

 completely. The diminution of the absorption of light thereby served as 

 a measure of the enzyme activity of the test solutions. 



Intravenous injections of rabbits with 6 mg. of enzyme protein at 

 intervals of three days during two to three months produced anti- 

 enzyme immunity. The blood of immunized rabbits was introduced 

 into citrate solution (0.7 per cent sodium chloride containing 1.1 per 

 cent sodium citrate) in which the final ratio of blood to citrate solution 

 was three to one. 



To determine the anti-enzyme activity of the immune plasma the 

 following combinations were studied: enzyme protein without plasma; 

 enzyme protein -f-normal plasma; enzyme protein+immune plasma, 

 and immune plasma, without enzyme protein. The test solution 

 consisted of a mixture of 2y of enzyme protein (dissolved in 0.05 ml. 

 of 0.02 M phosphate of pH 7. 4)+ 1.2 ml. of plasma, incubated at 

 20° for 10 minutes. To this was added 5.2 ml. of 0.1 M phosphate of 

 pH 7.3+6.4 ml. of distilled water-fO.l ml. of 0.01 M sodium pyruvate 

 (final concentration=lXlO-^ M). At hour 0.1 ml. (0.265 mg.) of 

 dihydro-coenzyme I (final concentration^ 3.08X10"^ M) was added 



