164 A. L. SCHADE VOL. 12 (1953) 



EXPERIMENTAL 



Materials 



The dba mouse ascites thymoma was obtained through the generosity of Dr. T. Hauschka 

 of the Institute for Cancer Research, Philadelphia, Pennsylvania and maintained by serial passage 

 as ascites in dba mice. The Ehrlich ascites carcinoma and the Krebs-2 ascites carcinoma were kindly 

 provided by Dr. V. Evans and Dr. Dean Burk, respectively, of the National Cancer Institute, 

 Bethesda, Maryland and were maintained as ascites in strain C albino mice. 



Crystalline aldolase was prepared from rabbit muscle by the method of Warburg and Chris- 

 TiAN^. Crystalline phosphoglyceraldehyde dehydrogenase (oxidizing enzyme) was made from rabbit 

 muscle by the method of CoRi, Slein and Cori^. Partially purified isomerase containing a trace of 

 a-glycerophosphate dehydrogenase but free of aldolase and phosphoglyceraldehyde dehydrogenase was 

 prepared from rabbit muscle by the method of Meyerhof and Beck^". 



Monomagnesium fructose- 1,6-diphosphate (HDP) was purchased from the Schwarz Laboratories, 

 New York, N.Y. ; the dioxane addition compound of i-bromide 3-phosphoglyceraldehyde from the 

 Concord Laboratories, Cambridge, Massachusetts; and the diphosphopyridine nucleotide (DPN), 

 95% purity, from Nutritional Biochemicals, Cleveland, Ohio. Reduced DPN was prepared by the 

 method given in "Biochemical Preparations "^1. 



Methods 



The aldolase assay method employed was essentially that described by Warburg and Chris- 

 tian*. Each test was run in a cuvette containing: 0.75 ml HDP, pH 7.4 (50 mg/ml) ; 0.3 ml glycine 

 (20 mg/ml); 0.3 ml Na arsenate (54 mg/ml); 0.15 ml DPN, pH 7.4 (10 mg/ml); 1.2 ml cysteine- HCl, 

 pH 7.4 (7 mg/ml) ; and o.oi ml crystalline muscle oxidizing enzyme (25 mg/ml). Five minutes incuba- 

 tion time was allowed for activation of the muscle oxidizing enzyme by the cysteine before addition 

 of the sample to be assayed. The amount of sample used, o.i or 0.2 ml, effected azJ E 1 cm/34om/< of 0.050 

 to 0.500 in 10 minutes at room temperature {ca. 25° C). The aldolase activity of the sample is given 

 by W = A\n I II^jhovLV, corrected for dilution. Since E i cm/340 m/< as obtained in the Beckman spectro- 

 photometer is based on logarithms to the base 10, the readings are multiplied by the factor 2.3 to 

 convert to natural logarithms. One ml of a fluid with a H^ of i is considered to contain one aldolase 

 unit capable of splitting 0.08 fi moles HDP per hour. 



Isomerase determination followed, in general, the method described by Warburg and Chris- 

 tian*. A 0.03 ml sample to be assayed, or a dilution thereof, was incubated at room temperature with 

 2 ml of a solution of the dioxane addition compound of phosphoglyceraldehyde-i-Br, containing 5 mg 

 of the compound in 7 ml of o.oi M phosphate buffer with a final pH of 7.5. After 10 minutes the reac- 

 tion was stopped by the addition of 0.15 ml of A'' HCl and the mixture allowed to stand for five 

 minutes to destroy the isomerase. The amount of phosphoglyceraldehyde remaining unconverted was 

 determined enzymatically. i.o ml of o.i M pyrophosphate buffer, pH 7.5; 0.2 ml DPN (10 mg/ml); 

 0.3 ml Na arsenate (54 mg/ml) ; 0.5 ml HjO; and i.o ml of the neutralized isomerase test reaction mix- 

 ture were put into a cuvette. After the E 1 cm/340 m/< of this mixture was noted, 0.05 ml of an oxidizmg 

 enzyme solution, pH 7.4, (10 mg enzyme plus 3ooy cysteine • HCl, per ml) were added and the reaction 

 allowed to go to completion at room temperature (8-10 minutes). A 2 ml ahquot of the original phos- 

 phoglyceraldehyde solution plus 0.03 ml of water in place of the sample for test was assayed in a 

 similar manner to give the amount of substrate originally present. We arbitrarily designate the per- 

 centage of phosphoglyceraldehyde converted to dihydroxyacetone phosphate multiplied by 100 as the 

 isomerase unitage of the sample assayed. Amounts or dilutions of the sample were chosen which 

 converted 20 to 50% of the total phosphoglyceraldehyde in ten minutes. All results are expressed 

 as isomerase units in 0.03 ml of the original sample. On this basis, 0.03 ml of a blood plasma or ascitic 

 fluid with an isomerase activity of unity would convert 0.07 n moles of phosphoglyceraldehyde to 

 dihydroxyacetone phosphate in 10 minutes. 



a-Glyccrophosphate dehydrogenase was determined by the decrease in light absorption [A log 

 Jp//) after 3 1/2 minutes at 340 mn for i cm light path at 25° C of 3.1 ml of a solution containing, in 

 addition to the test sample, 200 y of reduced DPN, 30 mg of HDP, 1200 units of crystalline aldolase 

 and 50,000 units of isomerase. In carrying out the te§t, all reagents with the exception of the HDP 

 were brought to a volume of 2.7 ml in o.oi M phosphate buffer, pH 7.5, in a cuvette and read in a 

 Beckman spectrophotometer at 340 m//. Then, 0.4 ml of HDP .solution (30 mg HDP) at pH 7.5 were 

 added and, after 3 Yi minutes, a reading was again taken. By the use of large amounts of HDP, aldolase, 

 and isomerase, a non-limiting concentration of dihydroxyacetone phosphate was assured as a substrate 

 for reduction to a-glyccrophosphate by the reduced Dl^N under the influence of the dehydrogenase in 

 the added test sample. The -AEicm /340 m/< in 3 y, minutes was directly proportional to the volume of 

 test solution added provided the A E was not greater than 0.150. For convenience of graphical re- 

 presentation, all a-glycerophosphate dehydrogena.se activities are expressed on the basis of the J E 

 per 10 minutes per ml of biological fluid. 



References p. lyi. 



