Human erythrocyte pyruvate kinase. Total purification and evidence for its antigenic identity with L-type enzyme.
Erythrocyte pyruvate kinase (ATP:pyruvate 2-0-phosphotransferase, EC 2.7.1.40) has been purified 40 000 times from human erythrocytes, according to an original method. The whole purification procedure included toluene extraction, ammonium sulphate fractionation, DEAE-Sephadex batchwise chromatography and affinity chromatography on a Dextran Blue-Sepharose column with specific elution by fructose 1,6-diphosphate. The final preparation had specific activity of 290 I.U./mg of proteins and the overall yield was about 30%. Pyruvate kinase showed only one protein band as judged by sodium dodecyl sulphate acrylamide gel electrophoresis. Pure enzyme was injected into rabbits and monospecific antiserum was obtained able to neutralize, per ml, 150 I.U. of erythocyte-type pyruvate kinase as well as of L-type enzyme. L-type and erythrocyte-type pyruvate kinases showed reactions of complete identity when tested in immunodiffusion against anti-erythrocyte type pyruvate kinase sera; in all cases a single precipitation line could be detected. L-type pyruvate kinase when mixed with anti-erythocyte pyruvate kinase serum suppressed all ability of that antiserum to react immunological with erythocyte enzyme. Finally the microcomplement fixation curves using anti-erythrocyte pyruvate kinase serum were identical for erythrocyte and L-type enzymes. From these results it appeared that no antigenic difference between L-type and erythocyte enzyme could be detected. Consequently the most likely hypothesis is that both these enzymes are coded by  the same single gene, the slight electrophoretic differences between them being due to post-synthetic tissue-specific changes.
D000941:Antigens; D003168:Complement Fixation Tests; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D005779:Immunodiffusion; D008722:Methods; D011770:Pyruvate Kinase / Q000276:immunology / Q000302:isolation & purification*
D000941:Antigens; D011770:Pyruvate Kinase