L-type pyruvate kinase from human liver. Purification by double affinity elution, electrofocusing and immunological studies.
L-type pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) was highly purified from adult human liver. This purification included ammonium sulphate fractionation, DEAE-Sephadex batchwise absorption and two CM-Sephadex chromatographies with selective elution by ligands; in the former chromatography pyruvate kinase was eluted by ATP, in the latter one by phosphoenolpyruvate and fructose 1,6-diphosphate. The last step of the purification procedure involved a hydroxyapatite column chromatography. This purification procedure allowed us to obtain 3.6 mg of protein with a specific activity 190 I.U./mg, i.e. a 1200-fold purification with an overall yield of about 8%. This preparation was homogenous as judged by immunodiffusion, acrylamide and sodium dodecyl sulphate acrylamide gel electrophoresis. Anti L-type pyruvate kinase antibodies were obtained from rabbits and the antigenic properties of L-type pyruvate kinase were studied. The enzyme appeared to be a tetramer (molecular weight 220 000-240 000) with subunits of similar molecular weight about 60 000). Two interconvertible major forms were found by isoelectrofocusing in a sucrose gradient and in an acrylamide slab gel: one had an isoelectric point of 5.85 +/- 0.09 and was the major enzymatic form after incubation with fructose 1,6-diphosphate or high concentrations or SH reagents. The other form (isoelectric point 6.28 +/- 0.03) was the major form of L-type pyruvate kinase in liver crude extract, and after incubation of purified enzyme with a proteic fraction isolated from liver extract by ammonium sulphate precipitation.
D002846:Chromatography, Affinity; D006801:Humans; D005779:Immunodiffusion; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D011770:Pyruvate Kinase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D007527:Isoenzymes; D046911:Macromolecular Substances; D011770:Pyruvate Kinase