Abstract
Abstract We describe a purification scheme for human erythrocyte pyruvate kinase. Following two ammonium sulfate fractionations, batchwise absorption on CM-cellulose, chromatography on CM-cellulose with fructose 1,6-diphosphate elution, and chromatography on DEAE-cellulose, a 30,000-fold purification was achieved with a yield of 8% from the original hemolysate. Antiserum against partially purified pyruvate kinase was prepared in rabbits and used to follow the subsequent purification of the enzyme. Catalase and immunoglobulin G were identified by immunodiffusion as components of the impure preparation. Isoelectric focusing of partially purified pyruvate kinase gave a single activity peak with an isoelectric point of 7.1 at 4°. The final product was homogeneous as judged by polyacrylamide disc gel electrophoresis and immunodiffusion. No free NH2-terminal amino acids could be detected. Sedimentation velocity analysis gave a major peak and a faster migrating, minor peak corresponding to 5% of the total. We suggest that the minor peak is due to aggregation of the major component. From sedimentation equilibrium analysis a molecular weight of 225,400 was obtained for the purified pyruvate kinase. The amino acid composition is presented.
Highlights
MethodsMaterials-Chemicals and reagents were purchased from the following sources: NADH, disodium ADP, the tetrasodium salt of n-fructose 1,6-diphosphate, Grade II, and crystallized bovine serum albumin from Sigma; the crystalline tricyclohexylammonium salt of phosphoenolpyruvate, Grade A, and rabbit muscle lactic dehydrogenase, Grade A, with a specific activity of608 i.u. per mg from Calbiochem; DEAE-cellulose (WhatmanDE-52) and CM-cellulose (Whatman CM-52) from Reeve-Angel; Sephadex G-25 coarse from Pharmacia; special enzyme grade, ultrapure ammonium sulfate from Mann; dansylr chloride fromPierce; previously coated silica gel thin layer sheets (Ql) fromQuantum; carrier ampholytes for isoelectric focusing from LKB; human y-globulin from the American Red Cross; completeFreund’s adjuvant from Difco; goat anti-human immunoglobulins from Hyland
We describe a purification schemefor human erythrocyte pyruvate kinase
Isoelectric focusing of partially purified pyruvate kinase gave a single activity peak with an isoelectric point of 7.1 at 4’
Summary
Materials-Chemicals and reagents were purchased from the following sources: NADH, disodium ADP, the tetrasodium salt of n-fructose 1,6-diphosphate, Grade II, and crystallized bovine serum albumin from Sigma; the crystalline tricyclohexylammonium salt of phosphoenolpyruvate, Grade A, and rabbit muscle lactic dehydrogenase, Grade A, with a specific activity of608 i.u. per mg from Calbiochem; DEAE-cellulose (WhatmanDE-52) and CM-cellulose (Whatman CM-52) from Reeve-Angel; Sephadex G-25 coarse from Pharmacia; special enzyme grade, ultrapure ammonium sulfate from Mann; dansylr chloride fromPierce; previously coated silica gel thin layer sheets (Ql) fromQuantum; carrier ampholytes for isoelectric focusing from LKB; human y-globulin from the American Red Cross; completeFreund’s adjuvant from Difco; goat anti-human immunoglobulins from Hyland. Materials-Chemicals and reagents were purchased from the following sources: NADH, disodium ADP, the tetrasodium salt of n-fructose 1,6-diphosphate, Grade II, and crystallized bovine serum albumin from Sigma; the crystalline tricyclohexylammonium salt of phosphoenolpyruvate, Grade A, and rabbit muscle lactic dehydrogenase, Grade A, with a specific activity of. 608 i.u. per mg from Calbiochem; DEAE-cellulose DE-52) and CM-cellulose (Whatman CM-52) from Reeve-Angel; Sephadex G-25 coarse from Pharmacia; special enzyme grade, ultrapure ammonium sulfate from Mann; dansylr chloride from. Freund’s adjuvant from Difco; goat anti-human immunoglobulins from Hyland. Anti-human IgG was prepared by immunizing rabbits with purified human myeloma IgG [12]. Reference standard dansyl amino acids were prepared by the method of Boulton and Bush [13]. Thrombocytes were prepared from fresh human blood [14]
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