Volume 13, number 1
MOLECULAR • CELLULAR BIOCHEMISTRY
October 30, 1976
INTERCONVERSION P H E N O M E N A BETWEEN TWO KINETIC FORMS OF CLASS A P Y R U V A T E KINASE FROM EHRLICH ASCITES TUMOR CELLS Juan E. F E L [ U * and Alberto SOLS
Instituto de Enzimologfa del Conse]o Superior de Investigaciones Cientfficas, Facultad de Medicina de la Universidad Aut6noma, Madrid-34, Spain (Received April 20, 1976)
Summary 1. The regulatory properties of two interconvertible kinetic forms of class A pyruvate kinase from Ehrlich ascites tumor cells have been studied with a partially purified enzyme preparation free of interfering enzymatic activities. 2. The hyperbolic form shows MichaelisMenten kinetics for P-pyruvatet, with high affinity for this substrate and low affinity for the inhibitory amino acids alanine and phenylalanine. The sigmoidal form displays positive cooperativity respect to P-pyruvate (n = 1.4), with lower affinity for this substrate and higher affinity for the inhibitory amino acids. 3. The equilibrium between the hyperbolic and the sigmoidal forms of the enzyme is affected by substrates and effectors. P-pyruvate, A D P and Fru-P2 shift the equilibrium to the hyperbolic form while ATP, alanine and phenylalanine stabilize the sigmoidal form. 4. Effector metabolites affect the molecular weight of the protein, acting on an equilibrium between dimers and tetramers. P-pyruvate and A D P associate the enzyme to a tetramer while ATP, alanine and phenylalanine favor the occurrence as a dimer. The positive modifier Fru-P2 did not associate the enzyme to the tetramer, even at 1 mM concentration. * Present address: Laboratoire de Chimie Physiologique, Facult6 de M6decine, Universit6 Catholique de Louvain, Avenue Hippocrate, 75 B-1200 Brussels t Abbreviations. DTE, dithioerythritol; Fru-P2, fructose 1,6bis-phosphate; P-pyruvate, phosphoenolpyruvate.
5. A tentative molecular model for pyruvate kinase A on the basis of the kinetic and aggregation interconversion is proposed.
Introduction Three major types of pyruvate kinases have been identified in mammalian tissues by electrophoretic 1'2 and immunological techniques 3. Work in this laboratory 4 has led to the identification of three classes of isozymes with qualitative differences in regulatory properties: the well known classes L (liver) and M (muscle) and a regulable new one which was designated as class A, because it was first observed in adipose tissue 5"6. This class A pyruvate kinase appears to be the same that the one designated in other laboratories as M2-PK 3, PK-III 7, and P K - K 8, and is the more widely distributed in tissues. Recently we reported 9 the characterization of the pyruvate kinase from Ehrlich ascites tumor cells as class A and its occurrence in two interconvertible forms with different regulatory properties that were designated as hyperbolic and sigmoidal forms from their saturation curves for P-pyruvate. Now we have purified this pyruvate kinase until elimination of interfering enzymes. With this preparation we have carried out a detailed study of the kinetic properties of the two interconvertible forms, of the interconversion process and the influence of the kinetic effectors on the degree of association of the enzyme, which can occur as dimers or
Dr. W. Junk b.v. Publishers - The Hague, The Netherlands
31
tetramers. A tentative molecular model is proposed to integrate the interconversions involving changes in both kinetic properties and quaternary structure.
Materials and Methods
Ascites tumor cells A hypertriploid strain of Ehrlich ascites carcinoma 9 was grown in 2 month old Swiss female mice, and harvested around the seventh day after inoculation. Cells were withdrawn from the sacrified animal, washed in an isotonic solution (140 mM NaC1 and 5 mM Tris-HC1, pH7.4) and centrifuged at low speed at 2-4 °C. When red cells contamination was apparent, the packed cells were resuspended in a hypotonic solution (40 mM NaC1 and 5 mM Tris-HC1, pH7.4) for 60 seconds, in order to lyse the erythrocytes, after which the cells were returned to isotonicity and centrifuged. This process was repeated two or three times. Contamination of our ascites cell suspensions by red cells was less than 5 per cent as estimated with a Neubauer chamber.
Preparation of extracts Washed cells were suspended in 3 volumes of the extraction medium (10 mM Tris-HC1, pH7.4, and 0.5 mM DTE), placed in a propylene centrifuge tube and frozen and thawed twice in liquid nitrogen. The resulting homogenate was centrifuged at 30,000 x g for 1 hour at 2-4 °C and the pellet discarded.
Purification of pyruvate kinase Pyruvate kinase was partially purified by ammonium sulfate fractionation of the extracts between 6 0 - 8 0 % saturation. The precipitated material was collected and dissolved in a minimal volume of solution A (10 mM Tris-HC1, pH7.4, 75 mM KC1, and 0.5 mM DTE) and passed through a Sephadex G-25 column (25 cm length and 2 cm diameter), equilibrated with the same solution. The eluted fractions with pyruvate kinase activity were pooled together, and P-pyruvate and Fru-P2 were added to a final concentration of 0.1 mM each. The sample of pyruvate k i n a s e t r e a t e d in this manner, in a volume smaller than 8 ml, was applied to a Sephadex G-200 column of 120 cm of length and 2 cm diameter equilibrated with solution A 2
including P-pyruvate and Fru-P2, at the above mentioned concentration. The eluted fractions from this last column with pyruvate kinase activity, were concentrated in an Amicon ultrafiltration cell, using a Diaflon PM30 membrane. The specific activity of this partially purified pyruvate kinase was 35 U/mg protein. The 50 times purified preparation was essentially free of the interfering enzymatic activities: aldolase (
MOLECULAR • CELLULAR BIOCHEMISTRY
October 30, 1976
INTERCONVERSION P H E N O M E N A BETWEEN TWO KINETIC FORMS OF CLASS A P Y R U V A T E KINASE FROM EHRLICH ASCITES TUMOR CELLS Juan E. F E L [ U * and Alberto SOLS
Instituto de Enzimologfa del Conse]o Superior de Investigaciones Cientfficas, Facultad de Medicina de la Universidad Aut6noma, Madrid-34, Spain (Received April 20, 1976)
Summary 1. The regulatory properties of two interconvertible kinetic forms of class A pyruvate kinase from Ehrlich ascites tumor cells have been studied with a partially purified enzyme preparation free of interfering enzymatic activities. 2. The hyperbolic form shows MichaelisMenten kinetics for P-pyruvatet, with high affinity for this substrate and low affinity for the inhibitory amino acids alanine and phenylalanine. The sigmoidal form displays positive cooperativity respect to P-pyruvate (n = 1.4), with lower affinity for this substrate and higher affinity for the inhibitory amino acids. 3. The equilibrium between the hyperbolic and the sigmoidal forms of the enzyme is affected by substrates and effectors. P-pyruvate, A D P and Fru-P2 shift the equilibrium to the hyperbolic form while ATP, alanine and phenylalanine stabilize the sigmoidal form. 4. Effector metabolites affect the molecular weight of the protein, acting on an equilibrium between dimers and tetramers. P-pyruvate and A D P associate the enzyme to a tetramer while ATP, alanine and phenylalanine favor the occurrence as a dimer. The positive modifier Fru-P2 did not associate the enzyme to the tetramer, even at 1 mM concentration. * Present address: Laboratoire de Chimie Physiologique, Facult6 de M6decine, Universit6 Catholique de Louvain, Avenue Hippocrate, 75 B-1200 Brussels t Abbreviations. DTE, dithioerythritol; Fru-P2, fructose 1,6bis-phosphate; P-pyruvate, phosphoenolpyruvate.
5. A tentative molecular model for pyruvate kinase A on the basis of the kinetic and aggregation interconversion is proposed.
Introduction Three major types of pyruvate kinases have been identified in mammalian tissues by electrophoretic 1'2 and immunological techniques 3. Work in this laboratory 4 has led to the identification of three classes of isozymes with qualitative differences in regulatory properties: the well known classes L (liver) and M (muscle) and a regulable new one which was designated as class A, because it was first observed in adipose tissue 5"6. This class A pyruvate kinase appears to be the same that the one designated in other laboratories as M2-PK 3, PK-III 7, and P K - K 8, and is the more widely distributed in tissues. Recently we reported 9 the characterization of the pyruvate kinase from Ehrlich ascites tumor cells as class A and its occurrence in two interconvertible forms with different regulatory properties that were designated as hyperbolic and sigmoidal forms from their saturation curves for P-pyruvate. Now we have purified this pyruvate kinase until elimination of interfering enzymes. With this preparation we have carried out a detailed study of the kinetic properties of the two interconvertible forms, of the interconversion process and the influence of the kinetic effectors on the degree of association of the enzyme, which can occur as dimers or
Dr. W. Junk b.v. Publishers - The Hague, The Netherlands
31
tetramers. A tentative molecular model is proposed to integrate the interconversions involving changes in both kinetic properties and quaternary structure.
Materials and Methods
Ascites tumor cells A hypertriploid strain of Ehrlich ascites carcinoma 9 was grown in 2 month old Swiss female mice, and harvested around the seventh day after inoculation. Cells were withdrawn from the sacrified animal, washed in an isotonic solution (140 mM NaC1 and 5 mM Tris-HC1, pH7.4) and centrifuged at low speed at 2-4 °C. When red cells contamination was apparent, the packed cells were resuspended in a hypotonic solution (40 mM NaC1 and 5 mM Tris-HC1, pH7.4) for 60 seconds, in order to lyse the erythrocytes, after which the cells were returned to isotonicity and centrifuged. This process was repeated two or three times. Contamination of our ascites cell suspensions by red cells was less than 5 per cent as estimated with a Neubauer chamber.
Preparation of extracts Washed cells were suspended in 3 volumes of the extraction medium (10 mM Tris-HC1, pH7.4, and 0.5 mM DTE), placed in a propylene centrifuge tube and frozen and thawed twice in liquid nitrogen. The resulting homogenate was centrifuged at 30,000 x g for 1 hour at 2-4 °C and the pellet discarded.
Purification of pyruvate kinase Pyruvate kinase was partially purified by ammonium sulfate fractionation of the extracts between 6 0 - 8 0 % saturation. The precipitated material was collected and dissolved in a minimal volume of solution A (10 mM Tris-HC1, pH7.4, 75 mM KC1, and 0.5 mM DTE) and passed through a Sephadex G-25 column (25 cm length and 2 cm diameter), equilibrated with the same solution. The eluted fractions with pyruvate kinase activity were pooled together, and P-pyruvate and Fru-P2 were added to a final concentration of 0.1 mM each. The sample of pyruvate k i n a s e t r e a t e d in this manner, in a volume smaller than 8 ml, was applied to a Sephadex G-200 column of 120 cm of length and 2 cm diameter equilibrated with solution A 2
including P-pyruvate and Fru-P2, at the above mentioned concentration. The eluted fractions from this last column with pyruvate kinase activity, were concentrated in an Amicon ultrafiltration cell, using a Diaflon PM30 membrane. The specific activity of this partially purified pyruvate kinase was 35 U/mg protein. The 50 times purified preparation was essentially free of the interfering enzymatic activities: aldolase (
