568th MEETING, ABERDEEN
747
phorylase a by O.lm~-AM.P:the much greater activation that they obtained is probably due to the much lower glycogen concentrations used in their studies. Under the conditions of the experiments reported in Fig. 1 , the specific activity of phosphorylase b is about one-fifth that of phosphorylase a. This is largely accounted for by the low affinity for AMP of phosphorylase b : 0.1 mM-AMP gives maximal activation of phosphorylase a, but about O . ~ ~ M - A M is required P to give 50% of full activation of phosphorylase b. Observations on the inhibitory effects of ATP and glucose 6-phosphate are shown in Table 1. It is not feasible to use the changing-pH assay with ATPpresent at concentrations much above 1m, but Table 1 shows clearly that both substances will strongly inhibit the AMP-dependent activity of phosphorylase b at the concentrations at which they occur in resting muscle. The kinetics of inhibition by both substances appear complex, showing some co-operativity. Together these inhibitors neither act synergistically nor d o they compete, since, as is shown in Table 1, the observed activity in the presence of mixtures of the two is close to that predicted for their independent action over most of the range of inhibitor concentrations studied. When similar studies were carried out with glucose 6-phosphate and ADP, the inhibition by one inhibitor was less marked when the second was present. We are grateful to Dr. Philip Cohen for advice and encouragement in this work. J. M. S. holds a University of Malaya studentship. Cori, C. F., Cori, G. T. & Green, A. A. (1943) J. Biol. Chem. 151, 39-55 Hesrin, S. (1949) J. Bid. Chem. 179, 943-949 Karpatkin, S., Helmreich, E. & Cori, C. F. (1974) J. Biol. Chem. 239, 3139-3146 Lowry, 0. H., Schulz, D. W. & Passonneau, J. V. (1964) J. Biol. Chem. 239, 1947-1953 Morgan, H. E. & Parmeggiani, A. (1964) J. Biol. Chem. 239,2440-2445 Palter, K. & Lukton, A. (1973) Anal. Biochem. 53, 613-623 Taylor, C., Cox, A. J., Kernohan, J. C. & Cohen, P. (1975) Eur. J. Biochem. 51,105-1 15
The Free Radical in Ribonucleotide Reductase from Escherichiu coli BRITT-MARIE SJOBERG* and ASTRID GRASLUNDt *Medical Nobel Institute, Department of Biochemistry I, Karolinska Institute, S-104 01 Stockholm, Sweden, and tDepartment of Biophysics, University of Stockholm, Arrhenius Laboratory, S-10405 Stockholm, Sweden
Ribonucleotide reductase from Escherichia coli consists of two non-identical subunits, protein B1 and protein B2, both needed for enzymic activity (Brown et al., 1969). Protein B2 contains two atoms of iron and an organic free radical in its active form (Atkin et al., 1973; Ehrenberg & Reichard, 1972). The limited amounts of ribonucleotide reductase in E. coli cells have hitherto obstructed detailed studies on the free radical of the enzyme. Recently, an overproducing strain was constructed (0.Karlstrom, J. Collins, W. Lindenmaier, B.-M. Sjoberg & S. Eriksson, unpublished work). It has enabled a careful analysis of the nature of this organic free radical. The strain that we used, KK546, is lysogenic for a defective, heat-inducible hybrid 3, bacteriophage, which in its DNA also contains the structural genes for both subunits of ribonucleotide reductase. Strain KK546 is normally cultured at 33°C. On heat induction of the strain at 42°C an effective transcription over the A-phage genome is mediated. Production of phage-A-specific proteins is then allowed to take place at 37°C for a few generations, after which time the cells are harvested by centrifugation at #"C. Ribonucleotide reductase constitutes 5-10% of the total protein content ofsuchcells, avalue approx. 20 times that normally found in E. coli strains. Owing to this high content of proteins B1 and B2 in heat-induced strain KK546, the free radical can bestudieddirectly
Vol. 5
748
BIOCHEMICAL SOCIETY TRANSACTIONS
in frozen thick suspensions of whole cells, making any purification of the protein B2 unnecessary. Isotope-substitution experiments were used to see if the general features of the doublet e.p.r. (electron-paramagnetic-resonance) spectrum of protein B2 could be influenced. Spectra were usually recorded at 77 K in cell suspensions from 60ml cultures of strain KK546. A striking change of the normal doublet spectrum ('H-spectrum) to a narrower singlet line ("H-spectrum) was observed when cells were grown in 93 %-"H-labelled medium instead of the normal ('H) medium. The spectral features are consistent with a >C-H free-radical fragment in the protein. We also investigated if the free radical of protein B2 could be localized to a specific amino acid residue. In this case cultures were grown in 93 %-'H-labelled medium as described above and different non-deuterated amino acids were also included in excess. Only in the presence of tyirosine was the singlet 'H-spectrum reversed to the normal "H-spectrum again. Finally, we grew cells in normal ('H) medium with the inclusion of specifically "H-labelled tyrosines and found that the "H-spectrum was obtained in the presence of [BB-2H2]tyrosine. These experiments conclusively demonstrate that the unpaired electrons which give rise to the e.p.r. signal of protein B2 are localized to the benzyl carbon atom of a tyrosine residue with hyperfine coupling to one of the benzyl protons. Purified protein B2 has previously (Brown et al., 1969) always been obtained in a partly inactivated form, which needed re-activation (Atkin et al., 1973). This procedure involved removal of iron from the protein, which also destroyed the free radical, and a subsequent addition of Fe(II), which reconstituted both enzymic activity and the e.p.r. signal. The high concentration of ribonucleotide reductase in heat-induced strain KK546 permitted a new milder, purification procedure for both subunits (S. Eriksson, B.-M. Sjoberg, S. Hahne & 0. Karsltrom, unpublished work), one great advantage being that the enzymic activity of protein B2 was fully preserved throughout the purification. Such preparations contained one free radical and two atoms of iron per proteinB2 molecule, and it was also shown that the radical content did not decrease during the purification procedure. The relation between the formation and properties of the free radical and the iron remains to be elucidated, as well as the role of the free radical in the substrate reaction. Atkin, C. L., Thelander, L., Fleichard, P. & Lang, G. (1973) J. Biol. Chem. 248,7464-7472 Brown, N. C., Canellakis, 2:. N., Lundin, B., Reichard, P. & Thelander, L. (1969) Eur. J. Biochern. 9,561-573
Ehrenberg, A. & Reichard, 1'. (1972) J. Biol. Chem. 247, 3485-3488
A Method to Detect Protein-Protein Interactions LARS BACKMAN, VITHALDAS SHANBAG and GOTE JOHANSSON Department of Biochemistry, Uniuersity of Umed, 9 9 0 1 87 Umed, Sweden
Several functional complexes of enzymes that catalyse a sequence of reactions are known, for example the a-0x0 acid dehydrogenase complexes (Reed & Cox, 1966). Methodological problems have probably limited the study of complexes thus far to investigation of relatively strong complexes. Systems involving weak interactions between enzymes could also be of great physiological iimportance. A simple equilibrium method to detect protein-protein interactions, proposed by Bethuene and Kegeles (1961), is to study the mutual influence of the proteins on their respectivecountercurrent distribution in liquid-liquid biphasic systems. Aqueous biphasic systems developed by Albertsson (1971) are very suitable for this purpose. In a biphasic system each protein is characterized by a partition coefficient,K, defined as c,/q where c,, and cI are the concentrations of the partitioned protein of upper and 1977
747
phorylase a by O.lm~-AM.P:the much greater activation that they obtained is probably due to the much lower glycogen concentrations used in their studies. Under the conditions of the experiments reported in Fig. 1 , the specific activity of phosphorylase b is about one-fifth that of phosphorylase a. This is largely accounted for by the low affinity for AMP of phosphorylase b : 0.1 mM-AMP gives maximal activation of phosphorylase a, but about O . ~ ~ M - A M is required P to give 50% of full activation of phosphorylase b. Observations on the inhibitory effects of ATP and glucose 6-phosphate are shown in Table 1. It is not feasible to use the changing-pH assay with ATPpresent at concentrations much above 1m, but Table 1 shows clearly that both substances will strongly inhibit the AMP-dependent activity of phosphorylase b at the concentrations at which they occur in resting muscle. The kinetics of inhibition by both substances appear complex, showing some co-operativity. Together these inhibitors neither act synergistically nor d o they compete, since, as is shown in Table 1, the observed activity in the presence of mixtures of the two is close to that predicted for their independent action over most of the range of inhibitor concentrations studied. When similar studies were carried out with glucose 6-phosphate and ADP, the inhibition by one inhibitor was less marked when the second was present. We are grateful to Dr. Philip Cohen for advice and encouragement in this work. J. M. S. holds a University of Malaya studentship. Cori, C. F., Cori, G. T. & Green, A. A. (1943) J. Biol. Chem. 151, 39-55 Hesrin, S. (1949) J. Bid. Chem. 179, 943-949 Karpatkin, S., Helmreich, E. & Cori, C. F. (1974) J. Biol. Chem. 239, 3139-3146 Lowry, 0. H., Schulz, D. W. & Passonneau, J. V. (1964) J. Biol. Chem. 239, 1947-1953 Morgan, H. E. & Parmeggiani, A. (1964) J. Biol. Chem. 239,2440-2445 Palter, K. & Lukton, A. (1973) Anal. Biochem. 53, 613-623 Taylor, C., Cox, A. J., Kernohan, J. C. & Cohen, P. (1975) Eur. J. Biochem. 51,105-1 15
The Free Radical in Ribonucleotide Reductase from Escherichiu coli BRITT-MARIE SJOBERG* and ASTRID GRASLUNDt *Medical Nobel Institute, Department of Biochemistry I, Karolinska Institute, S-104 01 Stockholm, Sweden, and tDepartment of Biophysics, University of Stockholm, Arrhenius Laboratory, S-10405 Stockholm, Sweden
Ribonucleotide reductase from Escherichia coli consists of two non-identical subunits, protein B1 and protein B2, both needed for enzymic activity (Brown et al., 1969). Protein B2 contains two atoms of iron and an organic free radical in its active form (Atkin et al., 1973; Ehrenberg & Reichard, 1972). The limited amounts of ribonucleotide reductase in E. coli cells have hitherto obstructed detailed studies on the free radical of the enzyme. Recently, an overproducing strain was constructed (0.Karlstrom, J. Collins, W. Lindenmaier, B.-M. Sjoberg & S. Eriksson, unpublished work). It has enabled a careful analysis of the nature of this organic free radical. The strain that we used, KK546, is lysogenic for a defective, heat-inducible hybrid 3, bacteriophage, which in its DNA also contains the structural genes for both subunits of ribonucleotide reductase. Strain KK546 is normally cultured at 33°C. On heat induction of the strain at 42°C an effective transcription over the A-phage genome is mediated. Production of phage-A-specific proteins is then allowed to take place at 37°C for a few generations, after which time the cells are harvested by centrifugation at #"C. Ribonucleotide reductase constitutes 5-10% of the total protein content ofsuchcells, avalue approx. 20 times that normally found in E. coli strains. Owing to this high content of proteins B1 and B2 in heat-induced strain KK546, the free radical can bestudieddirectly
Vol. 5
748
BIOCHEMICAL SOCIETY TRANSACTIONS
in frozen thick suspensions of whole cells, making any purification of the protein B2 unnecessary. Isotope-substitution experiments were used to see if the general features of the doublet e.p.r. (electron-paramagnetic-resonance) spectrum of protein B2 could be influenced. Spectra were usually recorded at 77 K in cell suspensions from 60ml cultures of strain KK546. A striking change of the normal doublet spectrum ('H-spectrum) to a narrower singlet line ("H-spectrum) was observed when cells were grown in 93 %-"H-labelled medium instead of the normal ('H) medium. The spectral features are consistent with a >C-H free-radical fragment in the protein. We also investigated if the free radical of protein B2 could be localized to a specific amino acid residue. In this case cultures were grown in 93 %-'H-labelled medium as described above and different non-deuterated amino acids were also included in excess. Only in the presence of tyirosine was the singlet 'H-spectrum reversed to the normal "H-spectrum again. Finally, we grew cells in normal ('H) medium with the inclusion of specifically "H-labelled tyrosines and found that the "H-spectrum was obtained in the presence of [BB-2H2]tyrosine. These experiments conclusively demonstrate that the unpaired electrons which give rise to the e.p.r. signal of protein B2 are localized to the benzyl carbon atom of a tyrosine residue with hyperfine coupling to one of the benzyl protons. Purified protein B2 has previously (Brown et al., 1969) always been obtained in a partly inactivated form, which needed re-activation (Atkin et al., 1973). This procedure involved removal of iron from the protein, which also destroyed the free radical, and a subsequent addition of Fe(II), which reconstituted both enzymic activity and the e.p.r. signal. The high concentration of ribonucleotide reductase in heat-induced strain KK546 permitted a new milder, purification procedure for both subunits (S. Eriksson, B.-M. Sjoberg, S. Hahne & 0. Karsltrom, unpublished work), one great advantage being that the enzymic activity of protein B2 was fully preserved throughout the purification. Such preparations contained one free radical and two atoms of iron per proteinB2 molecule, and it was also shown that the radical content did not decrease during the purification procedure. The relation between the formation and properties of the free radical and the iron remains to be elucidated, as well as the role of the free radical in the substrate reaction. Atkin, C. L., Thelander, L., Fleichard, P. & Lang, G. (1973) J. Biol. Chem. 248,7464-7472 Brown, N. C., Canellakis, 2:. N., Lundin, B., Reichard, P. & Thelander, L. (1969) Eur. J. Biochern. 9,561-573
Ehrenberg, A. & Reichard, 1'. (1972) J. Biol. Chem. 247, 3485-3488
A Method to Detect Protein-Protein Interactions LARS BACKMAN, VITHALDAS SHANBAG and GOTE JOHANSSON Department of Biochemistry, Uniuersity of Umed, 9 9 0 1 87 Umed, Sweden
Several functional complexes of enzymes that catalyse a sequence of reactions are known, for example the a-0x0 acid dehydrogenase complexes (Reed & Cox, 1966). Methodological problems have probably limited the study of complexes thus far to investigation of relatively strong complexes. Systems involving weak interactions between enzymes could also be of great physiological iimportance. A simple equilibrium method to detect protein-protein interactions, proposed by Bethuene and Kegeles (1961), is to study the mutual influence of the proteins on their respectivecountercurrent distribution in liquid-liquid biphasic systems. Aqueous biphasic systems developed by Albertsson (1971) are very suitable for this purpose. In a biphasic system each protein is characterized by a partition coefficient,K, defined as c,/q where c,, and cI are the concentrations of the partitioned protein of upper and 1977
