On pages 131, 132, 136, and 137, the word conformational should be set off by quotation marks, rather than by parentheses.

Transcription

1 536 ERRATUM: HELMREICRIAND CORI PROC. N. A. S. ERRA TUM In the article entitled "The Role of Adenylic Acid in the Activation of Phosphorylase," by Ernst Helmreich and Carl F. Cori, which appeared in the January issue of volume 51 (1964), pp , the concentration of glycogen given in Tables 4 and 5 should be M X 10-4 instead of M X On pages 131, 132, 136, and 137, the word conformational should be set off by quotation marks, rather than by parentheses.

2 THE ROLE OF ADENYLIC ACID IN THE ACTIVATION OF PHOSPHORYLASE* BY ERNST HELMREICH AND CARL F. CORI DEPARTMENT OF BIOLOGICAL CHEMISTRY, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, MISSOURI Communicated November 12, 1963 The question of how 5'-AMIP' increases phosphorylase activity has long been a puzzle.2 The conversion of muscle phosphorylase a to b by a specific phosphatase results in an absolute dependence of the enzyme on 5'-AMP for activity.3 Measurements of light scattering indicate that the molecular dissociation which accompanies the a--b transformation of muscle phosphorylase is not reversed by 5'- AMP.4 Phosphorylase b differs from a by the absence of phosphate groups bound to serine residues, but potato phosphorylase which also lacks these phosphate groups does not require 5'-ANIP for activity.5 More surprising still is the observation that 5'-AMP can substitute for phosphohexapeptide residues6 split off from phosphorylase a by trypsin.7 Muscle phosphorylase has a specific binding site for 5'-AMP,5 but no evidence could be obtained that the nucleotide participates in the reaction catalyzed by the enzyme.9 From the temperature dependence of the phosphorylase a reaction in the presence and absence of 5'-AMP it was calculated that the binding of 5'-AMP to the enzyme lowered the energy of activation of the catalytic system.10 Monod et al.ii have recently summarized evidence which emphasizes the importance of (conformational) changes of enzymes for their catalytic activity and have used phosphorylase as a specific example. The present results indicate that inorganic P and glycogen are bound more tightly in the presence of 5'-AMP and that the concentration of the substrates in turn influences the affinity of the enzyme for 5'-AMP. These effects express themselves kinetically in large changes in Km under conditions where there is little change in Vmax. Experimental Procedure.-Most of the measurements of the phosphorylase reaction were carried out in the direction, glycogen + inorganic P - glucose-l-p. In the past it had been difficult to determine initial rates, because at ph 6.7 equilibrium is reached when only 20% of the inorganic P has disappeared. These difficulties were overcome in a coupled assay system containing phosphoglucomutase, glucose-6-p dehydrogenase, and TPN. The auxiliary enzymes were added in large excess, so that there was only a short lag period before linear rates, proportional to the phosphorylase concentration, were attained. The concentrations of phosphorylase were so chosen that the rate of the reaction could conveniently be followed by taking readings at 340 mu every 2-3 min up to 20 min. The temperature was kept constant at 240 by means of a jacketed cellholder and a circulating waterbath. Reactions were started by addition of phosphate or, in the case of phosphorylase b, by addition of 5'-AMP. No reaction was observed with phosphorylase b, unless 5'-AMP was added. The concentration of the reactants in 1 ml of reaction mixture in cuvettes of 1 cm light path was: Tris-acetate, 45 mm; EDTA-Na2, 2 mm; Mgacetate, 10 mm; 2-mercaptoethanol, 1 mm; TPN 0.6 mm; glucose, 1,6-diP12 5 X 10-8 M; phosphoglucomutase 10 jig; glucose-6-p dehydrogenase 2 jig; phosphorylase jig, ph 7.5. Enzymes: Crystalline muscle phosphoglucomutase and crystalline yeast glucose-6-p dehydrogenase were from Boehringer and Sons. Excess (NH4)2S04 was removed by dialysis. Phosphorylase b and a were prepared from frozen rabbit skeletal muscle The phosphorylase preparations were recrystallized at least 4 times. In order to ensure absence of nucleotides, the preparations were treated as follows. Crystals were redissolved in 1-2 ml of a 0.1 M glycerophosphate, 0.1 mm EDTA solution, ph 6.8 at 300 and layered on a pellet of repeatedly washed Dowex-l-X-8 acetate. After gentle mixing for 10 min, the resin was removed by centrifugation. 131

3 132 BIOCHEMISTRY: HELMREICH AND CORI PROC. N. A. S. The supernatant solution (1-2 ml) was added to 0.30 ml of a 30% Norit A suspension in water. After again mixing for 5 min, the mixture was centrifuged, and the supernatant solution treated once more with Norit. The ratio E260/E280 was 0.53 after Norit treatment. The enzyme solution was stored under toluene. Dilutions 1: 100-1:300 were made with a solution of 0.01 Al tris-acetate (ph 7), 0.1 mm EDTA, 0.1 mm 2-mercaptoethanol, and 1% egg albumin immediately preceding kinetic measurements. Dilute enzyme solutions were kept on ice, usually no longer than 1 day. For some experiments phosphorylase a was prepared enzymatically by incubating charcoal-treated phosphorylase b with a 100-fold purified rabbit muscle phosphorylase b kinase preparation'5 in the presence of 3 X 10-3 M ATP and 1 X 10-2 M Mg-acetate. These preparations were diluted 1: 22,500 for kinetic measurements. Glycogen: Rabbit liver glycogen was purified as follows: to 100 ml of 5% aqueous glycogen solution were added 200 mg Norit A, and the suspension was mixed for 10 min at room temperature. The Norit was removed by centrifugation or by filtration, and the treatment with Norit was repeated once or twice. The glycogen was then reprecipitated twice with alcohol and dried. It has been reported that commercial samples of glycogen contain 5'-AMP.l6 In order to test the effectiveness of the charcoal treatment in removing nucleotides, 1 X 10-5 M C14-labeled 5'- AMP was added to the glycogen solution. No radioactivity was recovered after Norit treatment. If there had remained 1 X 10-8 M C14-AMP in the glycogen after Norit treatment, it would have been detected. Materials: TPN-Na was a product of Boehringer and Sons, Germany; we are indebted to Dr. H. U. Bergmeyer for a sample of TPN-Na (Lot no ) which was stated to be free of 5'-AMP by enzymatic tests. We have also tested this TPN preparation in our assay system, using 0.36 jg charcoal-treated phosphorylase a. With 0.24 umoles TPN per ml, the rate was O.D.340 units X min-'. At 5 times this concentration the rate was O.D. units X min-'. With 0.24,moles TPN, but in the presence of 5 X 10-4 M 5'-AMP, the rate was Hence, concentrations of 5'-AMP which could activate phosphorylase a, were not detected in this TPN preparation. Rabbit liver glycogen was purchased from Mann Research Laboratories, Inc. 5'-AMP-8-C14, specific activity 1.5 mc per mmole, was obtained from Tracerlab, Inc. Dowex-1, 5'-AMP, ATP, sodium salts of glucose-i-p and glucose-6-p, Tris-acetate, and 2 times recrystallized egg albumin were purchased from the Sigma Chemical Co. EDTA-Na2 was a product of Geigy Chemical Co., and 2-mercaptoethanol of Eastman Kodak Co. Norit A was from Pfanstiehl Laboratories, Inc. The Norit was washed successively with HCl and then with water until neutral. All other chemicals were ACS grade reagents. Double distilled or deionized H20 was used throughout. Results.-Kinetic measurements with phosphorylase a: Very few values for the Km of inorganic P for phosphorylase are recorded in the literature.2 Preliminary experiments with the coupled assay system were carried out with phosphorylase a prepared by the action of kinase on phosphorylase b. Two such preparations gave Km values for inorganic P of 15 and 9 X 10-3 M (Fig. 1, curves I and II). On addition of 5'-AMP to one of these preparations, the Km value decreased to 5 X 10-3 M (Fig. 1, curve III). A similar value was obtained with phosphorylase a crystallized directly from rabbit muscle (Fig. 1, curve IV). The experiment was repeated with another preparation of phosphorylase a crystallized from rabbit muscle with similar results. On addition of 5'-AMP the Km for inorganic P decreased from 15 to 5 X 10-3 M (Table 2). The fact that the values of Vmax for the curves in Figure 1 are nearly the same would indicate that at high phosphate concentration 5'-AMP is not needed for activity. Conversely, the increasing stimulation by 5'-AMP with decreasing phosphate concentration would indicate that the enzyme becomes more and more dependent on 5'-AMP for activity. These observations suggested that (conformational) changes of the enzyme at low substrate concentration were counteracted by 5'-AMP. That 5'-AMP can effect a reversal in the case of enzyme in-

4 VOL. 51, 1964 BIOCHEMISTRY: HELMREICH AND CORI 133 activation by dilution is indicated by 600 the experiment in Table 1. It can be / seen that dilution of the enzyme with- l out protective protein resulted in a 40 / per cent loss of activity (a rate of versus 0.03 per min). Addition of 5'- / AMP restored activity, as shown by 420/ nearly equal rates whether or not the / dilution of the enzyme had been carried 360/ out with egg albumin as protective "/v / protein. Rate measurements with the / unprotected enzyme made 24 hr later 2 AlX miv. indicated that the enzyme, after the / rapid initial change, was relatively sta- 80 ble. It is also shown that egg albumin / added to the cuvette did not reactivate 120 / the enzyme. Other data in Table 1 relate to the 60A sensitivity of the enzyme to 5'-AMP. Based on the increment over the rate C 0'5 It 1.5 2'0 25 without 5'-AMP, a concentration of 1 I/ P X I0-3 X 10-6 M 5'-AMP gave gave about 1/2 FIG. 1.Km of inorganic P for phosphorylase. a. Curves I and II refer to two batches of maximal stimulation of activity. At phosphorylase a prepared from the same phos- 0.5 mm inorganic P as compared to 5 phorylase b stock solution with kinase, ATP, and Mg++ and tested without added 5'-AMP. ma\j, the rate without 5'-AMP had Curve III (0) refers to enzyme preparation II decreased 90 per cent and that with tested in the presence of 5 X 10-4 M 5'-AMP. Curve IV (U) refers to phosphorylase a crystal- 5'-AMP 57 per cent. Consequently, lized directly from rabbit muscle and tested in the stimulation by 5'-AMP becomes the presence of 5 X 10-4 M 5'-AMP, after appropriate dilution so as to give the same ormuch greater at the low substrate dinate intercept as the other curves. The concentration. Similar observations glycogen concentration was 0.1%. The rate v represents the linear part of the curve and is exwere made recently by Dr. 0. H. pressed as optical density change per minute. Lowry 17 Previous observations were made with a standard test system with 16 mm natural or synthetic glucose-i-p as substrate. Under these conditions most of the TABLE 1 ACTIVATION OF PHOSPHORYLASE a BY 5'-AMP --- Without Egg Albumin - - With Egg Albumin (0.1%) -_ Hr after % activity % activity dilution no AMP + AMP without AMP no AMP + AMP without AMP * (1 X 10-6M) "t (1 X 10-7 M) it (1 X 10-8 M) cc 0.022t * At 5 X 10-4 M inorganic P this value was 17, based on rates of and 0.018, without and with 5'-AMP respectively. t Egg albumin added to the cuvette. One part of a charcoal-treated phosphorylase a solution was diluted with egg albumin, as described under Experimental Procedure. Another part was diluted identically, except that egg albumin was omitted. Both samples were kept on ice. For enzymatic tests 0.1 ml aliquots containing 2.7 erg of enzyme were added to the cuvettes. Inorganic P, 5'-AMP, and glycogen were 5 X 10-3 M, 5 X 10-5 M, and 0.1%, respectively, except when stated otherwise. A TPN preparation shown to be free of 5'-AMP was used at a concentration of 0.20 mm. The rates are given es optical density changes per minute at 340 my.

5 134 BIOCHEMISTRY: HELMREICH AND CORI PROC. N. A. S. phosphorylase a preparations showed an activity without 5'-AMIP which was per cent of that in the presence of 5'-AMP. Recently, fractions of very high specific activity were obtained by gradient elution of phosphorylase a from DEAE cellulose columns. The peak fractions were not stimulated at all by 5'-AMP in the above test, or stimulated only 5-10 per cent. On aging, these preparations became increasingly responsive to stimulation by 5'-AMP.'5 Phosphorylase a preparations freshly made from phosphorylase b by kinase often show little stimulation by 5'-AMP (cf. curves II and III, Fig. 1). This applies also to phosphorylase formed in vivo. Thus, the phosphorylase present in diluted, charcoal-treated extracts of tetanized frog sartorius muscle often was stimulated no more than 5 per cent when 5'-AMP was added.'9 In de novo synthesis,20 polysaccharide chains are formed when the only reactants are phosphorylase a and glucose-i-p. Recently, Drs. Jllingworth and Brown kindly repeated this experiment, using Norit-treated phosphorylase a and chemically synthesized glucose-1-p. Formation of polysaccharide chains set in after the usual lag period. Addition of 5'-AMP greatly shortened the lag period. When the reaction became linear with time, the sample without 5'-AMP formed about 1.4 Mumoles inorganic P per ml per hr, as compared to 3.3.omoles for the sample with 5'-AMP. The same phosphorylase a preparation tested either with glucose-i-p (16 mm) or inorganic P (10 mm) in the presence of glycogen showed an activity of 68 and 62 per cent, respectively, of that obtained in the presence of 5'-AMP. These observations show that phosphorylase a has enzymatic activity in the absence of 5'-AMP. It then seems possible that the absolute requirement of phosphorylase b for 5'-AMP could be the result of a less rigid structure of catalytic site. The changes in molecular weight and in charges which are associated with the a-.~b transformation could be responsible for the differences between the two enzymes. Determination of Km with phosphorylase b: Preliminary data on the effect of 90 5'-AM\'IP on the velocity of the phos- 80 phorylase b reaction are shown in 70 /l C Figure 2 in the form of a double recip- 60 / / rocal plot. Within the range of 5'- AMP concentrations of 1 X 10-4 M /1V 50 - / /to 1 X 10-' M, the experimental 40 / points extrapolate to the ordinate, and 30 these were the concentrations chosen 20 /SSfor the final experiments. At lower I 0 concentrations the experimental points 0' ' do not yield an ordinate intercept. The I/AMP x 10-5 reason for this falling off in rate has FIG. 2.-Km of 5'-AMP for phosphorylase b. not been ascertained. It was also ob- The enzyme dilution was made with buffer served that the same enzyme solution containing glycogen so as to give a final concentration of 0.2%. Curves I and II refer on aging, as well as different preparato enzyme tested at concentrations of 1 X tions made from the same stock solu M and 1 X 10-2 M inorganic P, respectively. Km calculated from Curve I was tion, may give large variations in the 2.6 X 104 M. The portion of Curve II which Km value. For this reason it was intercepts the ordinate yields a Km value of 2.2 X 10-5 M. found necessary to complete all meas- its

6 VOL. 51, 1964 BIOCHEMISTRY: HELMREICH AND CORI 135 urements of a series on the same day and with the same enzyme solution. In the final series, 4 concentrations of inorganic P (1-10 X 10-3 M) were used at each of 4 concentrations of 5'-AMP (1.5-9 X 10-5 M), a total of 16 measurements of initial rate. From a plot of 1/v versus 1/S, depending on whether S represents the concentration of inorganic P or of 5'-AMP, one obtains the respective Km values. The data in Tables 2 and 3 represent such a series. As a measure of the consistency of the data, the 1/v versus 1/S plots were found to extrapolate to the same Vmax within close limits. Table 2 shows that the Km for inorganic P decreases progressively from 23 TABLE 2 Km OF ORTHOPHOSPHATE (P) AND Ki OF ARSENATE (As) AT DIFFERENT CONCENTRATIONS OF 5'-AMP AND 0.1% GLYCOGEN Phosphorylase b Phosphorylase a of 5'-AMP Km (P) Ki (As) Km (P) M X 1O05 M X 10-5 M X 10-3 M X to 1.5 X 10-3 M as the concentration of 5'-AMP is increased over its effective range. This result could be expressed by saying that the essential cofactor 5'-AMP has the effect of increasing the affinity of the enzyme for inorganic P. Further insight is provided by the experiments with arsenate (Table 2). Arsenate acts as a competitive inhibitor of phosphate. When the enzyme concentration is of the order of a few,ug per ml, the reaction leading to the formation of free glucose (presumably via glucose-l-arsenate) is too slow to be measured. Table 2 shows that arsenate has an affinity for the enzyme which is similar to that of phosphate and that the inhibitor constant (Ki) is influenced in a similar manner by the concentration of 5'-AMP, as is the Km of phosphate. In this case it is clearly the binding affinity of the competitor for the active site which is influenced by the concentration of 5'-AMP. The experiment in Figure 2 anticipates the results shown in greater detail in Table 3, namely, that the Km value for 5'-AMP decreases as the concentration of inorganic P is increased, without a significant change in Vax. Determinations of the Km of 5'-AMP with glucose-i-p as substrate are included in Table 3. At TABLE 3 Km OF 5'-AMP AND Ki OF ATP FOR PHOSPHORYLASE b AT DIFFERENT CONCENTRATIONS OF PHOSPHATE AND 0.1% GLYCOGEN of phosphate Km (S-AMP) Ki (ATP) Ratio Substrate M X 10-3 M X 10-5 M X 10-3 Ki/Km Orthophosphate "I Glucose-i-P * it IC * Values reported in the literatures

7 136 BIOCHEMISTRY: HELMREICH AND CORI PROC. N. A. S. high substrate concentrations the Km values for 5'-AMP approach the apparent dissociation constant of 6.6 X 10-5 M reported for phosphorylase b.8 Parmeggiani and Morgan 21 have reported that ATP inhibits phosphorylase b by competing with 5'-AMP. In Table 3 the inhibitor constant, K,, has been calculated at 3 different concentrations of inorganic P. The appropriate Km values for 5'-AMP at these phosphate concentrations were used in this calculation. From the relative constancy of the ratio, Ki/Km, it can be concluded that the concentration of substrate influences the binding affinity of the enzyme for 5'-AMP and for its competitor in a similar manner. The numerical value of this ratio shows that ATP is a rather weak competitive inhibitor. Kmfor glycogen: The (conformational) changes of the enzyme induced by 5'-AMP at one substrate binding site raised the question as to what effects 5'-AMP would have on the second substrate binding site, that for glycogen. The Km value for glycogen in the direction of degradation could be determined with the coupled assay system. The range of concentrations of glycogen ( mg per 100 ml corresponding to 1 X 10-4 to 10 X 10-4 M end group) was so chosen that initial linear rates were measured even at the lowest concentration. 22 TABLE 4 Km OF GLYCOGEN AND Km OF 5'-AMP FOR PHOSPHORYLASE b AT 1 X 10-2 M INORGANIC P of 5'-AMP Km (glycogen) of glycogen Km (5'-AMP) M X 105- M X 10-5 M X 10-5 M X * * From Fig. 2, curve II. The concentration of glycogen is expressed as moles of terminal glucose units at the nonreducing end of the chains. In Table 4, columns 1 and 2, the Km value for glycogen decreased from 14 to 4.3 X 10-5 M as the concentration of 5'-AMP was increased from 3 to 9 X 10-5 M. This can be compared with a similar decrease in the Km value for inorganic P within the same range of 5'-AMP concentrations (cf. Table 2). Also shown in Table 4 is the reverse experiment, i.e., the influence of the concentration of glycogen on the Km of 5'-AMP. Analogous effects were obtained by changing the concentration of inorganic P (cf. Table 3). These results indicate that the same reciprocal relationship exists between the binding sites for glycogen and 5'-AMP as between the binding sites for inorganic P (or glucose-1-p) and 5'-AMP. Interaction of substrate binding sites: Two possible variations in the combination of inorganic P, glycogen, and 5'-AMP have so far been examined. In Tables 2 and 3 the concentration of glycogen was kept constant and in Table 4 that of inorganic P was kept constant, while the concentration of the other 2 substances was varied. It remained to keep the concentration of 5'-AMP constant and to vary the concentration of the 2 substrates. The experiments are shown in Table 5. At 1 X 10-4 M and 6 X 10-5 M 5'-AMP, neither the Km for glycogen nor that for inorganic P was influenced significantly by varying the concentration of the respective substrate partner. Moreover, the Km values were close to those predicted from Tables 4 and 2, respectively. In these experiments Vmax did not remain constant (Table 5). Thus, within the range of substrate concentrations

8 VOL. 51, 1964 BIOCHEMISTRY: HELMREICH AND CORI 137 TABLE 5 Km OF INORGANIC P AND Km OF GLYCOGEN FOR PHOSPHORYLASE b AT FIXED 5'-AMP CONCENTRATIONS of inorganic P Km (glycogen) Vmas of glycogen Km (P) Vma. M X 10-3 M X 10-6 min-' M X 1O-5 M X 10-' min' 1 X 1O-4 M 5-AMP 1 X 10-4 M 5'-AMP X 10-5 M5'-AMP 6 X 10-5 M5-AMP 'I Different enzyme preparations were used for the 2 experiments. chosen, no interaction between the 2 substrate binding sites could be demonstrated. Discussion.-Phosphorylase b was one of the first of a class of enzymes which were shown to need a cofactor for activity. The function of the cofactor, where it has been investigated, seems to be similar to that described here, that is, the cofactor changes the affinity of the enzyme for its substrate From the data in Tables 2 and 3 it can be inferred that, as the concentration of 5'-AMP is increased, the Km for inorganic P becomes very small, and vice versa. It has not been possible, however, to replace the requirement of phosphorylase b for 5'-AMP for activity by very high substrate concentration. Phosphorylase a, on the other hand, is catalytically active in the absence of 5'-AMP. The stimulation of phosphorylase a activity by 5'-AMP increases as the concentration of substrate is decreased (cf. Fig. 1 and Table 1). This is a consequence of a change in Km for substrate under the influence of 5'-AMP and would lead to the prediction that at still lower substrate concentration phosphorylase a would appear to be inactive unless 5'-AMP is added. The two forms of the enzyme, a and b, could still be distinguished under these conditions, because the former is stimulated by much lower concentrations of 5'-AMP than the latter. It would be of great importance to know to what extent these findings based on experiments in vitro apply to intact muscle. The chief difficulty is lack of knowledge of the actual concentration of substrates in contact with the enzyme.26 If the concentration of inorganic P (about 2 mm in the intracellular water) and that of 5'-AMP (about 3 X 10-4 M)27 in resting frog muscle were taken at face value, then, based on the Km values in Tables 2 and 3, phosphorylase b should be more than 50 per cent active, but this is obviously not true because stimulation may result in a 100-fold increase in enzyme activity.26 Another factor to be considered is that phosphorylase as part of the structure of intact muscle may be less subject to conformational changes than under in vitro conditions. Summary.-The Km of muscle phosphorylase b for inorganic P and for glycogen decreased progressively as the concentration of 5'-AMP was increased. Conversely, the addition of either substrate in increasing concentration caused a decrease in the Km for 5'-AMP. These mutual interactions are presumably the result of (conformational) alterations of the enzyme protein. No interaction between the two substrate binding sites could be demonstrated when the concentration of 5'-AMP was held constant and that of the substrates was varied,

9 138 BIOCHEMISTRY: HELMREICH AND CORI PROC. N. A. S. Muscle phosphorylase a differs from phosphorylase b in being active in the absence of 5'-AMP and in being stimulated by much smaller concentrations of 5'-AMP. The degree of stimulation of phosphorylase a by 5'-AMP depends on the substrate concentration. This is a consequence of a decrease in Km for substrate when 5'-AMP is added while there is no significant change in Vmax. The dependence of muscle phosphorylase on 5'-AMP for activity, absolute in the case of phosphorylase b and relative in the case of phosphorylase a, is explained in either case by increased affinity of the enzyme for substrate. We wish to thank Dr. Maria Michaelides for the preliminary results, and Mr. Roger Sherman for excellent technical assistance. * Supported in part by the Nutrition Foundation and by research grants Al and AM from the National Institutes of Health, USPHS. Results of this work have been reported in part at the Ciba Symposium on Glycogen Metabolism, July 23, 1963, London, England. 1 Abbreviations are as follows: 5'-AMP, 5'-adenosine monophosphate; inorganic P, orthophosphate; Tris, Tris(hydroxymethyl) aminomethane; EDTA, ethylenediamine-tetraacetate. 2 Brown, D. H., and C. F. Cori, in The Enzymes, ed. P. D. Boyer, H. A. Lardy, and K. Myrback (New York: Academic Press, Inc., 1961), 2nd ed., vol. 5, pp Cori, G. T., and C. F. Cori, J. Biol. Chem., 158, 321 (1945). 4We are indebted to Dr. Carl Frieden for these measurements. 6 Lee, Y. P., Biochim. Biophys. Acta, 43, 18, 25 (1960). 6Fischer, E. H., D. J. Graves, E. S. Crittenden, and E. G. Krebs, J. Biol. Chem., 234, 1698 (1959). 7Keller, P. J., J. Biol. Chem., 214, 135 (1955). 8 Madsen, N. B., and C. F. Cori, J. Biol. Chem., 224, 899 (1957). 9 Cohn, M., and G. T. Cori, J. Biol. Chem., 175, 89 (1948). 10 Madsen, N. B., and C. F. Cori, Biochim. Biophys. Acta, 15, 516 (1954). "Monod, J., J. P. Changeux, and F. Jacob, J. Mol. Biol., 6, 306 (1963). 12 We are indebted to Dr. H. T. Narahara for this preparation. 13 Fischer, E. H., and E. G. Krebs, J. Biol. Chem., 231, 65 (1958). 14 Illingworth, B., and G. T. Cori, Biochem. Preparations, 3, 1 (1953). 15 Danforth, W. H., and E. Helmreich, in preparation. 16 Fischer, E. H., and E. G. Krebs, in Methods in Enzymology, ed. S. P. Colowick and N. 0. Kaplan (Academic Press, 1962), vol. 5, pp When glycogen is treated with strong alkali, as in the Pflueger method, most of the 5'-AMP would be expected to be destroyed. 17 Private communication from Dr. 0. H. Lowry. 18 Private communication from Dr. B. Illingworth. '9 Danforth, W. H., E. Helmreich, and C. F. Cori, these PROCEEDINGS, 48, 1191 (1962). 20 Illingworth, B., D. H. Brown, and C. F. Cori, these PROCEEDINGS, 47, 469 (1961). 21 Parmeggiani, A., and H. E. Morgan, Biochem. Biophys. Res. Comm., 9, 252 (1962). 22 It is difficult to determine a Km value for glycogen because the reaction rate with either glucose-l-p or inorganic P remains constant only within certain limits of chain elongation or chain shortening. As the glycogen concentration is decreased, this limit is quickly exceeded, and the rate of the reaction falls off. An apparent Km value corresponding to 1 X 10-4 M end group was previously obtained with glucose-i-p as substrate. The true Km value would be lower to the extent to which some of the end groups failed to react. 23 Kornfeld, R., and D. H. Brown, J. Biol. Chem., 237, 1772 (1962). 24 Hathaway, J. A., and D. E. Atkinson, J. Biol. Chem., 238, 2875 (1963). 26 Frieden, C., J. Biol. Chem., 238, 3286 (1963). 26 Helmreich, E., S. Karpatkin, and C. F. Cori, in Ciba Symposium on Glycogen Metabolism (1963), in press. 27 Lange, G., Biochem. Z., 326, 172 (1955).