studies on ligand binding during the conversion of phosphorylase b to phosphorylase a

Eur. .J. Biochem. 48, 571 -578 (1974)

Studies on Ligand Binding during the Conversion of Phosphorylase b to Phosphorylase a David J. BROOKS, Stephen J. W. BUSBY, and George K. RADDA Department of Biochemistry, Oxford University

(Received May 2/June 28, 1974)

1. Phosphorylase b may be specifically labelled on one fast reacting sulphydryl group per subunit with 4-iodoacetamido-salicylic acid without loss of enzymic activity.

2. The fluorescence intensity of the covalently linked acetamido-salicylate is altered on the binding of substrate and effector ligands.

3. These fluorescence change were used to derive the “apparent” binding constants of the different ligands and to observe the interaction between them.

4. No change is observed in the fluorescence of the acetamido-salicylate label when phosphorylase b is converted into phosphorylase a. When the conversion is carried out in the presence of either AMP or glucose 6-phosphate there are fluoresence changes which reflect the differential ligand binding to the two forms of phosphorylase. The fluorescence method in the presence of ligands therefore provides a continuous assay for the b to a conversion.

5. Studies on the rate of interconversion revealed that the ratio of activities of non-phosphorylated phosphorylase b kinase at pH 6.8 and 8.4 is over 100 compared to a ratio of 3 for phosphorylated kinase.

6. Experiments carried out in the presence of glucose 6-phosphate directly demonstrated the presence of an intermediate active form of phosphorylase which binds glucose 6-phosphate tightly unlike fully phosphorylated phosphorylase a.

7. The association of phosphorylase a dimers to tetramers was detected in a mixture of acetamido- salicylate-phosphorylase and 4-nitrobenzo-2-oxa-l,3-diazole phosphorylase. The quenching of the fluorescence of the former by the latter species in the mixed tetramer enabled us to confirm that tetramer formation was slower than the appearance of phosphorylase a activity.

The interaction of a number of ligands with phosphorylase may be important in the physiological regulation of glycogen breakdown [l]. There are a number of difficulties in defining the importance of such interactions under physiological conditions. First, the two forms of the enzyme (phosphorylase a and b) have different affinities for given ligands. Second, while extensive kinetic and some equilibrium binding studies have pointed to the possibility of multiple effector and substrate binding sites, the nature of the interaction between different ligands is not fully under- stood [2- 41. Third, there is kinetic evidence that in

Ahbreviution. Cyclic AMP, adenosine cyclic 3’ : 5‘-mono- phosphate.

Enzymes. Phosphorylase b and phosphorylase a (EC 2.4.1.1); phosphorylase b kinase (EC 2.7.1.38); phosphorylase h kinase kinase (cyclic AMP-dependent protein kinase) (EC 2.7.1.37).

the conversion of phosphorylase b to a, intermediate forms of phosphorylase have additional regulatory properties which may well be significant when phosphorylase is bound to the glycogen particle [ 5 ] . Finally, the nature of the intercations in the glycogen particle is only just beginning to be revealed [6].

In previous studies we have shown that a covalently attached spin label [7] or the fluorescent label 4-nitrobenzo-2-oxy-1,3-diazole [8] can be used to monitor the interaction of ligands with phosphorylase. The spin label has also been used to follow the conversion of phosphorylase b to phosphorylase a [9]. Because these latter studies require relatively high enzyme concentrations and involve measuring small changes in the electron spin resonance spectrum of the nitroxide radical they are not particularly suited for following the time course of the b to a conversion, particularly at the early stages of the reaction where the formation

Eur. J. Biochem. 48 (1974)

572 Conversion of Phosphorylase h to a

of hybrid forms of the enzyme are expected [lo]. The changes in the fluorescence of the 4-nitrobenzo-2-oxa- 1,3-diazole group are sufficient to follow the interaction with AMP[8] but are too small for the characterization of the interactions with other ligands or to observe the heterotropic interactions between different ligands.

In this study we introduce the use of 4-iodoacetamido-salicylic acid as an alkylating reagent for phosphorylase. The fluorescence of the acetamido-salicylate moiety provides a sensitive read-out following ligand interactions with the different forms of phosphorylase.

MATERIALS AND METHODS

Phosphorylase b was prepared according to the method of Fisher and Krebs [ l l ] substituting dithio- threitol for cysteine. Enzyme assays were done as before [8]. The enzyme (3 times recrystallised) was dialysed against a triethanolamine-HC1 buffer (50 mM) which contained KCI (100 mM) and EDTA (2 mM) at pH 7.5, and was labelled by incubation with one equivalent of 4-iodoacetamido-salicylic acid for 20 h at 25 "C.

Phosphorylase desensitized towards activation by AMP with 2,4-dinitrofluorobenzene was made by a modification of the method of Baumert et al. [12]. 20 mg/ml phosphorylase b (in triethanolamine-HC1 buffer given above) was incubated with 10 mM 2,4-dinitrofluorobenzene for 45 min at room temperature, the phosphorylase b activity falling to 20%. Excess reagent was removed by dialysis. After addition of 2-mercaptoethanol(20 mM) the solution was warmed to 30 "C for 60 min, followed by the removal of 2-mer-

captoethanol again by dialysis. The modified enzyme was 20 'i: activated by AMP but yielded a fully active phosphorylase a preparation after phosphorylation. In addition the reactive sulphydryl group remained available for labelling with 4-iodoacetamido-salicylic acid.

4-Nitrobenzo-2-oxa-l,3-diazole-phosphorylase was prepared by the method of Birkett et al. [13].

Pure phosphorylase b kinase and partially purified cyclic AMP-dependent protein kinase were prepared as described by Cohen [14].

2,4-Dinitrofluorobenzene and 4-iodoacetamido- salicylic acid were obtained from Koch Light labo- ratories. 7-Chloro-4-nitrobenzo-2-oxa-l,3-diazole was synthesized according to the method of Boulton et al. [15]. All other chemicals were obtained from Sigma Chemical Co. and were of the highest purity available.

Fluorescence measurements were done on a Hi- tachi-Perkin Elmer recording spectrofluorimeter (model MPF-2A) and ultracentrifugation runs were on a Beckman analytical ultracentrifuge (model E).

Fluorescence titrations utilising the quenching of light emission from acetamido-salicylate-phosphorylase h by ligands were carried out using 0.1 mg/ml enzyme (1 pM). As the minimum concentration of ligand added in the titrations was 30 pM, we were able to assume that the free ligand concentration was equal to the total ligand concentration added. In the case of AMP and phosphorylase a, we were not able to make this assumption and because of complications due to co-operativity, we were not able to calculate an exact value for the binding constant. In other cases, the apparent binding constant shown (Table 1) are the values of the total ligand concentration required to produce half of the total fluorescence quenching ob-

Table 1. Apparent binding constants andpevcentage quenching when various ligands ure titrated into phosphorylase a and b Binding followed by the quenching of the sa,licylate fluorescence of the labelled protein. 50 mM triethanolamine- KC1 buffer pH 7.5, 1 pM acetamido-salicylate-phosphorylase at 18 "C

Acetoamido-salicylate-phosphorylase b Acetoamido-salicylate-phosphorylase a

ligand kw,. quenching ligand k a w quenching

AMP ADP ATP IMP Glucose 1-phosphate Glucose 6-phosphate Phosphate 8-Glycerophosphate

mM

0.070 0.100 2 5

10 0.040 1 .0 5.0

YJ 20 15 14 14 12 12 14 14

AMP mM YJ 0.010 21

Glucose 6-phosphate 2 18

Glucose, gluconolactone, maltotetraose and glycogen N o effect ~ ~~


D. J. Brooks, S. J. W. Busby, and G. K. Radda 573

served. Corrections were made for dilution of the enzyme during the titration.

20 7;) of the fluorescence emission (excitation 320 nm; emission 400 nm) of acetamido-salicylate- phosphorylase b is due to protein fluorescence. Con- trol titrations were performed using unlabelled phosphorylase b to confirm that the fluorescence changes seen with the labelled enzyme were not due to changes in protein fluorescence.

The absorbance of a 1 %solution ofphosphorylase b was taken as 13.2 and a monomer molecular weight of 100000 was used [25].

RESULTS

Tlze Binding of Ligands to Phosphorylase Labelled with 4-lodoacetamido-salicylate

In this study phosphorylase b was reacted with one equivalent per subunit of 4-iodoacetamido-salicylate. The extent of labelling was assessed spectrophotomet- rically using the method of Malcolm and Radda [16]. The kinetics of the reaction between the enzyme and the reagent was studied in detail polarographically as was the reaction with iodoacetamide. The results of these studies have shown that one fast and two slow reacting sulphydryl groups per subunit reacted at pH 7.5 in triethanolamine/KCl buffer and the reaction rate constants are such that the rapidly reacting group can be specifically labelled. Since, however, the inter- pretal ion of the present results does not depend on the specificity of the labelling reaction the kinetic studies will be reported in a following paper particularly in view of the difficulties encountered to establish

Absorption

specificity for an enzyme as large as phosphorylase

The singly labelled acetamidosalicylate-phosphorylase h is a fully active dimer and sediments with an szO,,, value of 8.3 S. On excitation at 320 nm (Fig. 1) the salicylate label has a fluorescence emission maxi- mum at 400 nm. Binding of glucose 1 -phosphate (but not glucose or glycogen) and effector ligands to the labelled enzyme reduces the intensity of salicylate emission. In titrations with varying amounts of ligands the fluorescence changes can be used to obtain apparent binding constants for substrates and effectors. These binding constants together with the limiting fluorescence quenching produced by the ligands are summarized in Table 1. Both sets of values were in- dependent of pH in the range 6.8 to 8.4.

Fig. 2 shows the change in acetamido-salicylate fluorescence when labelled phosphorylase b and a are titrated by AMP. While the limiting quenching is the same in both cases the titration curves demonstrate tighter binding to the latter form of the enzyme in agreement with earlier equilibrium dialyses experiments [2,3]. The heterotropic interactions between glucose I-phosphate and AMP is demonstrated by the fluorescence titrations shown in Fig.3 and 4. Each ligand tightens the binding of the other in contrast to the interaction between glucose 6-phosphate and AMP (not shown) where glucose 6-phosphate weakens the binding of AMP.

2,4-Dinitrofluorobenzene-modified phosphorylase h, prepared using the procedure described above, has been reported not to bind AMP but to bind sugar phosphates in the same way as the native enzyme [12]. When phosphorylase is first modified with 2,4-dinitro-

~ 7 1 .

Emissior ,--',

I-

\, \. ,/' , \\, 200 240 280 320 360 400 440 480 520 560 600

Wavelength (nm)

Fig. 1. The absorption and emission bands of the acetamido- salicylate N-acetyl cysteine (full lines) and 4-nitrobenz-2-oxa- l,_?-diazole N-acetyl cysteine (broken lines). The absorption

and fluorescence spectra of the acetamido salicylate and 4-nitrobenzo-2-oxa-diazole moieties when attached to phosphorylase b are identical to the spectra shown in the figure


574 Conversion of Phosphorylase b to a

fluorobenzene and then labelled with the acetamido- salicylate group, addition of AMP, ADP or ATP leads to no change in the fluorescence of the salicylate probe, while the quenching by glucose 1-phosphate and glucose 6-phosphate is unaltered, as are their apparent binding constants to the enzyme. This result confirms that the AMP binding site of phosphorylase b may be blocked without affecting the sugar phosphate binding sites. Glucose 6-phosphate weakening of AMP binding to phosphorylase b is therefore not a result of simple competition for one binding site.

" 0 40 80 120 160 200

0 2 4 6 8 10 [AMP] for phosphorylase b (FM)

[AMP] for phosphorylase a (FM)

Fig. 2. Fluorescence changes as A M P binds to acetamido- .~alic~late-phosphor~la.~e h (0) and a ( m i . 50 mM triethanolamine- KCI buffer pH 7.5, 1 pM acetamido-salicylate-phosphorylase at 18 'C

The Kinetics of the Conversion of Phosphorylase b to a Catalysed by Phosphorylated Kinase

Acetamido-salicylate-phosphorylase b may be converted into the active a form; the conditions for the conversion are : 5 pM labelled phosphorylase 6, 1 nM phosphorylated-phosphorylase kinase, 5 mM MgCl,, 5 pM CaC1, and 50 pM ATP in 50 mM triethanolamine-HC1 buffer containing 100 mM KCl at pH 8.2. Under these conditions the interconversion takes about 5 min and no change in the salicylate fluorescence is seen. When the interconversion is carried out in the presence of 25 pM AMP a decrease in salicylate fluorescence is observed due to the tighter binding of AMP to the a form (see Table l), the uptake of AMP by the newly formed labelled phosphorylase a result- ing in fluoresence quenching (Fig. 5). Conversely, addition of glucose 6-phosphate (400 pM) to the reaction mixture produces an enhancement in the salicylate fluorescence during the b to a conversion (Fig. 5) . This is because glucose 6-phosphate is released from phosphorylase a , being weakly bound to phosphorylase a compared to phosphorylase h. The rate of the interconversion (as assayed by the appearance of phosphorylase a activity) is unaffected by AMP and glucose 6-phosphate at the concentrations used in the experiments described above.

The fluorescence change in the presence of AMP followed exactly the same time course as the appearance of activity, while when glucose 6-phosphate was present an initial lag in the fluorescence change was observed (Fig. 5). In activity assays (where activities are measured by taking aliquots of the reaction mixture and diluting them so that essentially no glucose 6-phosphate is present in the assay mixture) this lag

oov 2b 4'0 $0 8'0 I & 1;o 1 l O 1E;O lk 2io [AMP] (PW

Fig. 3. Fluorescence changes as A M P binds to acetamido- scilic~late-phosp/ior~la~~e b in the presence of various glucose 1-phosphate concentralions. 50 mM triethanolamine-KCI (A), 10 mM (O), 15 mM (0) and 20 mM (W)

buffer pH 7.5, 1 FM acetamido-salicylate-phosphorylase h at 18 "C. Concentration of glucose I-phosphate: 0 (O), 5 mM


D. J. Brooks, S. J . W. Busby, and G. K. Radda 575

does not appear. If the aliquots that are taken from the reaction mixture are assayed with and without glucose 6-phosphate (1 mM) it is found that glucose 6-phosphate inhibits activity only during the first few minutes of the conversion. This confirms the observation of Hurd et al. [lo] who suggested that the intermediate singly phosphorylated phosphorylase dimer binds glucose 6-phosphate (and AMP) relatively tightly. The fluorescence experiments provide a direct demonstration of this, since the lag seen in the fluorescence change in the presence of glucose 6-phosphate corresponds to the time period during which glucose

l 5 r

[Glucose 1 - phosphate] ( mM)

Fig. 4. Fluorescence changes as glucose 1-phosphate binds to ucetan~ido-.valicykcte-phosphorylase b in the presence of various A M P concentrations. 50 mM triethanolamine- KC1 buffer pH 7.5, 1 pM acetamido-salicylate-phosphorylase b at 18 "C. 0 AMP (O), 75 pM AMP (m) and 150 pM AMP (A)

6-phosphate inhibition is present. The fluorescence method, however, provides a more accurate measure of the time course for the appearance of a form of phosphorylase which is active but still binds both glucose 6-phosphate and AMP.

Fig.5 also shows that the initial velocity of the conversion at pH 8.2 is three times faster than at 6.5. The exact ratio of initial velocities at the two pH values varies with the phosphorylase concentration.

The Conversion of Phosphorylase b to a Catalysed by Non-Phosphorylated Kinase

Conversions were also studied at pH 6.8 and 8.2 using non-phosphorylated phosphorylase b kinase (Fig. 6). At pH 6.8 in the presence of AMP or glucose 6-phosphate the change in the fluoresence of labelled phosphorylase during the conversion showed a lag phase that corresponded to a similar delay in the appearance of phosphorylase a activity. Increasing the amount of kinase in the reaction mixture diminish- ed the duration of the lag while addition of cyclic AMP or preincubation of kinase with phosphorylase and ATP did not abolish the lag. Preincubation of kinase with ATP, magnesium and calcium abolished the lag phase in the activity changes.

At pH 8.2 the changes seen were similar to those observed when phosphorylated phosphorylase b kinase was used. In the presence of glucose 6-phosphate the lag phase was still apparent but it was not present when fluorescence was followed in the presence of AMP or in the activity assays.

.

50 0 V O 1 2 3 4 5

Time (m in )

Fig. 5. Fluorexence changes as A M P or glucose 6-phosphate hind to ucetamido-salicylate-phosphorylase during b to a conversion with phosphorylated phosphorylase b kinase at p H 6.8 or p H 8.2 (continuous lines). 50 mM triethanolamine- KCl buffer with no EDTA pH 6.8 or pH 8.2, 5 pM acetamido-


salicylate-phosphorylase b, 5 mM MgCI,, 5 pM CaCl,, 50 pM ATP, and 25 pM AMP or 400 pM glucose 6-phosphate. 1 nM phosphorylated phosphorylase b kinase was added at zero time. Percentage conversion of phosphorylase b to a at pH 8.2 (0) from activity measurements

576 Conversion of Phosphorylase b to a

- 100 I ."

V

0 5 10 15 20 25 30 35 40 Time ( m i n )

Fig. 6. Fluorescence changes us AMP or glucose 6-phosphate binds to ucetumido-sulicylute-phosphorylase during b to a conversion with non-phosphoryluted phosphorylase b kinuse at p H 6.8 or p H 8.2. Conditions as for Fig. 5 except 1 nM non-

Table 2. Summary of' the various lug effects during the conversion of'phosphoryluse b to a seen using the fluorescence of' acetamido-salicylate-phosphorylase in the presence of AMP or glucose 6-phosphate at p H 6.8 andpH 8.2 usingphosphoryluted or non-phosphoryluted phosphoryluse b kinase 50 mM triethanolamine- KCI buffer pH 8.2 and pH 6.8 at 18°C were used with no EDTA present, 5 pM acetamido- salicylate-phosphorylase b, 1.0 nM phosphorylase h kinase, 5 mM MgCI,, 5 pM CaC1, and 50 pM ATP, with 25 pM AMP or 400 pM glucose 6-phosphate present were concentrations used to follow the conversion by salicylate fluorescence

~~~

Kinase pH Fluorescence assay Activity assay

+ AMP + glucose (+ glucose 6-phos- 6-phosphate phate or AMP)

~ ~~~ ~~~

Activated 6.8 no lag lag no lag 8.4 no lag lag no lag

Non- 6.8 lag 1% lag activated 8.4 no lag lag no lag

The ratio of the initial activity of non-phosphoryl-

Table 2 summarises these observations. ated kinase at pH 8.2 to that at 6.8 was over 100.

The Kinetics of' the Formation of Phosphorylase a Tetramers

The rate of formation of the phosphorylasea tetramer has been previously followed using a noncovalent fluorescent probe 2-(N-methylanilino)-naph- thalene-6-sulphonate [8]. The use of this probe de- pended on a detailed comparison of the sedimentation

phosphorylated phosphorylase b kinase was used (continuous lines). Percentage conversion of phosphorylase b to a at pH 6.8 (0) from activity measurements

behaviour of the different aggregation states of the enzyme with the fluorescence characteristics of the probe. In this paper we report a more unequivocal method for following tetramer formation. The method utilises the fact that the fluorescence emission band on the acetamido-salicylate group on phosphorylase overlaps with the absorption band of the 4-nitrobenzo- 2-oxa-l,3-diazole group which can also be attached to the enzyme (Fig.1). If the two probes are closer than 3.0 nm apart, from theoretical considerations one may expect energy transfer to take place between them, with a consequent quenching of the acetamido- salicylate fluorescence. When a solution of acetamido- salicylate phosphorylase b (0.1 mgjml) is mixed with nitrobenzo-oxa-diazole-phosphorylase b (0.2 mgiml) no significant change in salicylate fluorescence is observed. After converting this mixture to phosphorylase a a 30% quenching of salicylate fluorescence is observed which can be reversed by the addition of glucose (50 mM). Glucose is known to disaggregate phosphorylase tetramers and does not affect the fluorescence of acetamido-salicylate-phosphorylase a (Table 1). No change in acetamido-salicylate fluorescence is observed if acetamido-salicylate-phosphorylase a (0.1 mg/ml) is added to 4-nitrobenzo-2-oxa- 1,3-diazole-phosphorylase a (0.2 mgiml). Quenching associated with the interconversion therefore can be attributed to energy transfer in the hybrid tetramers of phosphorylase a. Fig.7 shows the kinetics of the production of this quenching as phosphorylation takes place, using activated phosphorylase kinase at pH 8.5. The time course of the appearance of the quenching lags considerably behind that of the activity changes, but corresponds to the results obtained using the noncovalent probe.


D. J. Brooks, S. J . W. Busby, and G. K. Radda 577

40 t

/

1 I

1 2 3 4 Time (min)

96 Fig. I . The quenching OJ acetamido-salicylate Jluorescence by singlet energy transfer to 7-sulpha-4-nitrobenz-2-oxa-l,3-diazole moieties due to the tetramerisation of acetamido-salicylate- phosphorylase b and 7-sulpha-4-nitrobenz-2-oxa-l,3-diazole phosphorylase b dimers during phosphorylase h to a conversion. Tris- KCI buffer pH 8.5, 1 p M acetamido-salicylate-phosphorylase b. 2 p M nitrobenoxadiazole phosphorylase h, 5 mM MgCI,, 5 p M CaCl,, 50 p M ATP, and 1 nM phosphorylated phosphorylase b kinase. The inset illustrate the experiment. The 7-sulpha-4-nitrobenz-2-oxa-1,3-diazole is represented by 0, the acetamido-salicylate moiety by X and the arrow m- dicates conversion from the b to a form followed by tetramerisation

DISCUSSION

The acetamido-salicylate moiety attached to phosphorylase is a very sensitive indicator of the binding of substrate and effector ligands which have phosphate groups. Although at this stage of our studies the mechanism of quenching produced by these ligands remains unclear we believe the effect could be the result of a short range interaction between the phosphate groups of the ligand and the fluorescent side- chain rather than due to the conformational change that is induced by the ligands. The observation that the salicylate fluorescence is the same on phosphorylase b and a supports this hypothesis. This is in contrast to the behaviour of spin-labelled phosphorylase where b to a conversion decreases the mobility of the label almost as much as addition of AMP, while glucose 1 -phosphate binding produces very little change.

The binding constants deduced from ligandlenzyme fluorescence titrations are closely similar to those obtained from kinetic and spin label studies [19,20]. The observed cooperativities (both homotropic and heterotropic) are also compatible with kinetic data. Of particular interest are the observations on the marked heterotropic cooperativity between AMP and glucose l-phosphate and the tight binding of ADP

and glucose &phosphate to phosphorylase b (Table 1). Both ADP and glucose 6-phosphate are inhibitors and are present in resting muscle in concentrations of 2 mM for ADP and 200 pM for glucose 6-phosphate [21]. A large proportion of phosphorylase b will therefore have these ligands bound to it. On conversion to phosphorylase a glucose 6-phosphate will be released and AMP and ADP will be taken up as these two nucleotides bind more tightly to phosphorylase a than to phosphorylase b.

The continuous assay method for phosphorylase kinase which we have developed enabled us to study non-linear progress curves more precisely than was possible with the previous technique based on phosphorylase a activity measurements. The disadvantages of the fluorescence method are the need to use phosphorylase concentrations much lower than that of AMP and glucose 6-phosphate and the need to main- tain a low ADP concentration. The method is also very susceptible to interference by contaminating enzymes such as 5’adenylic acid deaminase and ATPases. While the pH 6.8 to 8.4 ratios for the activity of phosphorylated kinase are similar to those reported by Cohen [I41 in the case of non-phosphorylated kinase at pH 6.8 it is evident that autophosphorylation is appreciable [26] over a short time period even at low ATP concentrations. This effect is distinct from the lag phase observed by Kim and Graves [22], as preincubation of the kinase with phosphorylase b does not affect the activation lag period. Because of the contribution of autophosphorylation to activity, care must be taken when defining the pH 6.8 activity of non-phosphorylated kinase. The pH 8.4 to 6.8 activity ratios of over 100 are only seen when kinase is rapidly prepared since trace quantities of proteolytic enzymes will also active phosphorylase b kinase causing the ratio to drop.

It has been suggested [4] that, in vivo, the low pH 6.8 activity observed with non-phosphorylated phosphorylase b kinase is not sufficient to account for the rapid phosphorylase a production in glycogen particles following flash activation with calcium, magnesium and ATP and in muscle tissue on electrical stimulation [ 2 3 ] .

It is possible that in vivo and when in glycogen particles, the activity of phosphorylase kinase is in- creased when bound to glycogen [24] or other proteins. It is however difficult to relate the activity of non- phosphorylated kinase as seen here to that in vivo because of the high concentration found in muscle and the possibilities of phosphorylation during electrical stimulation.

Finally the intersubunit energy transfer experiments we described not only enable us to follow the formation of phosphorylase a tetramer (which is clearly irrelevant

Eur. J . Biochem. 48 (1974)

578 D. J. Brooks, S. J. W. Busby, and G. K. Radda: Conversion of Phosphorylase b to LI

to the activation process) but also provide a basis for relating the sulphydryl groups that are labelled to one another. The quantitative aspects of this relationship will be reported later but our data indicates that the labels (bound to sulphydryl groups), are relatively close to the tetramer interface as they are not more than 2.0 nm apart from one another.

This work was supported by the Science Research Council and is a contribution from the Oxford Enzyme Group. DJB and SJWB have been recipients of Medical Research Council Training Awards.

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D. J. Brooks, S. J. W. Busby, and G. K. Radda, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, Great Britain, OX1 3QU


studies on ligand binding during the conversion of phosphorylase b to phosphorylase a

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