- Email: [email protected]
An investigation of direct interaction between phosphorylase kinase and phosphorylase-b
AN INVESTIGATION OF DIRECT INTERACTION BETWEEN PHOSPHORYLASE KINASE AND PHOSP~~ORYLASE-b DIMITERDI%UTROV Central
Laboratory
of Biophysics. Bulgarian
Academy of Sciences,
Sofia 1113,Bulgaria
Abstract-l. The substrate molecule (phosphorylase-b) was modified by a fluorescence probe and the change of the Ruorcscence intensity in the presence of phosphorylase kmase was investigated. of the tluorescence was observed simultaneously. 2. The alteration of the degree of polarization 3. From the saturated curve, the dissociated constant and the number of bound phosphorylasc-b molecules on a mole of enzyme was calculated.
Phosphorylase kinase isolated from rabbit muscle catalyzes the transformation of phosphorylase-b into pb~sph~r~lase-~.
The
divalent
ions
of
Mg”
and
1971; Helmeyer er ul., 1970; Ozawa t’r II/.. 1967). glycogene and ATP (DcLange t’t al., 1968). have been found to have a regulatory effect on the enzyme. Simultaneously with this type of regulation phosphorylase kinase takes pert in the hormonal control of glycogen in the muscles. The enzyme is also influenced by adrenaline through adenyl cyclasc and cyclic A~lP~de~n~ent protein kinase. Phospho~l~s~ kinase is obtainable in a s&icientlq purified form to permit the study of its subunit structure (Cohen, 1973; Hayakawa er cr[., 1973). In this paper the binding of phosphorylase-b to phosphorylase kinase haf been investigated and evidence has been given for the formation of an enzyme substrate complex in the absence of ~cliv~t~n~ ions. By means of the fluorescence probe technique, taking into account the degree of polarization, the dissociation constant and the stoichiometry of the interaction have heen calculated. Cal*
(~r~~slr~rn
ef ul.,
homogenized in 2.5 vol of 4 mM EDTA, pH 7 in a Serval Omny Mixer. The suspension was centrifuged for 45’min at Cmg. The supernatant was brought to pH 6.1 with 1N acetic acid. The solution was centrifuged a second time for 45 min at 6cx)og. The precipitate obtained was dissolved in 30 ml of 0.1 M gly~rophosphat~. 4 mM EDTA buffer. pH 8.2. The suspension was diluted with 50mM glycerophosphate, 2 mM EDTA buffer (Cohen, 1973), after which it was centrifuged for 100 min at 78.0009. The supematant was collected. A cold solution of (NH&SO,+ (475 gl) was mlxed with the protein solution in a ratio of 1:2. After IOmin at 0°C the suspension obtained was centrifuged for 5 min at 1500~. The protein solution was passed through a column with Sepharose 4B (2.5/w)cm) for eliminating the foreign proteins. Samples of 5 ml were taken by using the Serva automatic collector.
Ten milligrams of phosphorylase-h were dissolved in lof)jd glycerophos~h~;e but&r pfi 6.8 and were diluted with 2 vol of NaWCO,, pH 9. Fluorescein isothi~yanate (FITC) (O.I- 1mg) was added to the solution and the mixture was left for 30min to react. The protein bound to FITC was separated from the unaffected dye on a column of Sephadex G25. Two colourcd bands were observed during eluation. That with a quicker movement corresponded to phosphorylase-b. and the other one to free FITC. The protein was dialysed for 16 hr with the same buffer for lhe elimination of loosely bound FITC molecules. Actirirx
Scpharosc 4B and Sephadex G25 produced by Pharmacia (Sweden) wcrc used in the experiments. The other chermcal preparation were: glucose-l-phosphate, glycogene, phosphorylase-b by Boehrinper (F.R.G.) and fluorcsceioe isothiocyanate adsorbed on kiesel8uhr by Serva (F R.G.I.
The enryme was purified from rabbit muscle according to the method modified by Brostrom 81 al. (1971) and Cohen (IY73). An amount of 600g of muscle tissue was
c!f ~h~~.s~~~r~~~se kinmv
The reaction mixture contained 0.2ml of 0.125 M Tris 0.125 M glycerophosphatc buff’er, pH 6.8, 0.02 ml 3 mM CaCI,. 0.02 ml 0.03 M MeCI,. 0.2 ml 0,005 M ATP. The latter s&tion was used fir initiating the reaction. After I.5 min 0.2 ml of the reaction mixture were decanted and diluted for the d~t~rmina&ion of the activity of phosphorylase-a. The reaction proceeded in the direction of glycogene synthesis (Krebs PI ~1.. 1966). The free phosphate in the solution was determined according to Weilmalherbe ef ol. (1952). The specific activity was 6.5 units/mg according to the method of Cohen (1973).
529
530
DIMITER DIMITRO%
The fluorescence investigations were carried out with a Zeiss M4 QIII spectrofluorimeter. A Xenon lamp was used as a source of excited light and necessary wavelength was separated from it by means of a filter.’ For polarization measurements a polarization filter was used. The degree of polarization was calculated by the formula p = ;.I,;;,
I where 1 and I, were the Ruorescence intensity in the direction parallel and perpendicular to the polarization plane. K = (1 ,/I,) is a constant. The direction of the exciting light was perpendicular to the direction of fluorescence measurement. The exciting light was with a maximum at 436nm (inpolarized) and that of fluorescence measured was at 525 nm. RESULTS
AND DISCUSSIOK
The precipitate obtained by sedimenting the protein solution with (NH&SO, and centrifuging at 15,000 g was dissolved in glycerophosphate buffer and its components were separated on a Sepharose 4B column. The results are shown in Fig. 1. The first protein maximum was non-transparent, and showed a high absorption at 280 nm, while the second and the third maxima showed a lower absorption and were completely transparent. The enzyme activity was concentrated at the second protein maximum. The activity connected with the first maximum was quite low, while that at the third maximum was entirely non-existent. These results coincide with those obtained by Cohen (t 973) and Hayakawa et al. (1973). The same authors have checked the purity of the second protein maximum and the high homogeneity of the protein fraction has been determined. The purity of the enzyme solution investigated was deter-
mined through electrophoresis of polyacryhunide gel and isoelectric focusing where single bands of the protein were obtained. Before each experiment the sample was passed through a Sepharose 4B column (i/60 cm) for eliminating aggregate forms. Fluorescence
i~~esti~atio~s
The binding of FITC to phosphorylase-b was effected according to the procedure described above. The measurements showed that about 4 or 5 moles of dye were adsorbed on 1 mole of protein. The concentration of FITC was determined by the absorption at 495 nm (Tietze er al., 1962) and that of phosphorlase-b as described by Lawry et af. (1951) or spectrophotometrically. After the chemical modification with FITC the activity of phosphorylase-b remained unchanged as determined by the reaction catalysed by phosphorylase kinase. The intensity of 1.2 x 10e7 M solution of phosphorylase-b was assumed to be 1007: and any other fluorescence was measured in comparison with it. The addition of phosphorylase kinase to solution of phosphorylase-b had a significant effect on the intensity of fluorescence. A reduction of this intensity was noticed after increasing the phosphorylase kinase concentration (Fig. 2, curve 1). All measurements were made on a constant volume so as to avoid errors due to delution of the solution. Figure 3 shows a typical saturation curve obtained by measuring the degree of polarization at a higher phosphorylase kinase concentration. The concentration of phosphorylase-b remained constant in the solution. This parameter (Fig. 2) changes simultaneously with the decrease of the fluorescence intensity. The increase of the degree of polarisation, according to the equation of Peryn (Wahl & Weber, 1967) is proportional to an increase of the relaxation
Fraction No. Fig. 1. Elution profile of phosphorylase kinase on a column of Sepharose 4B (2.5/90)cm equilibrated Active with 0.125 M glycerophosphate, 0.002 M EDTA buffer, pH 6.8, 4°C; 5 ml samples were taken. components. The specific activity = 6.5 units/mg- * according to the method of Cohen (1973).
Interaction
between
phosphorylase
kinase
mot
Ph.K.
mol
Ph-b
531
and phosphorylase-b
of fluorescence intensity (curve 1) and polarisation degree (curve 2) of FlTCpphosphorylase-b, as a function of phosphorylase kinase concentration. The concentration of phosphorylase-b
Fig. 2. Dependence
in the solution
was 1.2 x lo-’
M. Glycerophosphate
time of rotation diffusion-and indicates a decrease in the mobility of the protein molecule. A similar process by a constant viscosity gives ground to be interpreted as a protein-protein interaction. Gel jiltration
on Sepharose 48
The fluorescence measurements were paralleled by gel filtration experiments for the determination of the distribution of the 2 proteins. An analytical column (l/lOOcm) with Sepharose 4B was used for this purpose. Figure 4a shows the distribution of phosphorylase kinase. The distribution of phosphorylase-b obtained from the same column is shown in Fig. 4b. The protein concentration was measured by the fluorescence of bound FITC. The aggregate forms
buffer 0.05 M, pH 7.8. 30‘C vol 2 ml.
observed in the solution were eluated before the main maximum. Figure 4c shows the elution of a sample containing both proteins simultaneously. The continuous line corresponds to phosphorylase-b measured by the fluorescence at 525 nm. The protein distribution in this case is different from that in Fig. 4b. The main maximum is shifted forward and coincides with the maximum of phosphorylase kinase. According to some investigators (Cohen, 1973; Hayakava et al., 1973), the first maximum in the distribution of phosphorylase kinase is due to the aggregate forms of the enzyme (Fig. 4a). In our experiments part of the phosphorylase-b was observed also at this maximum (Fig. 4~). This result shows that the adsorption property of the enzyme is conserved also in the aggregate (where some lower molar activity was observed too, Fig. 1). The results described indicate the possibility of a direct interaction between phosphorylase kinase and phosphorylase-b and means that its parameters should be investigated. The equation of Ravitch & Weber (1972) permits the calculation of the amount of phosphorylase-b bound taking as a basis the anisotropy of the fluorescence investigated. 2P ~~ 3-P
A= I I
I 2
I 3
I
I
4
5
[Ml x IO7 Fig. 3. Titration curve: the polarisation of FITC-phosphorylase-b as a function added phosphrylase kinase. Phosphorylase-b concentration = 6.39 x lo-’ M. Experimental conditions: glycerophosphate buffer 0.05 M, pH 7.8, 30°C.
where P is the degree anisotropy
of polarisation
and A is the
Ai - A, ’ = iA,-A,)Ri
- A,
where Ai is the anisotropy observed and A, is anisotropy ofall the phosphorylase-h bound molecules. Af stands for all free molecules and /j’ is the amount
P
a"
04 02
I
2
3
4
i&l
Fig. 4. Elution profil on Sepharose 4B column (1 x I00 cm) equilibrate with 0.125 iV g~i~rophos~hate buher. 0.002 M EDTA pH 7.8,25‘C. (a) Phosphorylase kinasc 0.25 mt&ml (-- ). Phosphorylas~ kinase activity. The enzyme could be considers as a non-activated one. The specific activity of kinase is 6.5 uaits~m8- r. according the method of Cohen (1973). (b) ~~TC-la~ll~ phosphor~lase-b, 0.4 mg/ml. The number of dye binding molecule per mole protein is 2-3. ,I,. __.= 436 nm i,, = 525 nm. The other conditions are the same as in Pi8 a. (c) The mixture of both phosphorylase kinase and phosphorylase-b. Phosphorylase kinase 0.25 mgjml, f----j. phosph~~lase-b, 0.4 m8ml (----I. m Phos~~orylase kinase ~~iv~ty 5.2 units by
Fi8. 5. The fiction of bound ~hospborylase-b as a function of fhe ph~~p~o~~~e kinase added. The experimental conditions were the same as those in Fig. 2. [,~~I~~~~~ moles of phosphorylase kinase Per mole phosphorylase-b.
Meyer er al. (1970) d~c~~bed the isolation and characterized the protein-~lyc~~en c~rn~lex and sugg~t~ that this complex was a structural and functional unit of the cell. In vitro no ~t~cogen is present in the mixture and the phos~horylase-b conversion might be possible if a previous adsorption of the substrate molecule on the enzyme could be effected. This process may take place simuItaneously with that in the presence of ~lycog~n.
where R is the ratio of the quantum yield determine by the ~larization photometer. I;! and 1)” is the fluorescence intensity in the direction para1~~~ and normal to the ~larization plane. The results are given in
Fig. 5. From the equation of Weber er al. (1964) we have obtained for the stoic~iometr~ of the interaction the value of N = 2 and Kd = 1.03 x 10e6 M. This result corres~uds to the reaction schema (Krebs et ul., 1958) where two moles phosphorous-b was transformed to phosp~oryl~e-a. In the ~rnp~i~on with the Michaelis constant K, = 1.7 x 10-s M. K, has a smaller value. This is an interesting result as it may be ~surn~ that the transformation of the substrate mol~u~es is not a single process. In our inv~ti~~tio~
we observed the first sta of the conversion the adsorption of ~hospho~Ia~-b on the enzyme molecule. In t:ico the conversion reaction has perhaps another mech~~sm. It was found (Krebs et al,. 1964) that glycogen increases the unity of pho$~horyla~ kinase.
The interaction between rabbit muscle phosphorylase kinase and phosphor~Ia~-h has been investigated. By means of a fluorescence probe bound to the surface of the phos~horylase-b rnol~ul~ it has been demonstrated that the ~~larisat~on degree rises with the increase of the phosphory~~e kinase concentration The data from gel filtration on Sepharose 48 have also been presented. The interaction parameters have been calculated from the saturation curve oht~n~ by means of the equation of Ravitch & Weber. Ackn~~~~~~tfllcmen~~~Tbe author thanks Prof. Dr. F‘. KBrber. Prof. Dr. P. Sieemund and Dr. P. nietsch from the tnstitut fir Biochemye und Biophysik an dcr Freien ~niv~rsit~t in West Berlin for the possibility prov*ided of carrying out these inv~tigations and for their useful discussion.
RIWXR~M C. 0.. H~JNKELERF.
L. & KREBS E. G. (1971) The regulation of skeletal muscle phospho~la~ kinase by Ca. J. hiol. ~~~~~.246. l%t-~~9~~‘
Interaction
between
phosphorylase
COHENB. (1973) The subunit structure of rabbit-skeletalmuscle phosphorylase kinase and the molecular basis of its activation reaction. Eur. J. Biochem. 34, 1-14. DELANGER. J., KEMP R. J., RILEYW. D., COOPERR. A. & KREB~E. G. (1968) Activation of skeletal muscle phosphorylase kinase by adenosine triphosphate and adenosine 3,5-monophosphate. J. biol. Chem. 243, 22W2208. HAYAKAWAT., PERKINSJ. & KREBSE. G. (1973) Studies on the subunit structure of rabbit skeletal‘muscle phosphorylase
kinase.
Biochemistry
12. 567-573.
kinase
and phosphorylase-b
533
muscle phosphorylase-b kinase reaction. J. biol. Chem. 231, 73-83. KREBSE. G. & FISCHERE. H. (1956) Phosphorylase-b to a converting enzyme of rabbit skeletal muscle. Biochim. biophys. Acta 20, 15&157. OZAWA E., HASOIK. & EBASHIS. (1967) Reversible stimulation of muscle phosphorylase-b kinase by low concentation of calcium. J. Biochem. 61. 531. RAVITCHA. B. & WEBERG. (1972) The reversible association of lisozyme and thyroglobulin. J. biol. Chem. 247,
H;LME&R L. M. G., HASCHKEd. H., MEYERF. & FISCHER 68M85. E. G. (1970) Control of phosphorylase activity in a TIETZEF., MORTIMORG. E. & LAMAXN. R. (1962) Preparation and properties of fluorescent insulin derivatives. muscle glycogen particle. J. biol. Chem. 245, 6649-6656. Biochim. biophys. Acta 59, 336346. KREBSE. G., LOVE-D. C., BRATVOLDG. E., TRAYSERK. A., MEYERW. L. & FISCHERE. H. (1964) Purification WAHL P. & WEBERG. (1967) Fluorescence deoolarization and properties of rabbit skeletal muscle phosphorylase-b of rabbit gamma glodulin conjugates. J. m&c. Biol. 30, kinase. Biochemistry 3, 1022-1033. 371-382. KREBS E. G. (1966) Phosphorylase-b kinase from rabbit WEILMALHERBE H. & GREENR. H. (1952) Kalorimetrische muscle. Meth. Enzymol. 8, 543-546. Phosphatbestimmung mittels Ausschiitelung. Biochem. J. KREBSE. G., KENT A. B. & FISCHERE. H. (1958) The 49, 286.
Laboratory
of Biophysics. Bulgarian
Academy of Sciences,
Sofia 1113,Bulgaria
Abstract-l. The substrate molecule (phosphorylase-b) was modified by a fluorescence probe and the change of the Ruorcscence intensity in the presence of phosphorylase kmase was investigated. of the tluorescence was observed simultaneously. 2. The alteration of the degree of polarization 3. From the saturated curve, the dissociated constant and the number of bound phosphorylasc-b molecules on a mole of enzyme was calculated.
Phosphorylase kinase isolated from rabbit muscle catalyzes the transformation of phosphorylase-b into pb~sph~r~lase-~.
The
divalent
ions
of
Mg”
and
1971; Helmeyer er ul., 1970; Ozawa t’r II/.. 1967). glycogene and ATP (DcLange t’t al., 1968). have been found to have a regulatory effect on the enzyme. Simultaneously with this type of regulation phosphorylase kinase takes pert in the hormonal control of glycogen in the muscles. The enzyme is also influenced by adrenaline through adenyl cyclasc and cyclic A~lP~de~n~ent protein kinase. Phospho~l~s~ kinase is obtainable in a s&icientlq purified form to permit the study of its subunit structure (Cohen, 1973; Hayakawa er cr[., 1973). In this paper the binding of phosphorylase-b to phosphorylase kinase haf been investigated and evidence has been given for the formation of an enzyme substrate complex in the absence of ~cliv~t~n~ ions. By means of the fluorescence probe technique, taking into account the degree of polarization, the dissociation constant and the stoichiometry of the interaction have heen calculated. Cal*
(~r~~slr~rn
ef ul.,
homogenized in 2.5 vol of 4 mM EDTA, pH 7 in a Serval Omny Mixer. The suspension was centrifuged for 45’min at Cmg. The supernatant was brought to pH 6.1 with 1N acetic acid. The solution was centrifuged a second time for 45 min at 6cx)og. The precipitate obtained was dissolved in 30 ml of 0.1 M gly~rophosphat~. 4 mM EDTA buffer. pH 8.2. The suspension was diluted with 50mM glycerophosphate, 2 mM EDTA buffer (Cohen, 1973), after which it was centrifuged for 100 min at 78.0009. The supematant was collected. A cold solution of (NH&SO,+ (475 gl) was mlxed with the protein solution in a ratio of 1:2. After IOmin at 0°C the suspension obtained was centrifuged for 5 min at 1500~. The protein solution was passed through a column with Sepharose 4B (2.5/w)cm) for eliminating the foreign proteins. Samples of 5 ml were taken by using the Serva automatic collector.
Ten milligrams of phosphorylase-h were dissolved in lof)jd glycerophos~h~;e but&r pfi 6.8 and were diluted with 2 vol of NaWCO,, pH 9. Fluorescein isothi~yanate (FITC) (O.I- 1mg) was added to the solution and the mixture was left for 30min to react. The protein bound to FITC was separated from the unaffected dye on a column of Sephadex G25. Two colourcd bands were observed during eluation. That with a quicker movement corresponded to phosphorylase-b. and the other one to free FITC. The protein was dialysed for 16 hr with the same buffer for lhe elimination of loosely bound FITC molecules. Actirirx
Scpharosc 4B and Sephadex G25 produced by Pharmacia (Sweden) wcrc used in the experiments. The other chermcal preparation were: glucose-l-phosphate, glycogene, phosphorylase-b by Boehrinper (F.R.G.) and fluorcsceioe isothiocyanate adsorbed on kiesel8uhr by Serva (F R.G.I.
The enryme was purified from rabbit muscle according to the method modified by Brostrom 81 al. (1971) and Cohen (IY73). An amount of 600g of muscle tissue was
c!f ~h~~.s~~~r~~~se kinmv
The reaction mixture contained 0.2ml of 0.125 M Tris 0.125 M glycerophosphatc buff’er, pH 6.8, 0.02 ml 3 mM CaCI,. 0.02 ml 0.03 M MeCI,. 0.2 ml 0,005 M ATP. The latter s&tion was used fir initiating the reaction. After I.5 min 0.2 ml of the reaction mixture were decanted and diluted for the d~t~rmina&ion of the activity of phosphorylase-a. The reaction proceeded in the direction of glycogene synthesis (Krebs PI ~1.. 1966). The free phosphate in the solution was determined according to Weilmalherbe ef ol. (1952). The specific activity was 6.5 units/mg according to the method of Cohen (1973).
529
530
DIMITER DIMITRO%
The fluorescence investigations were carried out with a Zeiss M4 QIII spectrofluorimeter. A Xenon lamp was used as a source of excited light and necessary wavelength was separated from it by means of a filter.’ For polarization measurements a polarization filter was used. The degree of polarization was calculated by the formula p = ;.I,;;,
I where 1 and I, were the Ruorescence intensity in the direction parallel and perpendicular to the polarization plane. K = (1 ,/I,) is a constant. The direction of the exciting light was perpendicular to the direction of fluorescence measurement. The exciting light was with a maximum at 436nm (inpolarized) and that of fluorescence measured was at 525 nm. RESULTS
AND DISCUSSIOK
The precipitate obtained by sedimenting the protein solution with (NH&SO, and centrifuging at 15,000 g was dissolved in glycerophosphate buffer and its components were separated on a Sepharose 4B column. The results are shown in Fig. 1. The first protein maximum was non-transparent, and showed a high absorption at 280 nm, while the second and the third maxima showed a lower absorption and were completely transparent. The enzyme activity was concentrated at the second protein maximum. The activity connected with the first maximum was quite low, while that at the third maximum was entirely non-existent. These results coincide with those obtained by Cohen (t 973) and Hayakawa et al. (1973). The same authors have checked the purity of the second protein maximum and the high homogeneity of the protein fraction has been determined. The purity of the enzyme solution investigated was deter-
mined through electrophoresis of polyacryhunide gel and isoelectric focusing where single bands of the protein were obtained. Before each experiment the sample was passed through a Sepharose 4B column (i/60 cm) for eliminating aggregate forms. Fluorescence
i~~esti~atio~s
The binding of FITC to phosphorylase-b was effected according to the procedure described above. The measurements showed that about 4 or 5 moles of dye were adsorbed on 1 mole of protein. The concentration of FITC was determined by the absorption at 495 nm (Tietze er al., 1962) and that of phosphorlase-b as described by Lawry et af. (1951) or spectrophotometrically. After the chemical modification with FITC the activity of phosphorylase-b remained unchanged as determined by the reaction catalysed by phosphorylase kinase. The intensity of 1.2 x 10e7 M solution of phosphorylase-b was assumed to be 1007: and any other fluorescence was measured in comparison with it. The addition of phosphorylase kinase to solution of phosphorylase-b had a significant effect on the intensity of fluorescence. A reduction of this intensity was noticed after increasing the phosphorylase kinase concentration (Fig. 2, curve 1). All measurements were made on a constant volume so as to avoid errors due to delution of the solution. Figure 3 shows a typical saturation curve obtained by measuring the degree of polarization at a higher phosphorylase kinase concentration. The concentration of phosphorylase-b remained constant in the solution. This parameter (Fig. 2) changes simultaneously with the decrease of the fluorescence intensity. The increase of the degree of polarisation, according to the equation of Peryn (Wahl & Weber, 1967) is proportional to an increase of the relaxation
Fraction No. Fig. 1. Elution profile of phosphorylase kinase on a column of Sepharose 4B (2.5/90)cm equilibrated Active with 0.125 M glycerophosphate, 0.002 M EDTA buffer, pH 6.8, 4°C; 5 ml samples were taken. components. The specific activity = 6.5 units/mg- * according to the method of Cohen (1973).
Interaction
between
phosphorylase
kinase
mot
Ph.K.
mol
Ph-b
531
and phosphorylase-b
of fluorescence intensity (curve 1) and polarisation degree (curve 2) of FlTCpphosphorylase-b, as a function of phosphorylase kinase concentration. The concentration of phosphorylase-b
Fig. 2. Dependence
in the solution
was 1.2 x lo-’
M. Glycerophosphate
time of rotation diffusion-and indicates a decrease in the mobility of the protein molecule. A similar process by a constant viscosity gives ground to be interpreted as a protein-protein interaction. Gel jiltration
on Sepharose 48
The fluorescence measurements were paralleled by gel filtration experiments for the determination of the distribution of the 2 proteins. An analytical column (l/lOOcm) with Sepharose 4B was used for this purpose. Figure 4a shows the distribution of phosphorylase kinase. The distribution of phosphorylase-b obtained from the same column is shown in Fig. 4b. The protein concentration was measured by the fluorescence of bound FITC. The aggregate forms
buffer 0.05 M, pH 7.8. 30‘C vol 2 ml.
observed in the solution were eluated before the main maximum. Figure 4c shows the elution of a sample containing both proteins simultaneously. The continuous line corresponds to phosphorylase-b measured by the fluorescence at 525 nm. The protein distribution in this case is different from that in Fig. 4b. The main maximum is shifted forward and coincides with the maximum of phosphorylase kinase. According to some investigators (Cohen, 1973; Hayakava et al., 1973), the first maximum in the distribution of phosphorylase kinase is due to the aggregate forms of the enzyme (Fig. 4a). In our experiments part of the phosphorylase-b was observed also at this maximum (Fig. 4~). This result shows that the adsorption property of the enzyme is conserved also in the aggregate (where some lower molar activity was observed too, Fig. 1). The results described indicate the possibility of a direct interaction between phosphorylase kinase and phosphorylase-b and means that its parameters should be investigated. The equation of Ravitch & Weber (1972) permits the calculation of the amount of phosphorylase-b bound taking as a basis the anisotropy of the fluorescence investigated. 2P ~~ 3-P
A= I I
I 2
I 3
I
I
4
5
[Ml x IO7 Fig. 3. Titration curve: the polarisation of FITC-phosphorylase-b as a function added phosphrylase kinase. Phosphorylase-b concentration = 6.39 x lo-’ M. Experimental conditions: glycerophosphate buffer 0.05 M, pH 7.8, 30°C.
where P is the degree anisotropy
of polarisation
and A is the
Ai - A, ’ = iA,-A,)Ri
- A,
where Ai is the anisotropy observed and A, is anisotropy ofall the phosphorylase-h bound molecules. Af stands for all free molecules and /j’ is the amount
P
a"
04 02
I
2
3
4
i&l
Fig. 4. Elution profil on Sepharose 4B column (1 x I00 cm) equilibrate with 0.125 iV g~i~rophos~hate buher. 0.002 M EDTA pH 7.8,25‘C. (a) Phosphorylase kinasc 0.25 mt&ml (-- ). Phosphorylas~ kinase activity. The enzyme could be considers as a non-activated one. The specific activity of kinase is 6.5 uaits~m8- r. according the method of Cohen (1973). (b) ~~TC-la~ll~ phosphor~lase-b, 0.4 mg/ml. The number of dye binding molecule per mole protein is 2-3. ,I,. __.= 436 nm i,, = 525 nm. The other conditions are the same as in Pi8 a. (c) The mixture of both phosphorylase kinase and phosphorylase-b. Phosphorylase kinase 0.25 mgjml, f----j. phosph~~lase-b, 0.4 m8ml (----I. m Phos~~orylase kinase ~~iv~ty 5.2 units by
Fi8. 5. The fiction of bound ~hospborylase-b as a function of fhe ph~~p~o~~~e kinase added. The experimental conditions were the same as those in Fig. 2. [,~~I~~~~~ moles of phosphorylase kinase Per mole phosphorylase-b.
Meyer er al. (1970) d~c~~bed the isolation and characterized the protein-~lyc~~en c~rn~lex and sugg~t~ that this complex was a structural and functional unit of the cell. In vitro no ~t~cogen is present in the mixture and the phos~horylase-b conversion might be possible if a previous adsorption of the substrate molecule on the enzyme could be effected. This process may take place simuItaneously with that in the presence of ~lycog~n.
where R is the ratio of the quantum yield determine by the ~larization photometer. I;! and 1)” is the fluorescence intensity in the direction para1~~~ and normal to the ~larization plane. The results are given in
Fig. 5. From the equation of Weber er al. (1964) we have obtained for the stoic~iometr~ of the interaction the value of N = 2 and Kd = 1.03 x 10e6 M. This result corres~uds to the reaction schema (Krebs et ul., 1958) where two moles phosphorous-b was transformed to phosp~oryl~e-a. In the ~rnp~i~on with the Michaelis constant K, = 1.7 x 10-s M. K, has a smaller value. This is an interesting result as it may be ~surn~ that the transformation of the substrate mol~u~es is not a single process. In our inv~ti~~tio~
we observed the first sta of the conversion the adsorption of ~hospho~Ia~-b on the enzyme molecule. In t:ico the conversion reaction has perhaps another mech~~sm. It was found (Krebs et al,. 1964) that glycogen increases the unity of pho$~horyla~ kinase.
The interaction between rabbit muscle phosphorylase kinase and phosphor~Ia~-h has been investigated. By means of a fluorescence probe bound to the surface of the phos~horylase-b rnol~ul~ it has been demonstrated that the ~~larisat~on degree rises with the increase of the phosphory~~e kinase concentration The data from gel filtration on Sepharose 48 have also been presented. The interaction parameters have been calculated from the saturation curve oht~n~ by means of the equation of Ravitch & Weber. Ackn~~~~~~tfllcmen~~~Tbe author thanks Prof. Dr. F‘. KBrber. Prof. Dr. P. Sieemund and Dr. P. nietsch from the tnstitut fir Biochemye und Biophysik an dcr Freien ~niv~rsit~t in West Berlin for the possibility prov*ided of carrying out these inv~tigations and for their useful discussion.
RIWXR~M C. 0.. H~JNKELERF.
L. & KREBS E. G. (1971) The regulation of skeletal muscle phospho~la~ kinase by Ca. J. hiol. ~~~~~.246. l%t-~~9~~‘
Interaction
between
phosphorylase
COHENB. (1973) The subunit structure of rabbit-skeletalmuscle phosphorylase kinase and the molecular basis of its activation reaction. Eur. J. Biochem. 34, 1-14. DELANGER. J., KEMP R. J., RILEYW. D., COOPERR. A. & KREB~E. G. (1968) Activation of skeletal muscle phosphorylase kinase by adenosine triphosphate and adenosine 3,5-monophosphate. J. biol. Chem. 243, 22W2208. HAYAKAWAT., PERKINSJ. & KREBSE. G. (1973) Studies on the subunit structure of rabbit skeletal‘muscle phosphorylase
kinase.
Biochemistry
12. 567-573.
kinase
and phosphorylase-b
533
muscle phosphorylase-b kinase reaction. J. biol. Chem. 231, 73-83. KREBSE. G. & FISCHERE. H. (1956) Phosphorylase-b to a converting enzyme of rabbit skeletal muscle. Biochim. biophys. Acta 20, 15&157. OZAWA E., HASOIK. & EBASHIS. (1967) Reversible stimulation of muscle phosphorylase-b kinase by low concentation of calcium. J. Biochem. 61. 531. RAVITCHA. B. & WEBERG. (1972) The reversible association of lisozyme and thyroglobulin. J. biol. Chem. 247,
H;LME&R L. M. G., HASCHKEd. H., MEYERF. & FISCHER 68M85. E. G. (1970) Control of phosphorylase activity in a TIETZEF., MORTIMORG. E. & LAMAXN. R. (1962) Preparation and properties of fluorescent insulin derivatives. muscle glycogen particle. J. biol. Chem. 245, 6649-6656. Biochim. biophys. Acta 59, 336346. KREBSE. G., LOVE-D. C., BRATVOLDG. E., TRAYSERK. A., MEYERW. L. & FISCHERE. H. (1964) Purification WAHL P. & WEBERG. (1967) Fluorescence deoolarization and properties of rabbit skeletal muscle phosphorylase-b of rabbit gamma glodulin conjugates. J. m&c. Biol. 30, kinase. Biochemistry 3, 1022-1033. 371-382. KREBS E. G. (1966) Phosphorylase-b kinase from rabbit WEILMALHERBE H. & GREENR. H. (1952) Kalorimetrische muscle. Meth. Enzymol. 8, 543-546. Phosphatbestimmung mittels Ausschiitelung. Biochem. J. KREBSE. G., KENT A. B. & FISCHERE. H. (1958) The 49, 286.