- Email: [email protected]
The isozymes of glycogen phosphorylase in human and rabbit tissues. II. Electrofocusing in polyacrylamide gels
211
Clinica Chimica Acta, 57 (1974) 211-216 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6688
THE ISOZYMES OF GLYCOGEN PHOSPHORYLASE IN HUMAN AND RABBIT TISSUES. II. ELECTROFOCUSING IN POLYACRYLAMIDE GELS
D. PROUX,
M. VIBERT,
M.C. MEIENHOFER
and J.C. DREYFUS
Institut de Pathologie Mok?culaire*, 24, rue du Faubourg Saint Jacques, Paris 75014 (France) (Received
June 13,1974)
Summary We have investigated the isozymic pattern of glycogen phosphorylase by isoelectric focusing on polyacrylamide gel in various tissues of rabbit and man. 1. This study confirms the presence of three major isozymes of phosphorylase, i.e. muscle, liver and brain types, which can be separated by electrophoresis and the use of specific antisera. 2. It is confirmed that white blood cells and kidney cells contain mainly the liver type enzyme. Platelets and placenta extracts show a more complex pattern: brain and liver type phosphorylases are present, with an intermediary band which is probably a previously unrecognized hybrid between liver and brain types, since it disappears after incubation with an antiliver phosphorylase antiserum.
Introduction In a previous paper [ 11 we described the heterogeneity of human glycogen phosphorylase (EC 2.4.1.1) as observed by polyacrylamide gel electrophoresis, combined with immunological reactivity towards antisera directed against muscle or liver phosphorylase. This study led to the recognition of three major isozymes, i.e. muscle, liver and brain types. A further finding was that the liver form is prevalent in human tissues, while the brain type is the most common isozyme in the rabbit. Such a finding was unexpected: it was known [2] that phosphorylase of white blood cells is of the liver type. In contrast, platelet enzyme had been first regarded as being the muscle type [3] but this was not supported by genetic considerations, since platelet phosphorylase is normal in muscle phosphorylase deficiency [4-61. In addition we found no effect of antimuscle phosphorylase antiserum on the platelet enzyme [ 1,5] . * Universitd Paris V, Unit6 129 de l?nstitut toire asso&
national de la sant6 et de la recherche m6dicale, Laboraau Centxe National de la recherche scientifique.
212
The fact that platelets, placenta and fibroblasts showed the liver form seemed to be worthy of further investigation. Electrofocusing in a pH gradient seemed to provide a more sensitive tool to explore the problem. This paper describes the results obtained using this technique. Material and Methods Tissues Only fresh tissues were used. Most experiments were made on human tissues. Muscle, liver and brain were obtained by biopsy as in the previous study [l] . Placenta was obtained within 1 h after delivery. White blood cells and platelets were prepared according to conventional methods. Fibroblasts were grown from an embryonic human diploid cell line on Eagle medium supplemented with fetal calf serum. For comparison muscle, liver and brain were taken from rabbits. Methods Tissues or cells were frozen, then extracted by grinding in a Potter Elvehjem apparatus with cold water containing 80 mM mercaptoethanol. The homogenate was centrifuged at 23 000 X g for 10 min. Extracts were adjusted so that 0.1-0.4 unit of phosphorylase was applied to the gel. Enzyme activity was determined according to Cori et al. [ 71. Electrofocusing was performed according to Drysdale et al. [B] . Acrylamide concentration was 7.5% in an ampholine pH gradient 3.5-10. Six to twelve gels were run simultaneously. No glycogen was included in the gel, because it retarded the migration of phosphorylase to such an extent that no equilibrium was possible. Electrophoresis was performed for 16 h at 200 V. Intensity was initially 1 mA/gel, then dropped to 0.1 mA/gel. After completion of the run the gels were incubated for 24 h in a solution containing: glycogen 1% and glucose l-phosphate (G-1-P) 0.02 M; eventually adenosine monophosphate (AMP) 0.01 M and/or sodium sulfate 0.8 M were added. These reagents allow one to recognize the various types of the enzyme. The a form of phosphorylase of all sources is active in the presence of glycogen and G-1-P alone. The b form of muscle and brain types requires the addition of AMP. The b form of liver is inactive but is activated by the addition of sodium sulfate and AMP [9] . Staining took place at the end of the incubation period as follows: the gels were immersed in an iodine solution (0.01 M buffered at pH 5) for a few minutes, followed by rinsing with water to eliminate excess iodine, and then photographed. Antisera to rabbit liver phosphorylase were prepared in roosters. Muscle phosphorylase was crystallized according to Yunis et al. [lo]. Liver phosphorylase was purified using the technique of Appleman et al. [9] . Results (I) The results for muscle, liver and brain are typical and can be compared to those of conventional polyacrylamide electrophoresis (Figs 1 and 2).
213
Fig. 1. Human tissues.
Muscle Muscle extracts showed either one or two well separated bands, in man as well as in rabbit. When the cathodic band was present it stained with G-1-P alone as the substrate, while the anodic band reacted only in the presence of AMP. It is apparent that phosphorylase a migrated more slowly than phosphorylase b. Liver In contrast liver extracts did not stain without added AMP and Na, SO4 and it was not possible to define an isoelectric position for liver phosphorylase a. Liver phosphorylase b migrated more anodically than the muscle enzyme, but the difference was less in human than in rabbit tissues. In the best experiments, human liver phosphorylase was resolved into several discrete bands, which never happened with rabbit liver or crude muscle extracts, but was observed with purified rabbit muscle phosphorylase b.
214
PH
+AMP
Fig. 2. Rabbit
tissues.
Bra in Brain extracts showed either only one band which was more acidic than those of muscle and liver, or one or two more bands, the most basic of which was at the muscle position and the other intermediary to “muscle” and “brain” enzymes. The isoelectric points of the major bands of phosphorylase b from human muscle, liver and brain were 6.3 for muscle phosphorylase, between 6.3 and 6.1 for liver phosphorylase and 5.6 for brain phosphorylase. (II) In other cells or tissues the results were more complex. (a) In extracts of white blood cells the major band was of the liver type. In addition a small band was at the position of the brain enzyme. It must be noted that no attempt was made to separate granulocytes and lymphocytes. (b) The pattern of blood platelets and of placenta extracts was different from that observed in ordinary polyacrylamide gel electrophoresis. At least three bands were observed. The major one migrated like the brain enzyme. Two other bands at least could be seen: one was at the liver position and the other was intermediary. An antiserum against liver phosphorylase resulted in the disappearance of the two cathodic bands (Fig. 3).
Fig. 3. Effect of antiliver phosphorylase serum on various human tissue extracts. ns, normal serum; as, antiserum. Fig. 4. Human fibroblasts at different days of subculture (3 and 8 days, respectively).
(c) Fibroblasts (Fig. 4). were generally harvested at the confluent stage, after 6 or 7 days of subculture. They showed a pattern very similar to that of platelets or placenta: one band was of the liver type, one intermedi~, and the more anodie migrated like the brain type. In more recent experiments we have also examined extracts from fibroblasts harvested after 3 days of subculture, when cells were still dividing: only the brain enzyme was generally to be seen, sometimes with a faint intermediary band and no liver form, Discussion The technique of electrofocusing in a pH gradient allowed a more diseriminative analysis of phosphorylase isozyme distribution than single polyacrylamide gel electrophoresis. It was sensitive enough to make the analysis possible on a small sample of biopsy material or a fibroblast culture. The results were technically satisfactory but were sometimes difficult to interpret.
216
(1) The three major types, muscle, liver and brain, still clearly emerge, the brain type having a much lower isoelectric point (similar for example to aldolase or creatine kinase). Muscle-brain hybrids could also be recognized in heart (not shown) or brain extracts. The relative position of a and b forms can only be discussed for muscle. In muscle extracts the a form was more cathodic than phosphorylase b. The most likely interpretation was that a form of muscle phosphorylase was at least in part tetrameric, which slowed down its migration, and that the tetramer was unable to reach its true equilibrium position in the time allotted for electrophoresis. This time could not be increased since after 15 h the gradient began to lose its stability [ 111. (2) In other tissues which seemed to be almost entirely of the liver type upon polyacrylamide gel electrophoresis, new differences appeared. White blood cells and kidney extracts still showed a major liver band, but platelets, placenta and fibroblasts did not. Their major band was now of the brain type, while a fainter liver band and a well stained intermediary band showed up. An antiserum against liver phosphorylase inhibited both the liver and intermediary bands and left unchanged the most acidic isozyme. The intermediary band therefore seemed to be a hybrid between the liver and brain isozymes. Such a hybrid has not yet been described. It is likely that interactions between liver and brain forms can take place and slow down the migration on conventional polyacrylamide gel electrophoresis, especially when glycogen is included in the gel, which greatly improves the sensitivity of the method. (3) Fibroblasts grown from human skin showed a pattern similar to that of platelets or placenta when taken at the confluent phase: the liver type was present, with or without the intermediary band. In extracts from cells at the growing phase the liver type was absent and only the most acidic band could be seen (Fig. 4). A more specific isozyme, therefore, only appeared when the growth of the culture had stopped. In preliminary results, dexamethasone (10 pg/ml) seemed to accelerate the appearance of the liver type band. These results are compatible with the findings of Koster et al. [ 121, showing that total fibroblast phosphorylase activity is normal in a case of liver phosphorylase deficiency. References 1
D. Proux
2
E.H.
and J.C.
Williams
3
A.A.
Yuois
4
J.C.
Dreyfus
5
J.P.
Grunfeld,
Dreyfus.
and J.B. and
Clin.
Field,
G.K.
Arimura.
Y.
Alexandre,
and D.
Ganeval,
Chim.
Acta,
Metabolism,
48
12
Biochem.
(1973)
(1963)
Biophys.
Biochem.
J. Chanard,
Res.
Biophys. M.
167-172
464 Commun..
Res.
Fardeau
33
Commun..
and
J.C.
(1968)
44
Dreyfus,
119
(1971) New
1364 Eng.
J. Med.,
286
(1972)
1237 6
P.D.
Leathwood
7
G.T.
Cori,
8
J.W.
Drysdale,
9
M.M.
10
A.A.
11
A.
12
and
Yunis,
E.G.
Chrambach.
Sci.,
209
J.F.
Koster,
E.G.
and
and
E.H.
G.R.
E.H.
40
Fisher,
261
Enzymol.,
Biochim.
Fisher,
Finlayson,
(1971)
Methods
Biophys.
Biochemistry,
J. Biol. L.E.M.
Chem., Miles.
1 (1955) Acta, 5 (1966)
235
(1960)
R. Sherins
200 229
(1971)
42
2101 3163 and
R.
Rodbard.
Ann.
N.Y.
Acad.
44
J. Fernandes. 53
Sci..
H. Franklin,
Krebs
Krebs
CU.
P.J. Keller,
and
P. Doerr.
(1973)
Ryman, and
P. Righetti
Appleman,
Commun.,
B.E.
B. Illingworth
(1973)
282
R.G.
Slee,
Th.
J.C.
van Berkel
and W.C.
Hulsmann,
Biochem.
Biophys.
Res.
Clinica Chimica Acta, 57 (1974) 211-216 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 6688
THE ISOZYMES OF GLYCOGEN PHOSPHORYLASE IN HUMAN AND RABBIT TISSUES. II. ELECTROFOCUSING IN POLYACRYLAMIDE GELS
D. PROUX,
M. VIBERT,
M.C. MEIENHOFER
and J.C. DREYFUS
Institut de Pathologie Mok?culaire*, 24, rue du Faubourg Saint Jacques, Paris 75014 (France) (Received
June 13,1974)
Summary We have investigated the isozymic pattern of glycogen phosphorylase by isoelectric focusing on polyacrylamide gel in various tissues of rabbit and man. 1. This study confirms the presence of three major isozymes of phosphorylase, i.e. muscle, liver and brain types, which can be separated by electrophoresis and the use of specific antisera. 2. It is confirmed that white blood cells and kidney cells contain mainly the liver type enzyme. Platelets and placenta extracts show a more complex pattern: brain and liver type phosphorylases are present, with an intermediary band which is probably a previously unrecognized hybrid between liver and brain types, since it disappears after incubation with an antiliver phosphorylase antiserum.
Introduction In a previous paper [ 11 we described the heterogeneity of human glycogen phosphorylase (EC 2.4.1.1) as observed by polyacrylamide gel electrophoresis, combined with immunological reactivity towards antisera directed against muscle or liver phosphorylase. This study led to the recognition of three major isozymes, i.e. muscle, liver and brain types. A further finding was that the liver form is prevalent in human tissues, while the brain type is the most common isozyme in the rabbit. Such a finding was unexpected: it was known [2] that phosphorylase of white blood cells is of the liver type. In contrast, platelet enzyme had been first regarded as being the muscle type [3] but this was not supported by genetic considerations, since platelet phosphorylase is normal in muscle phosphorylase deficiency [4-61. In addition we found no effect of antimuscle phosphorylase antiserum on the platelet enzyme [ 1,5] . * Universitd Paris V, Unit6 129 de l?nstitut toire asso&
national de la sant6 et de la recherche m6dicale, Laboraau Centxe National de la recherche scientifique.
212
The fact that platelets, placenta and fibroblasts showed the liver form seemed to be worthy of further investigation. Electrofocusing in a pH gradient seemed to provide a more sensitive tool to explore the problem. This paper describes the results obtained using this technique. Material and Methods Tissues Only fresh tissues were used. Most experiments were made on human tissues. Muscle, liver and brain were obtained by biopsy as in the previous study [l] . Placenta was obtained within 1 h after delivery. White blood cells and platelets were prepared according to conventional methods. Fibroblasts were grown from an embryonic human diploid cell line on Eagle medium supplemented with fetal calf serum. For comparison muscle, liver and brain were taken from rabbits. Methods Tissues or cells were frozen, then extracted by grinding in a Potter Elvehjem apparatus with cold water containing 80 mM mercaptoethanol. The homogenate was centrifuged at 23 000 X g for 10 min. Extracts were adjusted so that 0.1-0.4 unit of phosphorylase was applied to the gel. Enzyme activity was determined according to Cori et al. [ 71. Electrofocusing was performed according to Drysdale et al. [B] . Acrylamide concentration was 7.5% in an ampholine pH gradient 3.5-10. Six to twelve gels were run simultaneously. No glycogen was included in the gel, because it retarded the migration of phosphorylase to such an extent that no equilibrium was possible. Electrophoresis was performed for 16 h at 200 V. Intensity was initially 1 mA/gel, then dropped to 0.1 mA/gel. After completion of the run the gels were incubated for 24 h in a solution containing: glycogen 1% and glucose l-phosphate (G-1-P) 0.02 M; eventually adenosine monophosphate (AMP) 0.01 M and/or sodium sulfate 0.8 M were added. These reagents allow one to recognize the various types of the enzyme. The a form of phosphorylase of all sources is active in the presence of glycogen and G-1-P alone. The b form of muscle and brain types requires the addition of AMP. The b form of liver is inactive but is activated by the addition of sodium sulfate and AMP [9] . Staining took place at the end of the incubation period as follows: the gels were immersed in an iodine solution (0.01 M buffered at pH 5) for a few minutes, followed by rinsing with water to eliminate excess iodine, and then photographed. Antisera to rabbit liver phosphorylase were prepared in roosters. Muscle phosphorylase was crystallized according to Yunis et al. [lo]. Liver phosphorylase was purified using the technique of Appleman et al. [9] . Results (I) The results for muscle, liver and brain are typical and can be compared to those of conventional polyacrylamide electrophoresis (Figs 1 and 2).
213
Fig. 1. Human tissues.
Muscle Muscle extracts showed either one or two well separated bands, in man as well as in rabbit. When the cathodic band was present it stained with G-1-P alone as the substrate, while the anodic band reacted only in the presence of AMP. It is apparent that phosphorylase a migrated more slowly than phosphorylase b. Liver In contrast liver extracts did not stain without added AMP and Na, SO4 and it was not possible to define an isoelectric position for liver phosphorylase a. Liver phosphorylase b migrated more anodically than the muscle enzyme, but the difference was less in human than in rabbit tissues. In the best experiments, human liver phosphorylase was resolved into several discrete bands, which never happened with rabbit liver or crude muscle extracts, but was observed with purified rabbit muscle phosphorylase b.
214
PH
+AMP
Fig. 2. Rabbit
tissues.
Bra in Brain extracts showed either only one band which was more acidic than those of muscle and liver, or one or two more bands, the most basic of which was at the muscle position and the other intermediary to “muscle” and “brain” enzymes. The isoelectric points of the major bands of phosphorylase b from human muscle, liver and brain were 6.3 for muscle phosphorylase, between 6.3 and 6.1 for liver phosphorylase and 5.6 for brain phosphorylase. (II) In other cells or tissues the results were more complex. (a) In extracts of white blood cells the major band was of the liver type. In addition a small band was at the position of the brain enzyme. It must be noted that no attempt was made to separate granulocytes and lymphocytes. (b) The pattern of blood platelets and of placenta extracts was different from that observed in ordinary polyacrylamide gel electrophoresis. At least three bands were observed. The major one migrated like the brain enzyme. Two other bands at least could be seen: one was at the liver position and the other was intermediary. An antiserum against liver phosphorylase resulted in the disappearance of the two cathodic bands (Fig. 3).
Fig. 3. Effect of antiliver phosphorylase serum on various human tissue extracts. ns, normal serum; as, antiserum. Fig. 4. Human fibroblasts at different days of subculture (3 and 8 days, respectively).
(c) Fibroblasts (Fig. 4). were generally harvested at the confluent stage, after 6 or 7 days of subculture. They showed a pattern very similar to that of platelets or placenta: one band was of the liver type, one intermedi~, and the more anodie migrated like the brain type. In more recent experiments we have also examined extracts from fibroblasts harvested after 3 days of subculture, when cells were still dividing: only the brain enzyme was generally to be seen, sometimes with a faint intermediary band and no liver form, Discussion The technique of electrofocusing in a pH gradient allowed a more diseriminative analysis of phosphorylase isozyme distribution than single polyacrylamide gel electrophoresis. It was sensitive enough to make the analysis possible on a small sample of biopsy material or a fibroblast culture. The results were technically satisfactory but were sometimes difficult to interpret.
216
(1) The three major types, muscle, liver and brain, still clearly emerge, the brain type having a much lower isoelectric point (similar for example to aldolase or creatine kinase). Muscle-brain hybrids could also be recognized in heart (not shown) or brain extracts. The relative position of a and b forms can only be discussed for muscle. In muscle extracts the a form was more cathodic than phosphorylase b. The most likely interpretation was that a form of muscle phosphorylase was at least in part tetrameric, which slowed down its migration, and that the tetramer was unable to reach its true equilibrium position in the time allotted for electrophoresis. This time could not be increased since after 15 h the gradient began to lose its stability [ 111. (2) In other tissues which seemed to be almost entirely of the liver type upon polyacrylamide gel electrophoresis, new differences appeared. White blood cells and kidney extracts still showed a major liver band, but platelets, placenta and fibroblasts did not. Their major band was now of the brain type, while a fainter liver band and a well stained intermediary band showed up. An antiserum against liver phosphorylase inhibited both the liver and intermediary bands and left unchanged the most acidic isozyme. The intermediary band therefore seemed to be a hybrid between the liver and brain isozymes. Such a hybrid has not yet been described. It is likely that interactions between liver and brain forms can take place and slow down the migration on conventional polyacrylamide gel electrophoresis, especially when glycogen is included in the gel, which greatly improves the sensitivity of the method. (3) Fibroblasts grown from human skin showed a pattern similar to that of platelets or placenta when taken at the confluent phase: the liver type was present, with or without the intermediary band. In extracts from cells at the growing phase the liver type was absent and only the most acidic band could be seen (Fig. 4). A more specific isozyme, therefore, only appeared when the growth of the culture had stopped. In preliminary results, dexamethasone (10 pg/ml) seemed to accelerate the appearance of the liver type band. These results are compatible with the findings of Koster et al. [ 121, showing that total fibroblast phosphorylase activity is normal in a case of liver phosphorylase deficiency. References 1
D. Proux
2
E.H.
and J.C.
Williams
3
A.A.
Yuois
4
J.C.
Dreyfus
5
J.P.
Grunfeld,
Dreyfus.
and J.B. and
Clin.
Field,
G.K.
Arimura.
Y.
Alexandre,
and D.
Ganeval,
Chim.
Acta,
Metabolism,
48
12
Biochem.
(1973)
(1963)
Biophys.
Biochem.
J. Chanard,
Res.
Biophys. M.
167-172
464 Commun..
Res.
Fardeau
33
Commun..
and
J.C.
(1968)
44
Dreyfus,
119
(1971) New
1364 Eng.
J. Med.,
286
(1972)
1237 6
P.D.
Leathwood
7
G.T.
Cori,
8
J.W.
Drysdale,
9
M.M.
10
A.A.
11
A.
12
and
Yunis,
E.G.
Chrambach.
Sci.,
209
J.F.
Koster,
E.G.
and
and
E.H.
G.R.
E.H.
40
Fisher,
261
Enzymol.,
Biochim.
Fisher,
Finlayson,
(1971)
Methods
Biophys.
Biochemistry,
J. Biol. L.E.M.
Chem., Miles.
1 (1955) Acta, 5 (1966)
235
(1960)
R. Sherins
200 229
(1971)
42
2101 3163 and
R.
Rodbard.
Ann.
N.Y.
Acad.
44
J. Fernandes. 53
Sci..
H. Franklin,
Krebs
Krebs
CU.
P.J. Keller,
and
P. Doerr.
(1973)
Ryman, and
P. Righetti
Appleman,
Commun.,
B.E.
B. Illingworth
(1973)
282
R.G.
Slee,
Th.
J.C.
van Berkel
and W.C.
Hulsmann,
Biochem.
Biophys.
Res.