Phosphorylase from Dogfish Skeletal Muscle. Purification and a Comparison of Its Physical Properties to Those of Rabbit Muscle Phosphorylase

Philip Cohen, Theresa Duewer, Edmond H. Fischer

Research output: Contribution to journalArticle

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Abstract

Large amounts of glycogen phosphorylases b and a (EC 2.4.1.1) have been purified from skeletal muscles of the Pacific dogfish (Squalus sucklii) to a state of homogeneity as judged by the criteria of ultracentrifuge analysis and poly-acrylamide gel electrophoresis. An investigation of the molecular properties showed many similarities and certain distinct differences when compared to those of rabbit skeletal muscle phosphorylases b and a. Specific activities of dogfish phosphorylase b (62 units/mg with AMP, <1 without AMP) and a (62 and 48 units per mg with and without AMP) were comparable to those of the rabbit enzymes (88 and <1 units per mg for b, and 80 and 54 for a when measured with and without AMP, respectively). Other closely similar properties for dogfish and rabbit phosphorylase b, respectively, include the absorbancy indices A1% 280 (12.9 vs. 13.1), 260/280 nm ratios (0.56 vs. 0.53), the apparent specific volumes (0.746 vs. 0.746 ± 0.002), sedimentation coefficients (8.9 S vs. 8.8 S), subunit size, and molecular weights (100,000 ± 1000 and 200,000 ± 2000 for both enzymes). Dogfish and rabbit phosphorylase b both incorporated one molecule of phosphate per subunit in the b into a conversion, and dogfish phosphorylases b and a were as good substrates for rabbit muscle phosphorylase kinase and phosphatase, respectively, as were the rabbit muscle phosphorylases themselves. Each enzyme contained one molecule of pyridoxal 5'-phosphate per subunit and interactions between this cofactor and the proteins were very similar. Dogfish phosphorylase a was only 20-25% associated from dimer to tetramer under conditions where rabbit phosphorylase a was completely in a tetrameric form. Addition of NaCl to 0.5 m promoted further tetramerization (to ca. 40%), but had no effect on the subunit interactions of rabbit phosphorylase a, although dissociation of the latter toward dimer occurred above 1.5 M NaCl. Likewise, 2 mM AMP had no effect on the s20,w or molecular weights of dogfish phosphorylase b, whereas rabbit phosphorylase b was 75-80% associated to tetramer at 10° under these conditions. In contrast to the rabbit enzymes, neither a nor b (in the presence of Mg2+ and AMP) crystallized at 0°, suggesting an inverse correlation between solubility at low temperature and tetramerization. The Km for glycogen as primer also differed markedly for dogfish and rabbit phosphorylase b (0.12 and 0.018%, respectively). The subunit molecular weight obtained for phosphorylases from both species is 8% higher than had previously been reported for the rabbit muscle enzyme. The reasons for a revision in both the size of the polypeptide chain and the nature of the interactions between subunits is discussed, together with possible implications of the differences observed between the rabbit and dogfish enzymes.

Original languageEnglish
Pages (from-to)2683-2694
Number of pages12
JournalBiochemistry
Volume10
Issue number14
DOIs
Publication statusPublished - 1 Jul 1971

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Phosphorylase b
Dogfish
Phosphorylases
Phosphorylase a
Purification
Muscle
Skeletal Muscle
Physical properties
Adenosine Monophosphate
Rabbits
Muscles
Enzymes
Molecular weight
Dimers
Molecular Weight
Glycogen Phosphorylase
Squalus
Molecules
Pyridoxal Phosphate
Polyacrylates

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@article{336cc7c20e2c4134939eb8a3ed6aa76a,
title = "Phosphorylase from Dogfish Skeletal Muscle. Purification and a Comparison of Its Physical Properties to Those of Rabbit Muscle Phosphorylase",
abstract = "Large amounts of glycogen phosphorylases b and a (EC 2.4.1.1) have been purified from skeletal muscles of the Pacific dogfish (Squalus sucklii) to a state of homogeneity as judged by the criteria of ultracentrifuge analysis and poly-acrylamide gel electrophoresis. An investigation of the molecular properties showed many similarities and certain distinct differences when compared to those of rabbit skeletal muscle phosphorylases b and a. Specific activities of dogfish phosphorylase b (62 units/mg with AMP, <1 without AMP) and a (62 and 48 units per mg with and without AMP) were comparable to those of the rabbit enzymes (88 and <1 units per mg for b, and 80 and 54 for a when measured with and without AMP, respectively). Other closely similar properties for dogfish and rabbit phosphorylase b, respectively, include the absorbancy indices A1{\%} 280 (12.9 vs. 13.1), 260/280 nm ratios (0.56 vs. 0.53), the apparent specific volumes (0.746 vs. 0.746 ± 0.002), sedimentation coefficients (8.9 S vs. 8.8 S), subunit size, and molecular weights (100,000 ± 1000 and 200,000 ± 2000 for both enzymes). Dogfish and rabbit phosphorylase b both incorporated one molecule of phosphate per subunit in the b into a conversion, and dogfish phosphorylases b and a were as good substrates for rabbit muscle phosphorylase kinase and phosphatase, respectively, as were the rabbit muscle phosphorylases themselves. Each enzyme contained one molecule of pyridoxal 5'-phosphate per subunit and interactions between this cofactor and the proteins were very similar. Dogfish phosphorylase a was only 20-25{\%} associated from dimer to tetramer under conditions where rabbit phosphorylase a was completely in a tetrameric form. Addition of NaCl to 0.5 m promoted further tetramerization (to ca. 40{\%}), but had no effect on the subunit interactions of rabbit phosphorylase a, although dissociation of the latter toward dimer occurred above 1.5 M NaCl. Likewise, 2 mM AMP had no effect on the s20,w or molecular weights of dogfish phosphorylase b, whereas rabbit phosphorylase b was 75-80{\%} associated to tetramer at 10° under these conditions. In contrast to the rabbit enzymes, neither a nor b (in the presence of Mg2+ and AMP) crystallized at 0°, suggesting an inverse correlation between solubility at low temperature and tetramerization. The Km for glycogen as primer also differed markedly for dogfish and rabbit phosphorylase b (0.12 and 0.018{\%}, respectively). The subunit molecular weight obtained for phosphorylases from both species is 8{\%} higher than had previously been reported for the rabbit muscle enzyme. The reasons for a revision in both the size of the polypeptide chain and the nature of the interactions between subunits is discussed, together with possible implications of the differences observed between the rabbit and dogfish enzymes.",
author = "Philip Cohen and Theresa Duewer and Fischer, {Edmond H.}",
year = "1971",
month = "7",
day = "1",
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language = "English",
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pages = "2683--2694",
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Phosphorylase from Dogfish Skeletal Muscle. Purification and a Comparison of Its Physical Properties to Those of Rabbit Muscle Phosphorylase. / Cohen, Philip; Duewer, Theresa; Fischer, Edmond H.

In: Biochemistry, Vol. 10, No. 14, 01.07.1971, p. 2683-2694.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Phosphorylase from Dogfish Skeletal Muscle. Purification and a Comparison of Its Physical Properties to Those of Rabbit Muscle Phosphorylase

AU - Cohen, Philip

AU - Duewer, Theresa

AU - Fischer, Edmond H.

PY - 1971/7/1

Y1 - 1971/7/1

N2 - Large amounts of glycogen phosphorylases b and a (EC 2.4.1.1) have been purified from skeletal muscles of the Pacific dogfish (Squalus sucklii) to a state of homogeneity as judged by the criteria of ultracentrifuge analysis and poly-acrylamide gel electrophoresis. An investigation of the molecular properties showed many similarities and certain distinct differences when compared to those of rabbit skeletal muscle phosphorylases b and a. Specific activities of dogfish phosphorylase b (62 units/mg with AMP, <1 without AMP) and a (62 and 48 units per mg with and without AMP) were comparable to those of the rabbit enzymes (88 and <1 units per mg for b, and 80 and 54 for a when measured with and without AMP, respectively). Other closely similar properties for dogfish and rabbit phosphorylase b, respectively, include the absorbancy indices A1% 280 (12.9 vs. 13.1), 260/280 nm ratios (0.56 vs. 0.53), the apparent specific volumes (0.746 vs. 0.746 ± 0.002), sedimentation coefficients (8.9 S vs. 8.8 S), subunit size, and molecular weights (100,000 ± 1000 and 200,000 ± 2000 for both enzymes). Dogfish and rabbit phosphorylase b both incorporated one molecule of phosphate per subunit in the b into a conversion, and dogfish phosphorylases b and a were as good substrates for rabbit muscle phosphorylase kinase and phosphatase, respectively, as were the rabbit muscle phosphorylases themselves. Each enzyme contained one molecule of pyridoxal 5'-phosphate per subunit and interactions between this cofactor and the proteins were very similar. Dogfish phosphorylase a was only 20-25% associated from dimer to tetramer under conditions where rabbit phosphorylase a was completely in a tetrameric form. Addition of NaCl to 0.5 m promoted further tetramerization (to ca. 40%), but had no effect on the subunit interactions of rabbit phosphorylase a, although dissociation of the latter toward dimer occurred above 1.5 M NaCl. Likewise, 2 mM AMP had no effect on the s20,w or molecular weights of dogfish phosphorylase b, whereas rabbit phosphorylase b was 75-80% associated to tetramer at 10° under these conditions. In contrast to the rabbit enzymes, neither a nor b (in the presence of Mg2+ and AMP) crystallized at 0°, suggesting an inverse correlation between solubility at low temperature and tetramerization. The Km for glycogen as primer also differed markedly for dogfish and rabbit phosphorylase b (0.12 and 0.018%, respectively). The subunit molecular weight obtained for phosphorylases from both species is 8% higher than had previously been reported for the rabbit muscle enzyme. The reasons for a revision in both the size of the polypeptide chain and the nature of the interactions between subunits is discussed, together with possible implications of the differences observed between the rabbit and dogfish enzymes.

AB - Large amounts of glycogen phosphorylases b and a (EC 2.4.1.1) have been purified from skeletal muscles of the Pacific dogfish (Squalus sucklii) to a state of homogeneity as judged by the criteria of ultracentrifuge analysis and poly-acrylamide gel electrophoresis. An investigation of the molecular properties showed many similarities and certain distinct differences when compared to those of rabbit skeletal muscle phosphorylases b and a. Specific activities of dogfish phosphorylase b (62 units/mg with AMP, <1 without AMP) and a (62 and 48 units per mg with and without AMP) were comparable to those of the rabbit enzymes (88 and <1 units per mg for b, and 80 and 54 for a when measured with and without AMP, respectively). Other closely similar properties for dogfish and rabbit phosphorylase b, respectively, include the absorbancy indices A1% 280 (12.9 vs. 13.1), 260/280 nm ratios (0.56 vs. 0.53), the apparent specific volumes (0.746 vs. 0.746 ± 0.002), sedimentation coefficients (8.9 S vs. 8.8 S), subunit size, and molecular weights (100,000 ± 1000 and 200,000 ± 2000 for both enzymes). Dogfish and rabbit phosphorylase b both incorporated one molecule of phosphate per subunit in the b into a conversion, and dogfish phosphorylases b and a were as good substrates for rabbit muscle phosphorylase kinase and phosphatase, respectively, as were the rabbit muscle phosphorylases themselves. Each enzyme contained one molecule of pyridoxal 5'-phosphate per subunit and interactions between this cofactor and the proteins were very similar. Dogfish phosphorylase a was only 20-25% associated from dimer to tetramer under conditions where rabbit phosphorylase a was completely in a tetrameric form. Addition of NaCl to 0.5 m promoted further tetramerization (to ca. 40%), but had no effect on the subunit interactions of rabbit phosphorylase a, although dissociation of the latter toward dimer occurred above 1.5 M NaCl. Likewise, 2 mM AMP had no effect on the s20,w or molecular weights of dogfish phosphorylase b, whereas rabbit phosphorylase b was 75-80% associated to tetramer at 10° under these conditions. In contrast to the rabbit enzymes, neither a nor b (in the presence of Mg2+ and AMP) crystallized at 0°, suggesting an inverse correlation between solubility at low temperature and tetramerization. The Km for glycogen as primer also differed markedly for dogfish and rabbit phosphorylase b (0.12 and 0.018%, respectively). The subunit molecular weight obtained for phosphorylases from both species is 8% higher than had previously been reported for the rabbit muscle enzyme. The reasons for a revision in both the size of the polypeptide chain and the nature of the interactions between subunits is discussed, together with possible implications of the differences observed between the rabbit and dogfish enzymes.

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U2 - 10.1021/bi00790a005

DO - 10.1021/bi00790a005

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