Glycogen phosphorylase (EC 184.108.40.206) a and b forms of silky shark (Carcharhinus falciformis) were crystallized and characterized. Unique in the purification of phosphorylase from the muscle of this shark is the fractionation with ammonium sulfate at a temperature of 40°. The ease of crystallization from dilute buffer and the remarkable stability of shark phosphorylase a and b in the absence of AMP and thiol reducing compounds allowed an extensive study of their kinetic and association-dissociation properties. Shark phosphorylase a was found to exist in a dimeric form (mol wt = 1.7-2.0 X 105 g/mol; S20,w = 9 S) under conditions where rabbit phosphorylase a remains a tetramer. However, shark phosphorylase a can be tetramerized at high protein concentration (6 6 mg/ml) and cold temperature (≤ 12°). This latter property of the shark enzyme contrasts sharply with lobster phosphorylase a, a dimer (Cowgill, R. W. (1959), J. Biol. Chem. 234, 3146) which has been shown to resist tetramerization (Assaf, S. A., and Graves, D. J. (1969), J. Biol. Chem. 244, 5544). Gel electrophoresis of shark, rabbit, and human muscle phosphorylase b in the presence of sodium dodecyl sulfate and 2-mercaptoethanol showed that these enzymes are made up of subunits of identical molecular weight (9.5 X 104). Shark phosphorylase b, which exists as a dimer when dissolved in buffer and examined at a wide range of temperature and protein concentration, was found to undergo association in the presence of AMP, Pi, and AMP, or AMP and NaF. But when compared to rabbit phosphorylase b which tetramerizes easily, the shark b enzyme like the a form seems to prefer a dimeric quaternary structure. Phosphorylase from silky shark was found to be immunologically similar to that crystallized from lemon shark (Negaprion brevirostris) but immunologically different from that of rabbit or human. The amino acid compositions of silky and lemon shark phosphorylases were essentially the same except for a minor difference in isoleucine. The content of arginine, leucine, and proline in phosphorylase from the sharks was significantly lower than that found for rabbit. However, the isoleucine content was significantly less in the latter. Considering all the published amino acid compositions for muscle phosphorylase of homo-iotherms and poikilotherms, one finds that the enzyme from the latter contains much less arginine. The kinetic behavior of shark phosphorylase a and b at 30 and 0° was investigated. Although both enzyme forms have a similar maximal velocity at 30°, at 0° the maximal velocity of the a form was double that of the b. Shark phosphorylase b, whose activity is dependent on AMP when studied in the direction of glycogen synthesis, was found to be partially active when examined in the direction of glycogen degradation at high Pi and in the absence of AMP as reported for rabbit and lobster phosphorylase b. The shark b enzyme also exhibited a deviation in its kinetics when studied at a wide range of concentrations of AMP; it showed a greater activator affinity at the low micromolar AMP levels. Except for the high Km value of shark phosphorylase a and b toward Pi as found in lobster phosphorylase b, the Km values for substrates and activator are more in keeping with those reported by many workers for rabbit phosphorylase a and b.
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