Crystallization of pig skeletal phosphorylase b. Purification, physical and catalytic characterization.
A new method for purification and crystallization of pig skeletal muscle phosphorylase b is presented. The ease of crystallization in the presence of 1 mM AMP and 1 mM spermine has permitted the study of some physical, chemical and enzymatic properties of the enzyme. The crystalline pig phosphorylase b gave a single band on SDS polyacrylamide gels of the same mobility as rabbit muscle phosphorylase subunit. Ultracentrifugation experiments showed that pig phosphorylase b exists in a dimeric form (S20,w = 8.4 S). No association occurred at 20 degrees C under conditions where rabbit phosphorylase b can be tetramerized; pig phosphorylase b was only 30% associated from dimer to tetramer at 13 degrees C. Pig phosphorylase b is highly stable to freezing and its specific activity did not change appreciably upon prolonged storage in the cold. Pig and rabbit phosphorylases b have comparable Vmax and Km values towards the substrate and the activator. However, there is an essential difference between the two enzymes in that pig phosphorylase b is not significantly inhibited by glucose 6-phosphate, which is a powerful inhibitor of the rabbit enzyme. Two different crystal forms of pig phosphorylase b were obtained which are small for X-ray diffraction studies. Diffusion of spermine into tetragonal crystals of rabbit phosphorylase b resulted in a difference Fourier synthesis at 3 A resolution that showed no strong indication of specific binding.
D000818:Animals; D003460:Crystallization; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004355:Drug Stability; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010763:Phosphorylase b / Q000302:isolation & purification* / Q000378:metabolism; D006005:Phosphorylases / Q000302:isolation & purification*; D011485:Protein Binding; D011817:Rabbits; D013045:Species Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis; D013552:Swine
D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D010763:Phosphorylase b; D006005:Phosphorylases