Purification and characterization of native and proteolytic forms of rabbit liver phosphorylase kinase.
1. Two forms of phosphorylase kinase having mol. wt of 1,260,000 (form I) and 205,000 (form II) have been identified by gel filtration chromatography of rabbit liver crude extracts. 2. Form I was the majority when the homogenization buffer was supplemented with a mixture of proteinase inhibitors. This form has been purified through a protocol including ultracentrifugation, gel filtration and affinity chromatography on Sepharose-heparin. 3. Form II was purified by a combination of chromatographic procedures including ion exchange, gel filtration and affinity chromatography on Sepharose-Blue Dextran and Sepharose-histone. 4. Upon electrophoresis in the presence of sodium dodecyl sulfate two subunits of 69,000 and 44,000 were identified for this low molecular weight enzyme. Thus, a tetrameric structure comprising two subunits of each kind can be proposed. 5. Treatment of form I with either trypsin or chymotrypsin gave an active fragment having a molecular weight similar to that of form II. On the contrary, other dissociating treatments with salts, thiols and detergents failed in producing forms of lower molecular weight. 6. The similarities between proteolyzed forms I and II were stressed by their behavior in front of antibodies raised against the muscle isoenzyme of phosphorylase kinase. 7. The study of the effect of magnesium and fluoride ions on the activity of both forms showed an inhibitory effect of magnesium when its concentration exceeded that of ATP. 8. The inhibition could nevertheless be reverted by including 50 mM NaF in the reaction mixture. 9. Form I and form II could be distinguished by their pH dependence in the presence of an excess of magnesium ions over ATP, whereas the affinity for both substrates was not significantly different.
Departamento de Bioquímica y Biología Molecular, Universidad Autónoma de Barcelona, España.
J
J
Beleta
P
P
Benedicto
P
P
Aymerich
F J
FJ
Gella
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010447:Peptide Hydrolases / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D012969:Sodium Fluoride / Q000494:pharmacology; D014461:Ultracentrifugation
D000906:Antibodies; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D010764:Phosphorylase Kinase; D010447:Peptide Hydrolases; D008274:Magnesium