Quantification and removal of glycogen phosphorylase and other enzymes associated with sarcoplasmic reticulum membrane preparations.
The enzymatic characterization of sarcoplasmic reticulum membrane fragments from rabbit skeletal muscle presented in this paper shows that glycogen phosphorylase, as well as other enzymes (e.g., creatine kinase, myokinase, phosphorylase kinase, glycosidase, AMP-deaminase, phosphoglucomutase) are associated with these membrane preparations. Amongst these enzymes, the highest activity associated with sarcoplasmic reticulum membranes is that of glycogen phosphorylase, which is mostly (at least 95%) in its b form (dephosphorylated form), since its activity in sarcoplasmic reticulum membranes is largely dependent upon AMP. A protocol is presented to quantify the amount of phosphorylase bound to sarcoplasmic reticulum membranes from fluorimetric measurements of the content of its coenzyme, pyridoxal 5'-phosphate. The content of phosphorylase ranged from 0.03 to 0.37 mg phosphorylase per mg of membrane protein, in sarcoplasmic reticulum membrane preparations made following several of the protocols most commonly used and also depending upon the length of the starvation period of the animal before killing. We also show that dilution of sarcoplasmic reticulum membranes to 0.1-0.2 mg protein per ml in a buffer containing 50 mM Tes-KOH (pH 7.4), 0.1 M KCl and 0.25 M sucrose removes at least 95% of glycogen phosphorylase from these membrane fragments, as well as other enzymes like myokinase and glycosidase. On these grounds, we suggest to introduce a final dilution step as indicated above in protocols of sarcoplasmic reticulum membrane preparations.
Cuenda
A
A
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
Henao
F
F
Nogues
M
M
Gutiérrez-Merino
C
C
D000659:AMP Deaminase / Q000032:analysis; D000818:Animals; D008565:Membrane Proteins / Q000032:analysis; D009132:Muscles / Q000201:enzymology*; D010733:Phosphoglucomutase / Q000032:analysis; D006005:Phosphorylases / Q000032:analysis* / Q000302:isolation & purification; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D008565:Membrane Proteins; D006005:Phosphorylases; D000659:AMP Deaminase; D010733:Phosphoglucomutase