Ercan N, Gannon M C, Nuttall F Q
Department of Medicine, University of Minnesota, Minneapolis, 55417, USA.
Arch Biochem Biophys. 1996 Apr 15;328(2):255-64. doi: 10.1006/abbi.1996.0171.
Phosphorylase removes glucosyl units from the terminal branches of glycogen through phosphorolysis, forming glucose-1-P. It is present in two interconvertible forms, phosphorylase a and b. The a form is the active form and is rate limiting in glycogen degradation. The activities of phosphorylase a and of total phosphorylase as conventionally measured exceed the activities of glycogen synthase R (active form) and of total synthase by approximately 10- and 20-fold. Thus, unless phosphorylase a is inhibited or compartmentalized or its substrates are exceedingly low in vivo, net glycogen synthesis could not occur. In addition, following an administered dose of glucose, phosphorylase a activity changes little when glycogen is being synthesized, is stable, or is being degraded, suggesting an important role for allosteric effectors in regulation. Therefore, we have determined the effect of potential modifiers of enzyme activity at estimated intracellular concentrations. Purified liver phosphorylase a was used. Activity was measured in the direction of glycogenolysis, at 37 degrees C, pH 7.0, and under initial rate conditions. Both a Km and a near-saturating concentration of inorganic phosphate (substrate) were used in the assays. A physiological concentration of AMP was saturating. It decreased the Km for Pi by approximately 50% and stimulated activity. ADP, ATP, and glucose inhibited activity. Fructose-1-P inhibited activity only at a high and nonphysiological concentration. Glucose-6-P and UDP-glucose were not significant inhibitors. Inhibition of activity by ADP was little affected by the addition of AMP. However, AMP partially abolished the inhibitory effect of ATP and completely abolished the inhibitory effect of glucose. When AMP, ADP, ATP, glucose-6-P, UDP-glucose, glucose, and fructose-1-P were added together, the net effect was no change in phosphorylase a activity compared to the activity without any effectors. In addition, changes in glucose concentration did not affect activity. K glutamine modestly stimulated activity. Numerous other metabolites were tested and were without effect. The present data indicate that the known endogenous allosteric effectors cannot explain the smaller than expected in vivo phosphorylase a activity or the regulation of phosphorylase a activity.
磷酸化酶通过磷酸解作用从糖原的末端分支去除葡萄糖基单位，形成葡萄糖-1-磷酸。它以两种可相互转换的形式存在，即磷酸化酶a和b。a形式是活性形式，在糖原降解中起限速作用。传统测量的磷酸化酶a和总磷酸化酶的活性比糖原合酶R（活性形式）和总合酶的活性分别高约10倍和20倍。因此，除非磷酸化酶a在体内被抑制、分隔或其底物极低，否则净糖原合成不可能发生。此外，给予葡萄糖剂量后，当糖原合成、稳定或降解时，磷酸化酶a的活性变化很小，这表明变构效应剂在调节中起重要作用。因此，我们在估计的细胞内浓度下测定了酶活性潜在调节剂的作用。使用纯化的肝脏磷酸化酶a。在37℃、pH 7.0和初始速率条件下，朝着糖原分解方向测量活性。测定中使用了Km和接近饱和浓度的无机磷酸盐（底物）。生理浓度的AMP是饱和的。它使Pi的Km降低约50%并刺激活性。ADP、ATP和葡萄糖抑制活性。果糖-1-磷酸仅在高且非生理浓度下抑制活性。葡萄糖-6-磷酸和UDP-葡萄糖不是显著的抑制剂。ADP对活性的抑制作用几乎不受AMP添加的影响。然而，AMP部分消除了ATP的抑制作用并完全消除了葡萄糖的抑制作用。当AMP、ADP、ATP、葡萄糖-6-磷酸、UDP-葡萄糖、葡萄糖和果糖-1-磷酸一起添加时，与没有任何效应剂时的活性相比，磷酸化酶a的活性净变化为零。此外，葡萄糖浓度的变化不影响活性。K谷氨酰胺适度刺激活性。测试了许多其他代谢物，均无作用。目前的数据表明，已知的内源性变构效应剂无法解释体内磷酸化酶a活性低于预期或磷酸化酶a活性的调节。
