Abstract
Regulation of glycogenolysis in skeletal muscle is dependent on a network of interacting enzymes and effectors that determine the relative activity of the enzyme phosphorylase. That enzyme is activated by phosphorylase kinase and inactivated by protein phosphatase-1 in a cyclic process of covalent modification. We present evidence that the cyclic interconversion is subject to zero-order ultrasensitivity, and the effect is responsible for the "flash" activation of phosphorylase by Ca2+ in the presence of glycogen. The zero-order effect is observable either by varying the amounts of kinase and phosphatase or by modifying the ratio of their activities by a physiological effector, protein phosphatase inhibitor-2. The sensitivity of the system is enhanced in the presence of the phosphorylase limit dextrin of glycogen which lowers the Km of phosphorylase kinase for phosphorylase. The in vitro experimental results are examined in terms of physiological conditions in muscle, and it is shown that zero-order ultrasensitivity would be more pronounced under the highly compartmentalized conditions found in that tissue. The sensitivity of this system to effector changes is much greater than that found for allosteric enzymes. Furthermore, the sensitivity enhancement increases more rapidly than energy consumption (ATP) as the phosphorylase concentration increases. Energy effectiveness is shown to be a possible evolutionary factor in favor of the development of zero-order ultrasensitivity in compartmentalized systems.
Highlights
Regulation of glycogenolysis in skeletal muscle is cently the search has been for an equivalent and complimendependent on a network of interacting enzymes and tary sequence of regulatory reactions for the inactivation of effectors that determine the relative activity of the phosphorylase by its specific phosphatase, protein phosphaenzyme phosphorylase
We present evidence that thecyclic interconversion is subject to zero-order ultrasensitivity, and theeffect is responsible for the“flash” activationof phosphorylase by Ca2+in the presence of glycogen
The i n vitro experi- the kinetics of such systems are described by coupled equamental results are examined in termsof physiological tions rather than the simpler forms used for studying the conditions in muscle, and it is shown that zero-order kinetics of individual enzymes [6]
Summary
From the Departmentof Biochemistry, University of Minnesota, Minneapolis, Minnesota55455. Regu- covalently modified interconvertible enzyme systems have lation of the activity of phosphorylase has been a topic of been evaluated and found to provide significant advantage study for the several decades since its first description Much over those regulated by allosteric effector binding in of the emphasis of those studies has been directed toward terms of controlpattern flexibility, potential for multiple elucidation of the steps in the signal cascade: hormone + allosteric stimuli, and magnitude amplification [7]. EXPERIMENTALPROCEDURES where 6, =E ~ T / W Tc2 ;=EZT/WT(;Y = V,/V,, and theadded parameters, E ~ aTnd EZT, are the totalconcentrations of phosphorylase kinase and protein phosphatase-1, respectively In this case, the modified form of the enzyme W* is given by Analysis of the steady-state mixtures for adenine nucleotides was performed by HPLC [24]. Phosphorylase a [14], phosphorylase b [15], phosphorylase kinase [16], protein phosphatase-1 [17], and phosphatase inhibitor-2 [18]
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