The Hysteretic Properties of Glycogen Synthase I

Abstract

Glycogen-free synthase I from human polymorphonuclear leukocytes is activated by its own substrate, glycogen, in a slow, time-dependent proces (hysteretic activation). This lag in response to addition of glycogen depends on the concentration of glycogen, pH and temperature. At pH 7.4 and at a temperature of 30 "C, the half-time of activation, tl:2, decreases from 89 min at 0.004 mg/ml glycogen to 6 min at 25 mg/ml. The activation is accelerated by increasing temperature and pH, but is not influenced by enzyme concentration, glucose 6-phosphate, UDP, high ionic strength, EDTA, mercaptoethanol, glucose, sucrose or amylase limit dextrin. The K,,, for UDP-glucose (0.024 mM) and the activity ratio were unchanged during the activation process.The activation can be described by tit = uf + (vo -or) eCkr where ut, uf and uo are velocities at times t , 0 and c o and k is a complex rate constant. Evidence from ultracentrifugation and kinetic studies is presented to substantiate the hypothesis that the underlying mechanism is a simple bimolecular process : enzyme + glycogeneenzyme-glycogen complex, with the dissociation constant K, = 0.003 mg/ml. The hysteretic activation may become rate-limiting during experiments in vitro with synthase. The possibility of a physiological role in glycogen metabolism, perhaps in the form of a concerted hysteresis with H+ is discussed.The activity of glycogen synthase, the rate-limiting enzyme of glycogen synthesis (glycogen, + UDPglucose+glycogen,+ I + UDP), is controlled by covalent modification and by metabolite (allosteric) control [l]. In human polymorphonuclear leukocytes, the control is exerted at three levels with regard to degree of phosphorylation and value of kinetic constants. The kinetic constants for substrates and modifiers of the fully phosphorylated enzyme form (D for dependent on glucose-6-P for activity) are of such magnitude that this form of the enzyme can be considered inactive under intracellular conditions [2]. A recently discovered partially phosphorylated enzyme form (R for rheostatic) has been found to account for glycogen synthesis under several physiological conditions, the kinetic constants being of a magnitude compatible with control exerted by the intracellular concentration of glucose-6-P 13 -51. The third non-phosphorylated enzyme form, synthase 1, must from preliminary evidence [6] be considered fully active under all conditions. It can be formed in leukocytes under different experimental conditions, in particular when the inhibitory effect of glycogen on the D to 1 conversion is relieved during starvation [7], and/or when the activity of phosphorylase a reaches a critical, low level [S], suggesting the relief of an inhibitory effect on the R to I conversion [4].When we attempted to detail the kinetics of synthase I we became aware that glycogen, besides being both substrate and product in the enzymatic reaction and inhibitor of the D to I conversion, also confers stability on synthase I against inactivation, as described in the preceding paper [9]. In add...

Studies on the Allosteric Properties of Glycogen Synthase I

“…When it was attempted to perform a kinetic investigation on highly purified glycogen-free synthase I [5], it was found, however, that glycogen had to bind to a second site on the enzyme distinct from the catalytically active site before enzymatic activity appeared [6]. This activation is independent of UDPGlc and glucose-6-P, but depends on temperature, pH and glycogen concentration and requires times of minutes to hours before being complete.…”

“…A highly purified preparation of glycogen synthase I with adhering endogenous glycogen was obtained from human polymorphonuclear leukocytes as described in a previous publication [5]. The preparation has a specific activity of 9-11 U/mg protein and contains 5 -7 pg endogenous glycogen/mU.…”

“…The assay was terminated by spotting 100 p1 of the reaction mixture on squares of filter paper, which were immediately immersed in ice-cold 60% ethanol and treated as in the Thomas assay [8]. In experiments with MgC12, EDTA was omitted from the reaction mixture and MgClz added together with the UDP-Glc solution in order to avoid inactivation of the enzyme by Mg2+ [5] before assay. It has been ascertained in selected control experiments that EDTA and mercaptoethanol in the indicated concentrations do not influence the kinetic behaviour of the enzyme.…”

Purification and Properties of Glycogen Synthase I from Human Leukocytes

“…When glycogen is present a time-dependent activation is observed. In order to measure full activity, the enzyme is routinely incubated with 2% glycogen in the presence of 10 mM glucose-6-P (or 10 mM NazS04) for 2 h at 30 "C. Activation of synthase by glycogen will be further discussed in the following paper [16].…”

“…In addition, glycogen, besides being a substrate, also activates synthase I in a timedependent and concentration-dependent manner, as will be described in a following communication [16].…”