Inhibitory effect of polycations on phosphorylation of glycogen synthase by glycogen synthase kinase 3.

Several polycations were tested for their abilities to inhibit the activity of glycogen synthase kinase 3 (GSK-3). L-Polylysine was the most powerful inhibitor of GSK-3 with half-maximal inhibition of glycogen synthase phosphorylation occurring at approx. 100 nM. D-Polylysine and histone H1 were also inhibitory, but the concentration dependence was complex, and DL-polylysine was the least effective inhibitor. Spermine caused about 50% inhibition of GSK-3 at 0.7 mM and 70% inhibition at 4 mM. Inhibition of GSK-3 by L-polylysine could be blocked or reversed by heparin. A heat-stable polycation antagonist isolated from swine kidney cortex also blocked the inhibitory effect of L-polylysine on GSK-3 and blocked histone H1 stimulation of protein phosphatase 2A activity. Under the conditions tested, L-polylysine also inhibited GSK-3 catalyzed phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase and a 63 kDa brain protein, but only slightly inhibited phosphorylation of inhibitor 2 or proteolytic fragments of glycogen synthase that contain site 3 (a + b + c). L-Polylysine at a concentration (200 nM) that caused nearly complete inhibition of GSK-3 stimulated casein kinase I and casein kinase II, but had virtually no effect on the catalytic subunit of cAMP-dependent protein kinase. These results suggest that polycations can be useful in controlling GSK-3 activity. Polycations have the potential to decrease the phosphorylation state of glycogen synthase at site 3, both by inhibiting GKS-3 as shown in this study and by stimulating the phosphatase reaction as shown previously (Pelech, S. and Cohen, P. (1985) Eur. J. Biochem. 148, 245-251).

Rates of GDP-induced and GTP-induced depolymerization of glutamate dehydrogenase: a possible factor in metabolic regulation.

The rate of the depolymerization of beef liver glutamate dehydrogenase induced by coenzyme and the purine nucleotides guanosine 5'-diphosphate and guanosine 5'-triphosphate, which are potent inhibitors of enzymatic activity, has been measured by rapid light scattering techniques and by absorbancy changes with stop flow. It is shown that the rate constant for this process may vary from several milliseconds to several seconds depending upon the nucleotides used. The widely varying rate constants for the nucleotide-induced depolymerization may serve a role in determining the nature of the regulation of enzyme activity by nucleotides. Depolymerization induced by guanosine 5'-diphosphate in the presence of diphosphopyridine nucleotide is slower than in the presence of triphosphopyridine nucleotide as coenzyme, and this difference is apparently due to the isomerization of the enzyme as a result of diphosphopyridine nucleotide binding to a second, nonactive site. This binding, as well as binding of the coenzyme to the active site, may be conveniently measured by a purine nucleotide-induced spectral shift in the coenzyme absorption spectrum. It is also shown that complete depolymerization of the enzyme in the presence of guanosine 5'-triphosphate is accomplished by about half saturation of the coenzyme active sites (6-8 active "monomer").