Bovine lung cyclic GMP-dependent protein kinase exhibits two types of specific cyclic GMP-binding sites.

Cyclic GMP-dependent protein kinase from bovine lung exhibits two distinct and specific binding sites for cyclic GMP. Cyclic GMP bound to Site II dissociates very rapidly (t 1/2 less than 10 s at 0 or 30 degrees C) and requires the presence of ammonium sulfate in the wash buffer for its detection using cellulose ester filters. Both sites are highly specific for cyclic GMP and show affinities for cyclic GMP in the physiological range (less than 1 microM at 30 degrees C). Only cyclic GMP bound to the high affinity site (Site I) correlated with stimulation of histone kinase activity using arginine-rich histone as the substrate; however, both sites were involved with stimulation of histone kinase activity using mixed histones as the substrate. Stimulation of autophosphorylation by cyclic GMP was closely correlated with the binding of cyclic GMP to Site I.

Mechanism of activation of cyclic GMP-dependent protein kinase from rat liver by multiple modulators.

A number of polyanionic compounds, including DNA, RNA and polyglutamate, were shown to exhibit protein kinase stimulatory modulator activity as they were required for cyclic GMP to stimulate the phosphorylation of various cationic substrates by rat liver cyclic GMP-dependent protein kinase. Anionic proteins (casein, phosvitin) were phosphorylated poorly by the enzyme and their phosphorylation was not stimulated by the stimulatory modulators. Studies of the mechanism of action suggest that the modulators interact directly with the substrates to form a complex which is a better substrate than free histone. The observed effect of modulator is complex as it depends on the ratio of modulator to histone and the resultant state of the complex formed (better or poorer substrate than free histone). The observed effect is also dependent on the properties of the histone substrate as Michaelis-Menten kinetics are not observed in the phosphorylation of arginine-rich histone in the absence or presence of cyclic GMP.

Use of fluoride to inactivate phosphorylase a phosphatases from rat liver cytosol. Presence of fluoride-insensitive glycogen synthase-specific phosphatase.

The diverse metal requirements for activity of the phosphoprotein phosphatases (EC 3.1.3.16) concerned with glycogen metabolism in rat liver were postulated to reflect the diverse binding intensities of their essential metal(s). After inactivation by fluoride, three of these phosphatases had similar metal requirements in contrast to a fourth phosphatase. Further similarities led to a grouping of these enzymes into two general types. Phosphatases designated type 1 consisted of three enzymes which had the following properties; (1) preference for glycogen phosphorylase a as a substrate; (2) molecular weights in excess of 100 000; (3) conversion to an active 30 000 dalton 'subunit' form upon selective denaturation by 80% ethanol; (4) diverse degrees of stimulation by metals (Mg2+ and Mn2+); and (5) changes to an absolute dependence upon added Mn2+ (but not Mg2+) for activity of both the holoenzyme and the subunit after a demetallating treatment with fluoride in EDTA. The phosphatase designated type 2 exhibited the following properties; (1) preference for glycogen synthase D as a substrate; (2) molecular weight of 50 000; (3) no conversion to an active 30 000 dalton subunit form upon selective denaturation by 80% ethanol; (4) complete metal-dependence upon either Mg2+ or Mn2+; and (5) no change to an absolute dependence on added Mn2+ for activity after a demetallating treatment with fluoride in EDTA.