Wai Yiu Cheung

Calmodulin stimulates human platelet phospholipase A2

Abstract Calmodulin is a ubiquitous Ca 2+ -binding protein, mediating the effect of Ca 2+ on many enzyme systems and cellular reactions. Phospholipase A 2 (phosphatide-2-acyl-hydrolase, EC 3.1.1.4) which governs the level of arachidonic acid in human platelets, requires Ca 2+ for maximum activity. Results presented herein suggest that the stimulation of phospholipase A 2 by Ca 2+ is also mediated through calmodulin. This finding adds to the growing list of enzymes whose activities are regulated by calmodulin.

Human erythrocyte Ca2+-Mg2+-ATPase: Mechanism of stimulation by Ca2+

In the presence of ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA), a detergent-solubilized preparation of human erythrocyte membrane Ca2+-Mg2+-ATPase was separated by gel filtration from its activator protein, designated as calmodulin; this makes the enzyme dependent on an exogenous calmodulin for maximum activity. In the presence of Ca2+, ATPase co-migrated with calmodulin, and the enzyme activity was independent of an exogenous calmodulin, suggesting that the enzyme and calmodulin form an active holoenzyme. Lowering the Ca2+ level dissociates the two proteins, returning the enzyme activity to its basal level. A time course experiment indicated that the effect of Ca2+ on the enzyme·calmodulin complex was immediate and reversible. Calmodulin exerts its effect on ATPase primarily by increasing its V. Thus, the mode of stimulation of human erythrocyte Ca2+-Mg2+-ATPase by calmodulin appears similar to that of Ca2+-dependent adenylate cyclase and phosphodiesterase.

Ca++-dependent formation of brain adenylate cyclase-protein activator complex.

Abstract In the presence of EGTA (ethyleneglycol-bis-(β-aminoethyl-ether) N,N′-tetraacetic acid), a Lubrol-PX solubilized rat brain adenylate cyclase (E.C. 4.4.1.1) and its protein activator were separated from each other in a Sephadex G-200 column. No activator was associated with the eluted enzyme, which required an exogenous activator for maximum activity. On the other hand, in the presence of Ca++, some of the activator was eluted with the enzyme, which was independent of an exogenous activator for maximum activity. Because neither Ca++ nor EGTA affected the elution profile of the activator in the filtration column, these results suggest that the formation of the enzyme-activator complex is dependent on Ca++. Separate experiments indicated that the effect of Ca++ on the formation of the enzyme-activator complex was immediate and reversible. Because the activator appears to be in excess of the enzyme, adenylate cyclase activityin vivo could be modulated by the cellular flux of Ca++.

Rat brain adenylate cyclase: Fruther studies on its stimulation by a Ca2+-binding protein☆

Abstract Bovine or rat brain adenylate cyclase (EC 4.6.1.1) solubilized by Lubrol-PX, a nonionic detergent, requires a Ca2+-binding protein activator for full activity (Cheung et al., 1975, Biochem. Biophys. Res. Commun.66, 1055–1062). We now show that particulate rat brain adenylate cyclase also required the activator for maximum activity. A brain particulate fraction was extracted with a hypertonic NaCl solution containing [ethyl-enebis(oxyethylenenitrilo)] tetraacetic acid. This procedure removed preferentially the activator, making adenylate Cyclase activator deficient and, consequently, dependent on an exogenous activator for maximum activity. The activator increased the V of adenylate cyclase without affecting its apparent Km for ATP. In the presence of the activator, the enzyme was more stable against thermal inactivation, suggesting that the activator probably induced a conformational change to the enzyme. F− and 5′-guanylylimidodi-phosphate [GMP-p(NH)p] greatly stimulated brain adenylate cyclase. Adenylate cyclase activity obtained in the presence of the activator and F− was comparable to the summed activities of the two agents assayed separately, indicating that their effects were additive. Similarly, the effects of the activator and GMP-p(NH)p were additive. These results suggest that the action of the activator is independent of the other two ligands. Since the activator is present in excess over adenylate cyclase, the cellular flux of Ca2+ is believed to be important in modulating the enzyme activity. The role of the Ca2+/ activator is discussed with respect to cyclic AMP metabolism in brain.

An endogenous inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase.

Abstract Brain adenylate cyclase (EC 4.6.1.1) and cyclic nucleotide phosphodiesterase (EC 3.1.4.17) require an endogenous Ca2+-dependent activator protein for full activity (Cheung et al. (1975) Biochem. Biophys. Res. Commun. 66, 1055–1062). We now describe another brain factor which inhibited both brain adenylate cyclase and phosphodiesterase in vitro. The factor appeared to be a protein; it was inactivated by incubation with trypsin, but not with ribonuclease or deoxyribonuclease. Gel filtration with a calibrated column indicated a molecular weight of 80,000 and a Stokes radius of 3.85 nm. In the presence of Ca2+, the inhibitor interacted with the activator protein to form an inhibitor activator complex. This makes the activator unavailable to adenylate cyclase or phosphodiesterase, resulting in a decrease of enzyme activity.

Underestimation of cyclic 3',5'-nucleotide phosphodiesterase activity by a radioisotopic assay using an anionic-exchange resin.

Abstract The anionic-exchange resin technique utilizing isotopically labeled cyclic AMP (or cyclic GMP) and an auxiliary enzyme, 5′-nucleotidase, for the assay of phosphodiesterase (Thompson, M. J., and Appleman, M. M. (1971) Biochemistry10, 311) does not accurately measure the enzyme activity due to adsorption of the product (adenosine or guanosine) by the resin. Binding of adenosine or guanosine by the resin may lead to an underestimation of phosphodiesterase activity. Under comparable conditions, adsorption of guanosine by the resin is much larger than that of adenosine. Consequently, cyclic GMP phosphodiesterase activity is underestimated more than cyclic AMP phosphodiesterase activity.

Cyclic 3′,5′-nucleotide phosphodiesterase: A continuous titrimetric assay

Abstract A direct and continuous assay for cyclic 3′,5′-nucleotide phosphodiesterase has been developed. This method is based on the fact that the phosphate group of adenosine 3′,5′-phosphate has one titratable species whereas that of 5′-adenosine monophosphate has two. Hydrolysis of cyclic AMP to 5′-AMP by phosphodiesterase is accompanied by a stoichiometric generation of protons. The rate of addition of an alkaline solution to the reaction mixture to maintain a constant pH with a pH stat is thus stoichiometrically related to the rate of cyclic AMP hydrolysis. A reaction producing 10 mμmoles of H + or more per minute in 1.5 ml of reaction mixture is accurately measured by this technique. Duplicates are usually within 5% of each other. Results obtained by the titrimetric method correlate well with those obtained by conventional methods. This technique has been successfully used to assay phosphodiesterase of bovine brain in the purified as well as the crude stage.

Separate genetic regulation of cyclic nucleotide phosphodiesterase and its protein activator in cultured mouse fibroblasts.

Abstract Dibutyryl cyclic AMP stimulated the synthesis of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) but not its protein activator in normal mouse fibroblasts (3T3) and those virally transformed (3T3-SV 40). Stimulation appeared to be at the transcriptional level. These findings indicate that the increase of phosphodiesterase is not accompanied by its activator and that the two proteins are controlled by separate genes.

Marked reduction of cyclic GMP phosphodiesterase activity in virally transformed mouse fibroblasts

Dibutyryl cAMP induced the accumulation of both cAMP phosphodiesterase and cGMP phosphodiesterase in normal mouse fibroblast (3T3) and transformed fibroblast (3T3-SV 40). The increase was larger with cAMP phosphodiesterase. Although both enzyme activities were reduced in the transformed cells, the reduction of cGMP phosphodiesterase was more pronounced. The differential increase of cAMP phosphodiesterase by B 2 cAMP and the disparate reduction of cGMP phosphodiesterase in the transformed cell suggest that the two enzymes are regulated separately.

Stimulation of rat liver cyclic 3':5'-nucleotide phosphodiesterase by cyclic GMP is dependent on enzyme concentration.

Abstract A high-speed supernatant of rat liver extract displayed multiple forms of cyclic nucleotide phosphodiesterase (EC 3.1.4.17). One of the forms catalyzed the hydrolysis of cyclic AMP and cyclic GMP, with approximately comparable facility. One salient feature of the enzyme is that at micromolar concentrations, cyclic GMP stimulated the hydrolysis of cyclic AMP, but not vice versa. Another is that the activity of phosphodiesterase varied as a function of enzyme concentration in the assayed system: the enzyme activity was higher at low than at high enzyme concentrations. A concentrated enzyme was not stimulated by cyclic GMP but was stimulated by cyclic GMP upon dilution of the enzyme. Conversely, stimulation of the enzyme by cyclic GMP could be reversed by increasing the enzyme concentration. The cyclic GMP-stimulated cyclic AMP phosphodiesterase was partially purified by a continuous sucrose density gradient. The apparent change of phosphodiesterase activity as a function of enzyme concentration was also observed after partial purification by the sucrose density gradient. High enzyme concentrations favored the aggregated form of phosphodiesterase, whereas low concentrations favored the dissociated form. Dilution of the enzyme shifted the equilibrium toward the dissociated form, which presumably exposed the cyclic GMP regulatory site on the enzyme molecule.