Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of two diastereoisomeric p-nitrophenyl phosphodiesters of 2'',3''-secouridine and their affinity for phosphodiesterases.

The synthesis of the p-nitrophenyl esters of the 5'- and 3'-phosphates of the nucleoside analogue 2',3'-secouridine are described. Unlike the corresponding diesters of thymidine, these two compounds are diastereoisomers. Their affinity for phosphodiesterases types I and II were investigated. Both analogues were hydrolysed very slowly by snake venom phosphodiesterase but their affinity for the enzyme was similar to that of the p-nitrophenyl ester of thymidine 5'-monophosphate of which they were both competitive inhibitors with Ki approximately Km. Neither compound was hydrolysed by spleen phosphodiesterase but both competitively inhibited the p-nitrophenyl ester of thymidine 3'-monophosphate, with Ki's slightly higher than the Km. Although for each enzyme the Ki of the correct analogue phosphodiester (i.e. the 5'-derivative for snake venom and the 3'-derivative for spleen) was the lower, the absolute specificity seen for the normal substrates had been lost.     

3',5'-Cyclic nucleotide phosphodiesterase activity of the sulphatase A of ox liver

The sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver hydrolyses adenosine 3',5'-monophosphate (cyclic AMP) to adenosine 5'-phosphate at an optimum pH of approx. 4.3, close that for the hydrolysis of cerebroside sulphate, a physiological substrate for sulphatase A. The Km is 11.6 mM for cyclic AMP. On polyacrylamide gel electrophoresis sulphatase A migrates as a single protein band which coincides with both the arylsulphatase and phosphodiesterase activities, suggesting that these are due to a single protein. Cyclic AMP competitively inhibits the arylsulphatase activity of sulphatase A, showing that both activities are associated with a single active site on the enzyme. sulphatase A also hydrolyses guanosine 3',5'-monophosphate, but not uridine 3',5'-monophosphate nor adenosine 2',3'-monophosphate.     

Effects of 8-substituted analogs of cyclic adenosine 3'',5''-monophosphate on in vivo and in vitro syntheses of beta-galactosidase in Escherichia coli.

Several 8-substituted alkylthio and alkylamino cyclic adenosine 3',5'-monophosphate (cAMP) derivatives were tested for their ability to stimulate beta-galactosidase synthesis in Estherichia coli in vivo and in vitro and to inhibit the cAMP phosphodiesterase activity of E. coli. Stimulation of beta-galactosidease synthesis in vivo by cAMP derivatives decreased with increasing length of the unbranched carbon chain of the substituent. On the other hand, the stimulation in vitro was increased as the carbon chain elongated. The 8-decylthio- and 8-dodecylthio-cAMP compounds stimulated beta-galactosidase synthesis almost eight-fold compared with cAMP, whereas 8-undecyl-, 8-dodectyl-, and 8-tridecylamino-cAMP stimulated beta-galactosidase synthesis about threefold. However, in in vitro experiments with a phosphodiesterase-deficient strain of E. coli, the Crooks strain, the stimulatory effects of the derivatives disappeared, except for 8-dodecylthio cAMP which stimulated beta-galactosidase about 1.4- to 1.6-fold. All derivatives were quite resistant to hydrolysis by phosphodiesterase. Most derivatives competitively inhibited the hydrolysis of cAMP by phosphodiesterase.     

Human blood platelet 3': 5'-cyclic nucleotide phosphodiesterase. Isolation of low-Km and high-Km phosphodiesterase.

Human blood platelet contained at least three kinetically distinct forms of 3': 5'-cyclic nucleotide phosphodiesterase (3': 5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) (F I, F II, and F III) which were clearly separated by DEAE-cellulose column chromatography. Although a few properties of the platelet phosphodiesterases such as their substrate affinities and DEAE-cellulose profile resembled somewhat those of the three 3': 5'-cyclic nucleotide phosphodiesterase in rat liver reported by Russell et al. [10], there were pronounced differences in some properties between the platelet and the liver enzymes: (1) the platelet enzymes hydrolyzed both cyclic nucleotides and lacked a highly specific cyclic guanosine 3': 5'-monophosphate (cyclic GMP) phosphodiesterase and (2) kinetic data of the platelet enzymes indicated that cyclic adenosine 3': 5'-monophosphate (cyclic AMP) and cyclic GMP interact with a single catalytic site on the enzyme. F I was a cyclic nucleotide phosphodiesterase with a high Km for cyclic AMP and a negatively cooperative low Km for cyclic GMP. F II hydrolyzed cyclic AMP and cyclic GMP about equally with a high Km for both substrates. F III was low Km phosphodiesterase which hydrolyzed cyclic AMP faster than cyclic GMP. Each cyclic nucleotide acted as a competitive inhibitor of the hydrolysis of the other nucleotide by these three fractions with Ki values similar to the Km values for each nucleotide suggesting that the hydrolysis of both cyclic AMP and cyclic GMP was catalyzed by a single catalytic site on the enzyme. However, cyclic GMP at low concentration (below 10 muM) was an activator of cyclic AMP hydrolysis by F I. Papaverine and EG 626 acted as competitive inhibitors of each fraction with virtually the same Ki value in both assays using either cyclic AMP or cyclic GMP as the substrate. The ratio of cyclic AMP hydrolysis to cyclic GMP hydrolysis by each fraction did not vary significantly after freezing/thawing or heat treatment. These facts also suggest that both nucleotides were hydrolyzed by the same catalytic site on the enzyme. The differences in apparent Ki values for inhibitors such as cyclic nucleotides, papaverine and EG 626 would indicate that three enzymes were different from each other. Centrifugation in a continuous sucrose gradient revealed sedimentation coefficients F I and II had 8.9 S and F III 4.6 S. The molecular weight of these forms, determined by gel filtration on a Sepharose 6B column, were approx. 240 000 (F I and II) and 180 000 (F III). F III was purified extensively (70-fold) from homogenate, with a recovery of approximately 7%.     

Possible role of adenosine cyclic 3':5'-monophosphate phosphodiesterase in the morphological transformation of Chinese hamster ovary cells mediated by N6,O2-dibutyryl adenosine cyclic 3':5"-monophosphate.

We have demonstrated that in Chinese hamster ovary (CHO) cells, N6,O2'-dibutyryl adenosine cyclic 3':5'-monophosphate (dibutyryl cyclic AMP) has a remarkable morphogenetic effect in converting cells of a compact, epithelial-like morphology into a spindle-shaped, fibroblast-like form. Homogenates of CHO cells were found to contain two adenosine cyclic 3':5'-monophosphate (cyclic AMP) phosphodiesterase (EC 3.1.4.c) activities, which differ in apparent Km with respect to their substrate, cyclic AMP. These were designated cyclic AMP phosphodiesterase I, with a low Km of 2 to 5 muM and cyclic AMP phosphodiesterase II, with a high Km of 1 to 3 mM. Cyclic AMP phosphodiesterase I was competitively inhibited by N6-monobutyryl and dibutyryl cyclic AMP, with apparent Ki values of 40 to 60 muM and 0.25 to 0.35 mM, respectively. Experimental evidence demonstrates that the effect of exogenous dibutyryl cyclic AMP on cell morphology is a result of an increase in the endogenous level of cyclic AMP. This increase appears to be due largely to the inhibitory action of intracellular N6-monobutyryl cyclic AMP on cyclic AMP phosphodiesterase I, which results in a decreased rate of degradation of intracellular cyclic AMP.     

Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives

Cyclic nucleotide derivatives have been used as a tool to characterize distinct catalytic sites on phosphodiesterase enzyme forms: the cGMP-stimulated enzyme from rat liver and the calmodulin-sensitive enzyme from rat or bovine brain. Under appropriate assay conditions, the analogues showed linear competitive inhibition with respect to cAMP (adenosine 3',5'-monophosphate) as substrate. The inhibition sequence of the fully activated cGMP-stimulated phosphodiesterase was identical to the inhibition sequence of the desensitized enzyme, i.e. the enzyme which has lost its ability to be stimulated by cGMP. The inhibition pattern could, therefore, not be attributed to competition with cGMP at an allosteric-activating site. Also, the inhibition sequence of the calmodulin-sensitive phosphodiesterase was maintained whether activity was basal or fully stimulated by calmodulin. When cAMP and cGMP, with identical chemical ligands substituted at the same position, were compared as inhibitors of the calmodulin-sensitive phosphodiesterase, the cGMP analogues were always the more potent suggesting that, for that enzyme, the catalytic site was sensitive to a guanine-type cyclic nucleotide structure. Comparing the two phosphodiesterases, it was possible to establish both similar and specific inhibitor potencies of cyclic nucleotide derivatives. In particular, the two enzymes exhibited large differences in analogue specificity modified at C-6, 6-chloropurine 3',5'-monophosphate or purine 3',5'-monophosphate.     

Interaction of 8-substituted derivatives and adenosine-3',5'-cyclophosphate esters with protein kinase from pig brain]

A synthesis of previously unknown 8-substituted derivatives and alkyl esters of cyclic adenosine-3',5'-monophosphate, containing reactive groups, was carried out. The interaction of the compounds obtained with a homogeneous preparation of protein kinase from pig brain was studied. It was found that all compounds, with the exception of neutral esters of 3',5'-AMP, activate the enzyme and competitively inhibit 3H-labelled 3',5'-cAMP binding by the regulatory subunit of protein kinase. The activating effect and affinity of 8-(beta-aminoethylamino)-3',5'-cAMP for protein kinase was 10 times lower than that for 3',5'-cAMP and other 8-substituted derivatives of the cyclic nucleotide. It was found that 8-(N-chloroacetylaminoethylamino)-3',5'-cAMP interaction with the enzyme is of irreversible type, which suggest covalent blocking of the nucleophilic group of the 3',5'-cAMP binding site of protein kinase. The data obtained indicate that the 3',5'-cAMP molecule is bound to the regulatory site of protein kinase in the syn-conformation. The previously made assumption on the crucial importance of the negative charge in the 3',5'-cyclophosphate system for the interaction of cyclic AMP with the regulatory subunit of protein kinase has been thus confirmed.     

Effect of adenosine 3',5'-cyclic monophosphate derivatives on alpha-amylase release, protein kinase and cyclic nucleotide phosphodiesterase activity from rat parotid tissue.

Several 8-substituted derivatives of cyclic AMP were tested for their effects on alpha-amylase release. None of the 8-substituted compounds were more active than N6,O2-dibutyryl- or N6-monobutyryl adenosine 3',5'-monophosphate in causing alpha-amylase release. The rat parotid was found to contain a high (105 muM) and a low (1.15 muM) Km cyclic AMP phosphodiesterase activity. All of the 8-substituted cyclic AMP compounds inhibited the hydrolysis of 1 muM cyclic AMP. However, there was only a partial correlation between the ability to cause alpha-amylase release and inhibit cyclic AMP hydrolysis. Extracts of parotid tissue contained a cyclic AMP-dependent protein kinase activity. None of the compounds were as effective as cyclic AMP in activating the protein kinase. As in the case of inhibition of cyclic AMP hydrolysis, the ability of the 8-substituted cyclic AMP compounds to increase protein kinase activity did not correlate with their effects on alpha-amylase release. It is concluded that factors in addition to the in vitro inhibition of cyclic AMP hydrolysis and activation of protein kinase are important in determining the net result of the 8-substituted cyclic AMP compounds on parotid gland function. These additional factors might include differences in the rate of uptake and differences in rats of conversion to compounds with modified activity.     

Cyclic adenosine 3':5'-monophosphate phosphodiesterase. Distinct forms in human lymphocytes and monocytes.

Adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase activity of normal human peripheral blood leukocyte suspensions containing 90% lymphocytes and 10% monocytes showed anomalous kinetic behavior indicative of multiple enzyme forms. Kinetic analyses of purified lymphocyte (99%) or monocyte preparations (95%) indicated that only one type of phosphodiesterase was present in each cell type. None of the preparations contained any detectable guanosine 3':5'-monophosphate (cyclic GMP) hydrolytic activity. The lymphocyte enzyme had an apparent Km congruent to 0.4 muM for cyclic AMP and Vmax congruent to 0.5 picomoles/min/10(6) cells. These kinetic parameters were confirmed by several cell purification techniques used alone and sequentially. Sedimentation velocity analyses indicated that the higher Km monocyte enzyme had a molecular weight near 45,000 and that the lower Km lymphocyte enzyme most likely had a molecular weight near 98,000. A variety of procedures led to a loss of the higher molecular weight, high affinity enzyme leaving only the enzyme of 45,000 daltons with a much lower substrate affinity. A long term, stable human lymphoblastoid cell line had cyclic AMP phosphodiesterase activity that was similar to the lymphocyte enzyme by both physical and kinetic criteria. Lymphocyte cyclic AMP phosphodiesterase appears to be a soluble enzyme whose pH and temperature optima and cationic requirements are similar to those of other mammalian phosphodiesterases. The distinct cyclic AMP phosphodiesterase forms of these cells may possibly represent the basic, active subunit of mammalian cyclic nucleotide phosphodiesterases. We hypothesize that the extremely high affinity cyclic AMP phosphodiesterase of normal lymphocytes plays an important role in the regulation of normal function in these cells, and also in the rapid proliferative responses characteristic of the stimulated lymphocyte.     

Inhibitory effect of 8-substituted adenosine derivatives on Ca++ and modulator protein-dependent phosphodiesterase activity.

8-Substituted adenosine and cyclic AMP derivatives exhibited some negative Cotton effects in circular Dichroism at B_2u band in pH 7.5 solution, suggesting that these derivatives take syn conformation. The adenosine derivatives, as well as cyclic AMP derivatives, competitively inhibited the cyclic AMP hydrolyzing activity in Ca⁺⁺ and modulator protein-dependent phosphodiesterase preparation from hog brain cortex. The inhibitory potential of an adenosine derivative was lower than that of the cyclic AMP derivative having the same substituent by the lack of the phosphate moiety for which affinity was 0.5 kcal / mol. These results may suggest that the cyclic AMP hydrolyzing site on the enzyme requires the syn conformation of purine riboside.     

8-Substituted derivatives of adenosine 3',5'-cyclic phosphate require an unsubstituted 2'-hydroxyl group in the ribo configuration for biological activity.

Derivatives of adenosine 3',5'-cyclic phosphate (cAMP) with modifications in both the 2' and the 8 positions were synthesized and their enzymic activities as activators of cAMP-dependent protein kinase and as substrates for and inhibitors of cAMP phosphodiesterases were determined. Three types of derivatives were investigated: 8-substituted derivatives of O2'-Bt-cAMP, 8-substituted derivatives of 9-beta-D-arabinofuranosyladenine 3',5'-cyclic phosphate (ara-cAMP), and 8-substituted derivatives of 8,2'-anhydro-9-beta-D-arabinofuranosyladenine 3,'5'-cyclic phosphate (8,2'-anhydro-cAMP). The 8-substituted O2'-Bt-cAMP derivatives were synthesized by acylation of the preformed 8-substituted cAMP (8-HS-cAMP, 8-MeS-cAMP, and 8-PhCH2S-cAMP). 8-Br-O2'-tosyl-cAMP was sued as an intermediate for the preparation of 8,2'-anhydro-cAMP derivatives (8-HO-, 8-SH-, 8-H2N-, and 8-H3 CHN derivatives of 8,2'-anhydro-cAMP). 8-Substituted ara-cAMP derivatives were obtained by ring opening of 8-HO-8,2'-anhydro-cAMP with H+/H2O, NH3/MeOH, or MeONa/MeOH (to yield the 8-HO-, 8-H2N-, and 8-MeO-ara-cAMP derivatives). All of these doubly modified derivatives of cAMP are less than one-hundredth as active as cAMP at activating protein kinase and did not serve as substrates for the phosphodiesterase. These data show that the general inactivity of 2' derivatives of cAMP with kinase was not overcome by addition of an 8-substituent, even though many 8-substituted derivatives of cAMP activate the kinase more efficiently than does cAMP itself. In addition they show that while 2'-modification were tolerated by the phosphodiesterase, addition of an 8-substituent countermanded the allowable 2'-modification. The 8-substituted derivates of 02'-Bt-cAMP were found in general to be slightly better inhibitors of phosphodiesterase than the parent compounds containing no o2'-Bt substitution. As a group, the 8-substituted ara-cAMP derivatives were poorer inhibitors of phosphodiesterase than 8-substituted cAMP derivatives while the 8,2'-anhydro-cAMP derivatives were much poorer inhibitors than the 8-substituted ara-cAMP derivatives.     

Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity.     

Transient kinetics of a cGMP-dependent cGMP-specific phosphodiesterase from Dictyostelium discoideum

Chemotactic stimulation of Dictyostelium discoideum cells induces a fast transient increase of cGMP levels which reach a peak at 10 s. Prestimulation levels are recovered in approximately 30 s, which is achieved mainly by the action of a guanosine 3',5'-monophosphate cGMP-specific phosphodiesterase. This enzyme is activated about fourfold by low cGMP concentrations. The phosphodiesterase has two distinct cGMP-binding sites: a catalytic site and an activator site. cAMP does not bind to either site; inosine 3',5'-monophosphate (cIMP) binds only to the catalytic site, whereas 8-bromoguanosine 3',5'-monophosphate (c-b8-GMP) preferentially binds to the activator site. For detailed kinetical measurements we have used [3H]cIMP as the substrate and c-b8-GMP as the activator. c-b8-GMP activated the hydrolysis of [3H]cIMP by reducing the Km, whereas the Vmax was not altered. The hydrolysis of [3H]cIMP was measured at 5-s intervals by using a new method for the separation of 5'-nucleotides from cyclic nucleotides. The hydrolysis of [3H]cIMP by nonactivated enzyme or by preactivated enzyme was linear with time, which indicates that a steady state is reached at the catalytic site within 5 s after addition of the substrate. In contrast, the hydrolysis of [3H]cIMP immediately after activation by 0.1 microM c-b8-GMP was not linear with time, but increased in a quasi-exponential manner with a time constant of 21 s. This suggests that a steady state at the activator site is only reached in 30-45 s after addition of the activator. The on-rate of activation (k1) was 3 X 10(5) M-1s-1 for c-b8-GMP and 1.4 X 10(5) M-1s-1 for cGMP. The off-rate of activation (k-1) was 0.03 s-1 for both c-b8-GMP and cGMP. The significance of these kinetic constants for the chemoattractant-mediated cGMP response in vivo is discussed.     

Binding of adenosine 3',5'-monophosphate dependent protein kinase regulatory subunit to immobilized cyclic nucleotide derivatives.

Several cyclic nucleotide derivatives with aminoalkyl side chains attached to the purine ring were synthesized and their interactions with adenosine 3',5'-monophosphate (cAMP) dependent protein kinase were studied before and after immobilization to CNBr-activated Sepharose 4B. The soluble N6-substituted derivatives were as effective as cAMP itself in activating protein kinase and were more effective than 8-substituted cAMP derivatives, whereas the 2-substituted cAMP derivatives and the cGMP derivatives were the least effective. All of the synthetic derivatives tested were poor substrates for beef heart phosphodiesterase being hydrolyzed at rates less than 2% for that of cAMP itself. Utilizing methodology developed to evaluate the affinity of protein kinase for immogilized cyclic nucleotides it was found that all of the immobilized cyclic nucleotides interacted with protein kinase in a biospecific manner as judged by the following criteria: (1) the immobilized cyclic nucleotides competed with cAMP for the binding sites on protein kinase; (2) the analogous spacer-arm did not compete; and (3) the effects of enzyme concentration, MgATP, and cleavage of the cyclic phosphate ring on the interactions of protein kinase with the immobilized cyclic nucleotides were the same as previously shown for free cAMP. In addition, the immobilized ligands were bound with the same order of effectiveness as the analogous soluble ligand. The observed Ka for the activation of 0.005 muM protein kinase by N6-H2N(CH2)2-cAMP was increased from 0.23 to 3 muM by the process of immobilization. This increase was unaffected by the coupling density and spacer-arm length. The observed Kb for 0.10 muM protein kinase binding to immobilized N6-H2N(CH2)2-cAMP was increased as the molecular sieving exclusion limit of the matrix used was decreased indicating that at least part of this decrease in apparent affinity upon immobilization is due to exclusion of the enzyme from a portion of the matrix and therefore of the immobilized ligand molecules.     

A slowly dissociating form of the cell surface cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum.

Experiments using a phosphodiesterase-minus mutant of Dictyostelium discoideum indicate that ligand-induced loss of cell surface cyclic adenosine 3':5'-monophosphate binding sites (down regulation) can be evoked with concentrations of cyclic adenosine 3':5'-monophosphate as low as 10(-8) M. The loss of receptor sites is observed after 5 min of cell preincubation with cyclic adenosine 3':5'-monophosphate and can be as extensive as 75 to 80%. This decrease in binding sites is correlated with the appearance of a slowly dissociating cyclic adenosine 3':5'-monophosphate binding component. Radioactive cyclic adenosine 3':5'-monophosphate bound to this form of receptor cannot be competed for by nonradioactive cyclic adenosine 3':5'-monophosphate or adenosine 5'-monophosphate and is not accessible to hydrolysis by cyclic adenosine 3':5'-monophosphate phosphodiesterase. The extent of appearance of this binding component is dependent upon the concentration of cyclic adenosine 3':5'-monophosphate used to elicit the down regulation response and the temperature of the incubation medium.     

Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics.

An extract of rat liver or human platelet displayed three cyclic 3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a continuous sucrose density gradient when assayed with millimolar adenosine 3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The three fractions obtained from each nucleotide were not superimposable. The molecular weights corresponding to the three activity peaks of cAMP phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000; and III, 140,000. In both tissues, fraction I was barely detectable when assayed with micromolar concentrations of either nucleotide, presumably because fraction I has low affinity for cAMP and cGMP. Any one of the three forms upon recentrifugation on the gradient generated the others, indicating that they were interconvertible. The multiple forms appear to represent different aggregated states of the enzyme. The ratio of the three forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the formation of fraction I. Low enzyme concentration favored the equilibrium towards fraction I, while high enzyme concentration favored fraction III. When phosphodiesterase activities in the extract of rat liver, human platelets, or bovine brain were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar cAMP or cGMP. The specific activity with millimolar cAMP was higher with low than with high protein concentrations, suggesting that the dissociated form catalyzed the hydrolysis of cAMP faster than that of the associated form. In contrast, the specific activity with millimolar cGMP was lower with low than with high protein concentrations. Supplementing the reaction mixture with bovine serum albumin to a final constant protein concentration did not affect the activity, suggesting that the concentration of the enzyme rather than that of extraneous proteins affected the enzyme activity. A change in enzyme concentration affected the kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X 10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while a high enzyme concentration yielded a nonlinear plot, and apparent Km values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X 10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low enzyme concentration favored fraction I, the dissociated form, whereas a high enzyme concentration favored fraction III, the associated form, these kinetic constants suggest that the dissociated form exhibits a high Km and the associated form exhibits a low Km. In contrast, a high enzyme concentration gave a linear kinetic plot for cGMP phosphodiesterase, while a low enzyme concentration gave a nonlinear plot...     

A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase.

Nebularine undergoes hydration at the active site of adenosine deaminase, in a reaction analogous to a partial reaction in the displacement of ammonia from adenosine by water, to generate an inhibitory complex that captures much of the binding affinity expected of an ideal transition-state analogue. Enzyme affinities of several compounds related to nebularine 1,6-hydrate, and to its stable analog 2'-deoxycoformycin, were compared in an effort to identify the structural origins of strong binding. Binding of the stable transition-state analog inhibitor 2'-deoxycoformycin was rendered 9.8 kcal/mol less favorable by removal of substituent ribose, 9.7 kcal/mol less favorable by inversion of the 8-hydroxyl substituent of the diazepine ring, and 10.0 kcal/mol less favorable by removal of atoms 4-6 of the diazepine ring. Binding of the unstable transition-state analog nebularine hydrate was rendered at least 9.9 kcal/mol less favorable by removal of the 6-hydroxyl group and 10.2 kcal/mol less favorable by removal of atoms 1-3 of the pyrimidine ring. In each case, the enzyme exhibited only modest affinity (Kd greater than or equal to 10(-2) M) for the "missing piece", indicating that incorporation of 2 binding determinants within a single molecule permits an additional 7-12 kcal/mol of intrinsic binding energy to be manifested as observed binding energy. These results are consistent with earlier indications that adenosine deaminase may use 10.5 kcal/mol of the intrinsic free energy of binding of the two substrates to place them in positions appropriate for reaction at the active site, overcoming the unfavorable entropy change of -35 eu for the equilibrium of 1,6-hydration of purine ribonucleoside and reducing the equilibrium constant for attainment of the transition state in deamination of adenosine. Thus, adenosine deaminase may achieve up to 8 orders of magnitude of its catalytic power by converting the nonenzymatic, bimolecular, hydration reaction to a monomolecular reaction at its active site. Several new 6-substituted 1,6-dihydropurine ribonucleosides, prepared by photoaddition of formate and by low-temperature addition of organolithium reagents to a derivative of purine ribonucleoside, exhibited Ki values of 9-1400 microM against adenosine deaminase, in accord with the active site's considerable tolerance of bulky leaving groups in substrates. Inhibition by one diastereomer of 6-carboxy-1,6-dihydropurine ribonucleoside was found to be time-dependent, progressing from a weakly bound to a more strongly bound complex.     

Protein inhibitor of cyclic adenosine 3':5'-monophosphate phosphodiesterase in retina.

A protein acting as inhibitor of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.1.) activity was found in the ox retina tissue. An inhibitor from one tissue (ox retina) effectively cross-inhibited a phosphodiesterase from another tissue (rat brain), indicating a lack of tissue specificity. Kinetic analysis showed that inhibition was independent of the time of preliminary incubation of the inhibitor with enzyme but dependent on its concentration in the reaction mixture. An inhibitor decreased the V of the enzyme and had no effect on its Km for cyclic adenosine-3':5'-monophosphate. The inhibitory effect was more pronounced with cyclic adenosine-3':5'-monophosphate than with cyclic guanosine-3':5'-monophosphate used as substrates of the reaction. The extractable form of the phosphodiesterase of the retina rod outer segments was much more sensitive to the inhibitory action than the membrane-bound one. The binding of labeled cyclic adenosine-3':5'-monophosphate to the inhibitory protein was shown not to occur. The inhibitor was sensitive to trypsin treatment, indicating that it was a proten attempt was mode to purify the inhibitory factor. Gel filtration indicated that the inhibitor had a molecular weight of 38 000.     

Cyclic 3':5'-nucleotide phosphodiesterase. Stimulation of bovine brain cytoplasmic enzyme by lysophosphatidylcholine.

Brain cytoplasmic cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) requires an endogenous Ca2+-binding protein for ful activity. We now show that lysophosphatidylcholine also effectively enhances activator-deficient phosphodiesterase activity. Stimulation by both ligands was immediate and reversible; both rendered the enzyme more thermally labile, decreased the energy of activation, and increased the Vmax of phosphodiesterase without affecting its apparent Km for adenosine 3'5'-monophosphate. However, the cofactor requirements of the two ligands were different. Although the protein activator gave a greater stimulation than lysophosphatidylcholine, the simultaneous presence of the two gave a stimulation comparable to lysophosphatidylcholine, suggesting that the effect of the latter was predominant. Phosphodiesterase was also stimulated by oleic acid, cardiolipin, and phosphatidylinositol, albeit to a less extent.