Kinetics and regulation of sarcoplasmic reticulum ATPase.

The measurement of ATP binding to the sarcoplasmic reticulum membrane reveals that the calcium pump possesses one high affinity (Kd = 2--3 muM) site. Competition with substrate analogs show the high specifity of that site. At high ATP concentration another class of site can be detected with a much higher dissociation constant (Kd approximately 500 muM). This class of sites is of low specificity and ATP is easily displaced by other polyphosphates. The steady state rate of ATP cleavage is measured in the presence of ATP analogs. It is shown that the catalysis is due to the high affinity site. The activation of the hydrolysis rate at high substrate concentration may be related to the effect of binding of ATP to the weak sites. The effect of ATP analogs for various ATP concentration supports this hypothesis.     

Kinetic Analysis of Corn Mitochondrial F(1)-ATPase

The activation and catalytic mechanism of corn mitochondrial F₁ were examined for the two distinct forms of the enzyme which appear upon storage in ammonium sulfate or glycerol. Apparently irreversible differences in the stability of the two active forms were found. Nucleosidetriphosphate induced activation of the enzyme was found to produce lasting effects on subsequent catalysis. These effects varied with both the nucleotide used for activation, and the hydrolyzed species. The substrate and metal specificity were examined with the ATP activated enzyme. Mg²⁺ and Ca²⁺ were found to be the most effective at promoting ATP hydrolysis. The substrates were hydrolyzed in the order GTP > ITP > ATP regardless of which nucleotide was used for activation. While ATP and GTP hydrolysis exhibited kinetics typical of other ATPases, ITP showed a transition from negative to positive cooperativity at low substrate concentrations. Bicarbonate was found to affect primarily the kinetics of ATP hydrolysis. AMP-PNP proved to be a potent inhibitor with respect to ATP hydrolysis. The results are discussed in terms of possible catalytic mechanisms and the similarities of the corn mitochondrial F₁ to other ATPases.     

Catalytic mechanism of nucleoside diphosphate kinase investigated using nucleotide analogues, viscosity effects, and X-ray crystallography.

Nucleoside diphosphate (NDP) kinases display low specificity with respect to the base moiety of the nucleotides and to the 2'-position of the ribose, but the 3'-hydroxyl is found to be important for catalysis. We report in this paper the enzymatic analysis of a series of derivatives of thymidine diphosphate (TDP) where the 3'-OH group was removed or replaced by fluorine, azido, and amino groups. With Dictyostelium NDP kinase, kcat decreases 15-200-fold from 1100 s-1 with TDP, and (kcat/Km)NDP decreases from 12 x 10(6) to 10(3) to 5 x 10(4) M-1 s-1, depending on the substrate. The poorest substrates are 3'-deoxyTDP and 3'-azido-3'-deoxyTDP, while the best modified substrates are 2',3'-dehydro-3'-deoxyTDP and 3'-fluoro-3'-deoxyTDP. In a similar way, 3'-fluoro-2',3'-dideoxyUDP was found to be a better substrate than 2',3'-dideoxyUDP, but a much poorer substrate than 2'-deoxyUDP. (kcat/Km)NDP is sensitive to the viscosity of the solution with TDP as the substrate but not with the modified substrates. To understand the poor catalytic efficiency of the modified nucleotides at a structural level, we determined the crystal structure of Dictyostelium NDP kinase complexed to 3'-fluoro-2',3'-dideoxyUDP at 2.7 A resolution. Significant differences are noted as compared to the TDP complex. Substrate-assisted catalysis by the 3'-OH, which is effective in the NDP kinase reaction, cannot occur with the modified substrate. With TDP, the beta-phosphate, which is the leaving group when a gamma-phosphate is transferred to His122, hydrogen bonds to the 3'-hydroxyl group of the sugar; with 3'-fluoro-2',3'-dideoxyUDP, the beta-phosphate hydrogen bonds to Asn119 and moves away from the attacking Ndelta of the catalytic His122. Since all anti-AIDS nucleoside drugs are modified at the 3'-position, these results are relevant to the role of NDP kinase in their cellular metabolism.     

Rate of chase-promoted hydrolysis of ATP in the high affinity catalytic site of beef heart mitochondrial ATPase

Incubation of [gamma-32P]ATP with a molar excess of the soluble, homogeneous ATPase from beef heart mitochondria (F1) results in binding of substrate primarily in a single, very high affinity (KA = 10(12) M-1) catalytic site and in a slow rate of hydrolysis characteristic of single site catalysis. Subsequent addition of millimolar concentrations of nonradioactive ATP as a cold chase, sufficient to fill catalytic sites on the enzyme, results in an acceleration of hydrolysis of bound radioactive ATP of as much as 10(6)-fold, that is, to Vmax rates (Cross, R.L., Grubmeyer, C., and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12101-12105). For this reason, it was proposed that the high affinity catalytic site is a normal catalytic site on the molecule. Recently, Bullough et al. (Bullough, D.A., Verburg, J.G., Yoshida, M., and Allison, W.A. (1987) J. Biol. Chem. 262, 11675-11683) reported that when 5 to 20 microM concentrations of nonradioactive ATP were added as a cold chase to an enzyme-substrate complex consisting of F1 and ATP bound to the high affinity catalytic site, hydrolysis of the chase was commensurate with the turnover rate of the enzyme, whereas the hydrolysis of bound ATP was considerably slower. These authors suggested that the high affinity catalytic site on F1 is not a normal catalytic site. This paper shows, in experiments with a rapid mixing-chemical quench apparatus, that hydrolysis of ATP bound in the high affinity catalytic site is accelerated to Vmax rates following addition of 5 microM ATP as a cold chase. Hydrolysis of bound ATP appears to precede that of the chase. The weight of the available evidence continues to support the original suggestion that the high affinity catalytic site of beef heart F1 is a normal catalytic site.     

Nucleotide hydrolytic activity of isolated intact rat mesenteric small arteries.

Segments of isolated intact rat mesenteric small arteries were incubated in physiological bicarbonate buffer in the presence of nano- to millimolar concentrations of ATP. ATP was hydrolysed, and when the vessel was transferred from one incubation to another, the enzyme activity was transferred with the vessel, consistent with the presence of an ecto-ATPase. The substrate, ATP, was shown to induce a modification of the hydrolytic activity which occurred the more rapidly the higher the concentration of ATP. The modified system hydrolysed ATP with a decreased substrate affinity. As the substrate induced a modification of the hydrolytic activity, steady-state velocity measurements for determination of kinetic parameters could not be obtained. Nevertheless, it was possible to compare the modification caused by ATP and UTP, and to compare the hydrolysis rates measured with [32P]ATP, [32P]UTP and [32P]GTP. It was concluded that the hydrolytic activity of the vessels did not distinguish between the nucleoside triphosphates (NTPs). In a histidine buffer, the activity was shown to be activated by micromolar concentrations of either Ca2+ or Mg2+, and not to be influenced by inhibitors of P-type, F-type and V-type ATPases. Functional removal of the endothelium before assay did not reduce the measured NTP hydrolysis. At millimolar concentrations of trinucleotide the hydrolysis rate was 10-15 mumol per min per gram of tissue or 0.11-0.17 mumol per min per 10(6) vascular smooth muscle cells. This value is equivalent to the maximal velocity obtained for the Ca2+ or Mg(2+)-dependent NTPase released to the medium upon 2 s of sonication of the vessels (Plesner, L., Juul, B., Skriver, E. and Aalkjaer, C. (1991) Biochim. Biophys. Acta 1067, 191-200). Comparing the characteristics of the released NTPase to the characteristics of the activity of the intact vessel, they showed a strong resemblance, but the substrate-induced modification of the enzyme was seen only in the intact preparation.     

Increased substrate selectivity during transition from Ca2+-activated to K+,EDTA-activated nucleoside triphosphatase activity of heavy meromyosin

A comparison of kinetic parameters (Km(app) and V) of hydrolysis by heavy meromyosin of natural (ATP and ITP) and modified nucleoside triphosphates showed that in the K+, EDTA-ATPase conformation the enzyme exhibited a higher selectivity towards the structure of the substrate nucleoside moiety than in the case of the Ca2+-stimulated nucleoside triphosphatase activity. In the presence of Ca2+, all the N1- and N6-substituted analogs of ATP as well as ITP, etheno-ATP and the dialdehyde derivative of ATP were hydrolyzed at a high rate irrespective of their markedly decreased affinity for heavy meromyosin. In the presence of K+, EDTA the ATPase activity showed a tendency for a total decrease of the analog affinity for nucleoside triphosphates, i.e., the impossibility of tight binding of the substrate phosphate residues to the protein in the absence of bivalent cations, which was concomitant with an increase in the hydrolysis rate. However, it was found that only in N1-substituted analogs any appreciable changes in the substrate properties were absent. All the other nucleoside triphosphates tested (N6-carboxy-methoxy-ATP, N6-(N'-acetylaminoethoxy)-ATP, etheno-ATP, ITP and the dialdehyde derivative of ATP having a rupture in the ribose ring) lost their ability to be hydrolyzed by heavy meromyosin. The experimental results as well as the literature data are suggestive of differences in the spatial structure of the active center in two different myosin conformations associated with a high catalytic activity, i.e., K+, EDTA-ATPase and Ca2+-ATPase.     

Regulation of the chloroplast H+-ATPase by light. The involvement of Mg2+ ions

The involvement of Mg2+ ions in the light-dependent regulation of the chloroplast H+-ATPase was studied in both type C and osmotically shocked type A chloroplasts. The following results were obtained. ATPase activity measured under dark, partially uncoupling conditions, following light activation with dithiothreitol and pyocyanine, was markedly enhanced by the presence of Mg2+ in the activation stage. This Mg2+ effect required concentrations in the millimolar range, was rather slow (time range of minutes), reversible, rather unspecific and did not involve changes in the affinity to dithiothreitol. Dark deactivation of the ATPase in the absence of substrate was accelerated by Mg2+. The dark effect of Mg2+ also required millimolar concentrations, but was fast (time range of seconds), highly specific for Mg2+, and did not involve thiol oxidation. The major effect of the absence of Mg2+ from the light-activation stage or of its presence in the dark interval between activation and assay was the induction of an 'abnormal' sensitivity to uncouplers: after these treatments ATP hydrolysis was not stimulated but rather inhibited by NH4Cl or other uncouplers. The pretreatments in the light without Mg2+ or dark with Mg2+ did not affect the membrane proton permeability, nor the proton pumping coupled to ATPase activity. The results are discussed in terms of Mg2+-dependent regulation of the enzyme complex at the level of subunit interaction and its effect on the affinity to protons.     

Persistent Ca2(+)-induced activation of erythrocyte membrane Ca2(+)-ATPase unrelated to calpain proteolysis

Preincubation of human erythrocyte membranes with calcium in the submillimolar to millimolar concentration range resulted in an increase of the Ca2+ affinity and apparent maximum velocity of the Ca2(+)-stimulated Mg2(+)-dependent ATPase (Ca2(+)-ATPase). The activation was persistent, as it was not reversed when the Ca2(+)-preincubated membranes were washed with ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid-containing buffers. Magnesium was not required for the activation, whereas greater than 2 mM Mg2+ partially antagonized the activation by Ca2+. In some membrane preparations ATP was required in addition to Ca2+ for activation of the Ca2(+)-ATPase, but nonhydrolyzable analogs of ATP had the same effect. Calmodulin prevented the activation by Ca2+ over the same concentration range in which it interacts with the Ca2(+)-ATPase. Taken together the results obtained provided strong evidence that the Ca2+ activation of the enzyme was not due to proteolytic cleavage by endogenous calpain. Thus, activation by Ca2+ was not blocked by leupeptin (100-200 microM), did not require dithiothreitol, and occurred at Ca2+ concentrations greater than those required for activation of calpain I. Furthermore, Ca2+ activation did not result in change in the mobility the native 136-kDa species of the Ca2(+)-ATPase on SDS-gel electrophoresis. Moreover, solubilization of the Ca2(+)-pretreated membranes with Triton X-100 reversed the Ca2+ activation of the Ca2(+)-ATPase. On the other hand, Ca2(+)-pretreatment of the membranes modified the susceptibility of the Ca2(+)-ATPase to both cleavage and activation by exogenously added calpain I. We conclude that pretreatment of Ca2(+)-ATPase in erythrocyte membranes with millimolar Ca2+ activates the enzyme by inducing a persistent conformational change of the enzyme which is, however, subsequently reversed by detergent solubilization.     

Modifying the specificity of an RNA backbone contact.

The interaction between the MS2 bacteriophage coat protein homodimer and its cognate RNA hairpin is facilitated by 21 different RNA-protein contacts. In one of these contacts, the 2'-hydroxyl group at ribose -5 of the RNA acts as a hydrogen bond donor to Glu63 in one subunit of the protein. Previous experiments showed that substitution of ribose -5 with deoxyribose resulted in a 24-fold decrease in binding affinity between RNA and protein. Using a protein where the two MS2 monomers were fused to increase stability, the contribution of this contact to the overall binding affinity was investigated by site-directed mutagenesis. When Glu63 was substituted with glutamine, aspartate, or alanine, the binding affinity of the hairpin for the protein was weakened by 12 to 100-fold, similar to that observed with deoxyribose at position -5. However, the specificity of the three mutant proteins for RNAs with various modifications at the 2'-position of ribose -5 differed dramatically. While the Glu63Asp protein resembled the wild-type protein in preferring the 2'-hydroxyl group over a proton or a bulky 2'-substituent, both the Glu63Ala and Glu63Gln proteins preferred bulky 2'-substituents over the 2'-hydroxyl group by more than 100-fold. These experiments emphasize the ease with which the specificity of a protein-nucleic acid interaction can be changed at thermodynamically important sites.     

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: a new fluorescence displacement titration technique for characterizing the nucleotide binding site on the catalytic subunit

We determined the dissociation constant (Kd) of a series of nucleotides for the bovine skeletal muscle type II catalytic subunit by displacing lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) with increasing concentrations of competing nucleotide. The Kd of each nucleotide was calculated from the decreases in the fluorescence polarization of lin-benzo-ADP that accompany its displacement from the catalytic subunit. We found that modifications of the adenine moiety reduce nucleotide affinity for the enzyme. The effect was most pronounced with modifications at position 6 of the base. Replacement of the 3'-hydroxyl group of ribose with a hydrogen increased the affinity of the nucleotide; addition of phosphate to the 2'- or 3'-hydroxyl groups, on the other hand, decreased nucleotide affinity. MgATP and MgADP exhibited Kd's of about 10 microM. AMP, which contains a negatively charged alpha-phosphate, bound with reduced affinity (643 microM). Adenosine, which lacks a charged alpha-phosphate, bound with a higher affinity (32 microM). To learn more about the nature of the alpha-phosphate binding site, a series of uncharged and positively charged derivatives of the 5'-position on the ribose moiety was prepared. The uncharged derivatives bound with much greater affinity than the negatively charged AMP. The Kd's for 5'-tosyladenosine and 5'-iodo-5'-deoxyadenosine were 30 and 32 microM, respectively. Like the negatively charged AMP, positively charged derivatives also bound less tenaciously than the neutral species. The positively charged 5'-amino-5'deoxyadenosine, for example, exhibited a 15-fold higher Kd (506 microM) than the neutral congenors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction cycle of solubilized monomeric Ca2+-ATPase of sarcoplasmic reticulum is the same as that of the membrane form

Monomeric Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum dispersed in Triton X-100 is stoichiometrically phosphorylated from Pi in a Ca2+-depleted medium containing dimethyl sulfoxide and catalyzes efficient (80%) phosphoryl transfer to ADP following a jump in water activity in the presence of Ca2+. The Ca2+ concentration dependence of ATP synthesis was sigmoidal (nH = 1.7) and in the millimolar range (K0.5 = 0.3 mM), indicating the involvement of at least two low affinity Ca2+ binding sites. These results, taken together with the properties of the monomer in the forward direction of catalysis, show that the catalytic cycle of the detergent-solubilized monomer is essentially the same as that of the membrane enzyme. The substrate and ion specificity of the catalytic intermediates suggest that the monomer is capable of coupled vectorial transport of Ca2+.     

The role of magnesium ions and 2'-hydroxyl groups in the VS ribozyme-substrate interaction

The minimal substrate of the trans-cleaving Neurospora VS ribozyme has a stem-loop structure and interacts with the ribozyme by RNA tertiary interactions that remain only partially defined. The magnesium ion dependence of the catalytic parameters of a trans-cleaving VS-derived ribozyme were studied. The turnover number of the catalytic RNA was found to depend on the binding of at least three magnesium ions, with an apparent magnesium ion dissociation constant of 16mM, but K(M) was observed to be metal ion independent in the millimolar range. To address the role of 2'-hydroxyl groups of the VS substrate RNA in interactions with the ribozyme, 23 altered substrates, each with a single 2'-deoxyribonucleoside substitution, were synthesised and their kinetic properties in the VS ribozyme reaction were analysed. The removal of five 2'-hydroxyl groups, at positions G620, A621, U628, C629 and G630 inhibited the reaction, whereas at two sites, G623 and A639, reaction was stimulated by the modification. Substitution of G620 with a 2'-deoxynucleoside was expected to inhibit the reaction, in line with the critical role of this 2'-hydroxyl group in the transesterification reaction. Altered substrates in which a 2'-O-methyl nucleoside replaced A621, U628, C629 and G630 were prepared and characterised. Although removal of the hydroxyl group of A621 inhibited the turnover number of the ribozyme significantly, this activity was recovered upon 2'-O-methyl adenosine substitution, suggesting that the 2'-oxygen atom of this nucleoside forms an important contact within the ribozyme active site. A cluster of residues within the loop region of the substrate, were more modestly affected by 2'-deoxynucleoside substitution. In two cases, magnesium binding was impaired, suggesting that stem-loop I is a possible magnesium ion binding site.     

High Km soluble 5'-nucleotidase from human placenta. Properties and allosteric regulation by IMP and ATP

A human placental soluble "high Km" 5'-nucleotidase has been separated from "low Km" 5'-nucleotidase and nonspecific phosphatase by AMP-Sepharose affinity chromatography. The enzyme was purified 8000-fold to a specific activity of 25.6 mumol/min/mg. The subunit molecular mass is 53 kDa, and the native molecular mass is 210 kDa, suggesting a tetrameric structure. Soluble high Km 5'-nucleotidase is most active with IMP and GMP and their deoxy derivatives. IMP is hydrolyzed 15 times faster than AMP. The enzyme has a virtually absolute requirement for magnesium ions and is regulated by them. Purine nucleoside 5'-triphosphates strongly activate the enzyme with the potency order dATP greater than ATP greater than GTP. 2,3-Diphosphoglycerate activates the enzyme as potently as ATP. Three millimolar ATP decreased the Km for IMP from 0.33 to 0.09 mM and increased the Vmax 12-fold. ATP activation was modified by the IMP concentration. At 20 microM IMP the ATP-dependent activation curve was sigmoidal, while at 2 mM IMP it was hyperbolic. The A0.5 values for ATP were 2.26 and 0.70 mM, and the relative maximal velocities were 32.9 and 126.0 nmol/min, respectively. Inorganic phosphate shifts the hyperbolic substrate velocity relationship for IMP to a sigmoidal one. With physiological concentrations of cofactors (3 mM ATP, 1-4 mM Pi, 150 mM KCl) at pH 7.4, the enzyme is 25-35 times more active toward 100 microM IMP than 100 microM AMP. These data show that: (a) soluble human placental high Km 5'-nucleotidase coexists in human placenta with the low Km enzyme; (b) under physiological conditions the enzyme favors the hydrolysis of IMP and is critically regulated by IMP, ATP, and Pi levels; and (c) kinetic properties of ATP and IMP are each modified by the other compound suggesting complex interaction of the associated binding sites.     

Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate

The specific action of serine proteinases on protein substrates is a hallmark of blood coagulation and numerous other physiological processes. Enzymic recognition of substrate sequences preceding the scissile bond is considered to contribute dominantly to specificity and function. We have investigated the contribution of active site docking by unique substrate residues preceding the scissile bond to the function of prothrombinase. Mutagenesis of the authentic P(1)-P(3) sequence in prethrombin 2/fragment 1.2 yielded substrate variants that could be converted to thrombin by prothrombinase. Proteolytic activation was also observed with a substrate variant containing the P(1)-P(3) sequence found in a coagulation zymogen not known to be activated by prothrombinase. Lower rates of activation of the variants derived from a decrease in maximum catalytic rate but not in substrate affinity. Replacement of the P(1) residue with Gln yielded an uncleavable derivative that retained the affinity of the wild type substrate for prothrombinase but did not engage the active site of the enzyme. Thus, active site docking of the substrate contributes to catalytic efficiency, but it is does not determine substrate affinity nor does it fully explain the specificity of prothrombinase. Therefore, extended interactions between prothrombinase and substrate regions removed from the cleavage site drive substrate affinity and enforce the substrate specificity of this enzyme complex.     

Steady state kinetic studies of purified yeast plasma membrane proton-translocating ATPase

The plasma membrane H+-ATPase from bakers' yeast was purified and reconstituted with phosphatidylserine. The steady state kinetics of ATP hydrolysis catalyzed by the H+-ATPase were studied over a wide range of Mg2+ and ATP concentrations. Whereas MgATP was the substrate hydrolyzed, excess concentrations of either Mg2+ or ATP were inhibitory. The dependence of the steady state initial velocity of ATP hydrolysis on the concentration of MgATP at a fixed concentration of Mg2+ was sigmoidal rather than hyperbolic. This precluded mechanisms involving only activation and inhibition by Mg2+ and competitive inhibition by ATP. Two alternative interpretations of these results are: 1) the enzyme possesses multiple catalytic sites which interact cooperatively; or 2) the enzyme can exist in multiple conformational states which catalyze MgATP hydrolysis by parallel pathways. The rate laws for both mechanisms are identical so that the two mechanisms cannot be distinguished on the basis of the kinetic data. The data are well fit by the rate law for these mechanisms with the inclusion of competitive inhibition by Mg2+ and ATP and an independent inhibition site for Mg2+.     

Cell-free lutropin-dependent desensitization of the lutropin-sensitive adenylate cyclase of pig ovarian follicles is dependent on ATP.

Experiments were conducted to clarify the nucleotide requirements for lutropin (LH)-dependent adenylate cyclase desensitization in a cell-free membrane preparation derived from a thecal-cell-enriched component of preovulatory pig ovarian follicles. The follicular membranes were extensively washed in 2M-urea to remove endogenously bound GTP, and ATP devoid of GTP was utilized. Results conducted in the presence of 60 microM-GTP and various concentrations of ATP confirm the dependence of LH-stimulated adenylate cyclase activation and desensitization on millimolar concentrations of ATP. In experiments in which adenylate cyclase activation was supported by Mg2+, LH and adenosine 5'-[beta, gamma-imido]triphosphate, GTP did not support the desensitization response. Moreover, although GTP increased both basal and LH-stimulable adenylate cyclase activities in a concentration-dependent manner, the percentage desensitization was not significantly modified by the presence of 10nM-10mM-GTP. These results demonstrate that, even in the presence of exogenous GTP and Mg2+, activation of adenylate cyclase by saturating concentrations of LH in the presence of adenosine 5'-[beta, gamma-imido]triphosphate is not sufficient to initiate desensitization; millimolar concentrations of ATP are also required for the adenylate cyclase desensitization response.     

Allosteric-site and catalytic-site ligand effects on PDE5 functions are associated with distinct changes in physical form of the enzyme

Native phosphodiesterase-5 (PDE5) homodimer contains distinct non-catalytic cGMP allosteric sites and catalytic sites for cGMP hydrolysis. Purified recombinant PDE5 was activated by pre-incubation with cGMP. Relatively low concentrations of cGMP produced a Native PAGE gel shift of PDE5 from a single band position (lower band) to a band with decreased mobility (upper band); higher concentrations of cGMP produced a band of intermediate mobility (middle band) in addition to the upper band. Two point mutations (G659A and G659P) near the catalytic site that reduced affinity for cGMP substrate retained allosteric cGMP-binding affinity like that of WT PDE5 but displayed cGMP-induced gel shift only to the middle-band position. The upper band could represent a form produced by cGMP binding to the catalytic site, while the middle band could represent a form produced by cGMP binding to the allosteric site. Millimolar cGMP was required for gel shift of PDE5 when added to the pre-incubation before Native PAGE, presumably due to removal of most of the cGMP during electrophoresis, but micromolar cGMP was sufficient for this effect if cGMP was included in the native gel buffer. cGMP-induced gel shift was associated with stimulation of PDE5 catalytic activity, and the rates of onset and reversibility of this effect suggested that it was due to cGMP binding to the allosteric site. Incubation of PDE5 with non-hydrolyzable, catalytic site-specific, substrate analogs such as the inhibitors sildenafil and tadalafil, followed by dilution, did not produce activation of catalytic activity like that obtained with cGMP, although both inhibitors produced a similar gel shift to the upper band as that obtained with cGMP. This implied that occupation of the catalytic site alone can produce a gel shift to the upper band. PDE5 activation or gel shift was reversed by lowering cGMP with dilution followed by at least 1 h of incubation. Such slow reversibility could prolong effects of cGMP on PDE5 in cells after decline of this nucleotide. Reversal was also achieved by Mg⁺⁺ addition to the pre-incubation mixture to promote cGMP degradation, but Mg⁺⁺ addition did not reverse the gel shift caused by sildenafil, which is not hydrolyzed by PDE5. Upon extensive dilution, the effect of tadalafil, a potent PDE5 inhibitor, to enhance catalytic-site affinity for this inhibitor was rapidly reversed. Thus, kinetic effect of binding of a high-affinity PDE5 inhibitor to the catalytic site is more readily reversible than that obtained by cGMP binding to the allosteric site. It is concluded that cGMP or PDE5 inhibitor binding to the catalytic site, or ligand binding to both the catalytic site and allosteric site simultaneously, changes PDE5 to a similar physical form; this form is distinct from that produced by cGMP binding to the allosteric site, which activates the enzyme and reverses more slowly.     

The effects of octylglucoside on the interactions of chloroplast coupling factor 1 (CF1) with adenine nucleotides

The effects of octylglucoside (OcGlc) micelles, which stimulate a Mg-specific ATPase activity in chloroplast coupling factor 1 [Pick, U. and Bassilian, S. (1982) Biochemistry, 21, 6144-6152], on the interactions of the enzyme with adenine nucleotides have been studied. 1. OcGlc specifically accelerates the binding and the release of ADP but not of ATP or adenosine 5'[beta, gamma-imido]triphosphate (AdoPP[NH]P) from the tight-sites. The binding affinity for ADP and for ATP is only slightly decreased (twofold) by the detergent. ATP competitively inhibits the binding of ADP and vice versa in the presence or absence of OcGlc. 2.OcGlc-induced inactivation of CF1-ATPase is correlated with the release of bound nucleotides. In the absence of medium nucleotides ADP X CF1 is rapidly inactivated while ATP X CF1 and AdoPP[NH]P X CF1 are slowly inactivated by OcGlc in parallel with the release of bound nucleotide. In contrast, low concentrations of either ATP or ADP in the medium effectively protect against OcGlc inactivation while AdoPP[NH]P, whose binding to CF1 is inhibited by OcGlc, is ineffective even at millimolar concentrations. The results suggest that the occupancy of the tight-sites protects the enzyme against OcGlc-induced inactivation. 3. Mg ions specifically inhibit the release of bound ADP and the OcGlc-induced inactivation of CF1. High concentrations of medium ATP and ADP (K50 = 100 microM) also inhibit the OcGlc-induced release of bound nucleotides in an EDTA medium. In contrast, in the absence of OcGlc, medium ADP and ATP accelerate the release of bound adenine nucleotides. 4. Mg-ATP in the presence of OcGlc stimulates the release of bound ADP from CF1. Bound ATP is neither released nor hydrolyzed at the tight-sites under these conditions where medium ATP is rapidly hydrolyzed. Mg-ADP stimulates the release of bound ADP only in the presence of inorganic phosphate or of phosphate analogs, e.g. arsenate, pyrophosphate or selenate. 5. It is suggested that: (a) ATP and ADP bind to the same tight-sites, but OcGlc activation specifically accelerates the exchange of bound ADP at the site. (b) CF1 contains low affinity adenine nucleotide binding sites which may be the catalytical sites and which influence the tight-sites by cooperative interactions. (c) Mg-ATP in the presence of OcGlc induces a conformational change at the catalytical site which accelerates the release of ADP from the tight-site. The implications of these results to the role of adenine nucleotides in the regulation and mechanism of ATP hydrolysis by CF1 are discussed.     

Two pH optima of adenosine 5'-triphosphate dependent deoxyribonuclease from Bacillus laterosporus

The various catalytic activities of the ATP-dependent deoxyribonuclease (DNase) of Bacillus laterosporus have pH optima at 6.3 and 8.3. Although the pH profile of ATP-dependent DNase activity on duplex DNA is bell shaped with a maximum at about pH 8.3, ATP-dependent DNAse activity on single-stranded DNA has optima at pH 6.3 and 8.3. ATPase activities dependent on double-stranded and single-stranded DNA have a high bell-shaped peak with a maximum at pH 6.3 with a low and broad shoulder at about pH 8.3. ATP-independent DNase activity also has optima at pH 6.3 and 8.3. The ratio of the amount of ATP hydrolyzed per number of cleaved phosphodiester bonds in DNA increases with decrease in the pH value of the reaction. The ratios obtained at pH 8.3 and 6.3 were respectively about 3 and 22 with duplex DNA as substrate and 5 and 17 with single-stranded DNA as substrate. Formation of a single-stranded region of 15000-20000 nucleotides, which is linked to duplex DNA and about half of which has 3'-hydroxyl termini, was observed at about pH 6.3, but not at above pH 7.5. Furthermore, the optimum concentrations of divalent cations for the activity producing the single-stranded region and the activity hydrolyzing ATP were identical (3 mM Mn2+ or 5 mM Mg2+). Thus the two activities are closely related. These results indicate that the enzyme has two different modes of action on duplex DNA which are modulated by the pH.     

Conformational changes in the activation loop of the insulin receptor's kinase domain.

Low catalytic efficiency of basal-state protein kinases often depends on activation loop residues blocking substrate access to the catalytic cleft. Using the recombinant soluble form of the insulin receptor's kinase domain (IRKD) in its unphosphorylated state, activation loop conformation was analyzed by limited proteolysis. The rate of activation loop cleavage by trypsin is slow in the apo-IRKD. Bound Mg-adenine nucleoside di- and triphosphates increased the cleavage rate with half-maximal effects observed at 0.4-0.9 mM nucleotide. Adenosine monophosphate at concentrations up to 10 mM was not bound appreciably by the IRKD and had virtually no impact on activation loop cleavage. Amino-terminal and carboxy-terminal core-flanking regions of the IRKD had no statistically significant impact on the ligand-dependent or -independent activation loop cleavages. Furthermore, the core-flanking regions did not change the inherent conformational stability of the active site or the global stability of the IRKD, as determined by guanidinium chloride-induced denaturation. These measurements indicate that the intrasterically inhibitory conformation encompasses > or =90% of the ligand-free basal state kinase. However, normal intracellular concentrations of Mg-adenine nucleotides, which are in the millimolar range, would favor a basal-state conformation of the activation loop that is more accessible.