Negative Cooperative Behavior Research Articles

Will Increasing Government Subsidies Promote Open Innovation? A Simulation Analysis of China’s Wind Power Industry

Keeping open innovation both stable and sustainable can be difficult when it involves cooperation between large enterprises. Some empirical studies suggest that subsidy policies can play a positive role. This study addresses two key questions that follow from this observation: first, if the intensity of a subsidy policy is increased, can it play a greater role in strengthening the stability of cooperation between firms? Second, what other factors play a mediating role in this effect? Utilizing a dynamic game model, this paper analyses influential factors such as absorptive capacity, frequency of engagement and technical value on cooperative stability, and investigates the role of innovation policy in the process of cooperation through a random number-driven simulation. The findings indicate that only when the absorption capacity and technological value of both partners meet a certain threshold is the probability of positive cooperative behavior improved. Otherwise, increased subsidies tend to foster negative cooperative behavior instead.

Thermostable CITase from Thermoanaerobacter thermocopriae shows negative cooperativity

The biochemical properties of a putative thermostable cycloisomaltooligosaccharide (CI) glucanotransferase gene from Thermoanaerobacter thermocopriae were determined using a recombinant protein (TtCITase) expressed in Escherichia coli and purified to a single protein. The 171-kDa protein displayed maximum activity at pH 6.0, and enzyme activity was stable at pH 5.0-11.0. The optimal temperature was 60°C in 1h incubation, and thermal stability of the protein was 63% at 60°C for 24h. TtCITase produced CI-7 to CI-17, as well as CI-18, CI-19, and CI-20, which are relatively large CIs. Additionally, an unusual kinetic feature of TtCITase was its negative cooperative behavior in the dextran T2000 cleavage reaction. Based on our results, TtCITase can be applied to produce relatively large CIs at high temperature.

Negative Cooperative Binding of Thymidine, Ordered Substrate Binding, and Product Release of Human Mitochondrial Thymidine Kinase 2 Explain Its Complex Kinetic Properties and Physiological Functions.

Mitochondrial thymidine kinase 2 (TK2) catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC) and is essential for mitochondrial function in post-mitotic tissues. The phosphorylation of dT shows negative cooperativity, but the phosphorylation of dC follows classical Michaelis–Menten kinetics. The enzyme is feedback-inhibited by its end products deoxythymidine triphosphate (dTTP) and deoxycytidine triphosphate (dCTP). In order to better understand the reaction mechanism and the negative cooperative behavior, we conducted isothermal titration calorimetry (ITC) and intrinsic tryptophan fluorescence (ITF) quenching studies with purified recombinant human TK2. Cooperative binding was observed with dT but not dC by the ITC analysis in accordance with earlier enzyme kinetic studies. The phosphate donor adenosine triphosphate (ATP) did not bind to either dTTP-bound or dTTP-free enzymes but bound tightly to the dT– or dC–TK2 complexes with large differences in enthalpy and entropy changes, strongly suggesting an ordered binding of the substrates and different conformational states of the ATP and dT– and dC–TK2 ternary complexes. dTTP binding was endothermic; however, dCTP could not be shown to interact with the enzyme. ITF quenching studies also revealed tight binding of dT, dC, deoxythymidine monophosphate, deoxycytidine monophosphate, and dTTP but not adenosine 5′-diphosphate or ATP. These results strongly indicate an ordered sequential binding of the substrates and ordered release of the products as well as different conformational states of the active site of TK2. These results help to explain the different kinetics observed with dT and dC as substrates, which have important implications for TK2 regulation in vivo.

Fcab-HER2 Interaction: a Ménage à Trois. Lessons from X-Ray and Solution Studies

The crystallizable fragment (Fc) of the immunoglobulin class G (IgG) is an attractive scaffold for the design of novel therapeutics. Upon engineering the C-terminal loops in the CH3 domains, Fcabs (Fc domain with antigen-binding sites) can be designed. We present the first crystal structures of Fcabs, i.e., of the HER2-binding clone H10-03-6 having the AB and EF loop engineered and the stabilized version STAB19 derived by directed evolution. Comparison with the crystal structure of the Fc wild-type protein reveals conservation ofthe overall domain structures but significant differences in EF-loop conformations. Furthermore, we present the first Fcab-antigen complex structures demonstrating the interaction between the engineered Fcab loops with domain IV of HER2. The crystal structures of the STAB19-HER2 and H10-03-6-HER2 complexes together with analyses by isothermal titration calorimetry, SEC-MALS, and fluorescence correlation spectroscopy show that one homodimeric Fcab binds two HER2 molecules following a negative cooperative binding behavior.

Apparent cooperativity and apparent hyperbolic behavior of enzyme mixtures acting on the same substrate

It is well known that a negative cooperative behavior displayed by a monomeric enzyme may be associated with the simultaneous presence of two enzymes acting on the same substrate. In this paper, emphasis is given to the effect exerted by a rapid equilibrium between the enzyme forms in leading to a hyperbolic behavior, thus masking the presence of multiple enzyme forms.

Modulation of aldose reductase activity by aldose hemiacetals

BackgroundGlucose is considered as one of the main sources of cell damage related to aldose reductase (AR) action in hyperglycemic conditions and a worldwide effort is posed in searching for specific inhibitors of the enzyme. This AR substrate has often been reported as generating non-hyperbolic kinetics, mimicking a negative cooperative behavior. This feature was explained by the simultaneous action of two enzyme forms acting on the same substrate. MethodsThe reduction of different aldoses and other classical AR substrates was studied using pure preparations of bovine lens and human recombinant AR. ResultsThe apparent cooperative behavior of AR acting on glucose and other hexoses and pentoses, but not on tethroses, glyceraldehyde, 4-hydroxynonenal and 4-nitrobenzaldehyde, is generated by a partial nonclassical competitive inhibition exerted by the aldose hemiacetal on the reduction of the free aldehyde. A kinetic model is proposed and kinetic parameters are determined for the reduction of l-idose. ConclusionsDue to the unavoidable presence of the hemiacetal, glucose reduction by AR occurs under different conditions with respect to other relevant AR-substrates, such as alkanals and alkenals, coming from membrane lipid peroxidation. This may have implications in searching for AR inhibitors. The emerging kinetic parameters for the aldoses free aldehyde indicate the remarkable ability of the enzyme to interact and reduce highly hydrophilic and bulky substrates. General significanceThe discovery of aldose reductase modulation by hemiacetals offers a new perspective in searching for aldose reductase inhibitors to be developed as drugs counteracting the onset of diabetic complications.

Chemotactic Response of Escherichia Coli to Repellents, CoCl2 and NiCl2

Negative chemotaxis refers to the motion of microorganisms away from harmful chemicals. Soft agarose gel assay has been traditionally used to characterize the response to various repellents. In this study, we use the “chemical-in-plug” method to quantify the motion of Escherichia coli in the presence of repellents, NiCl2 and CoCl2, over a broad range of concentrations. These experiments were complimented with drift velocity measurements of individual bacteria in controlled gradients using a capillary assay. The latter also revealed the tumbling frequency and steady state clockwise bias for varying concentrations of repellents thereby providing insight into adaptation to repellents. The experimental technique yielded the motion of the bacteria in space and time and further related the motion to the evolving concentration profile of the repellent. Results show that the bacteria exhibit logarithmic sensing to the repellents, i.e., the drift velocity of E.coli is proportional to the logarithmic concentration gradient suggesting Weber law. The predictions of a standard population based model agreed with the observed linear behavior when the binding of the repellent to the receptor was sub-sensitive. This was borne out by a low value of Hill coefficient (n<<1) used to describe the binding characteristics of the receptors. The analysis shows that the binding characteristics for the repellents was sub-sensitive in contrast to an ultra-sensitive response observed for attractants suggesting a negative cooperative behavior of receptors. The above experiments suggest that negative cooperativity allows the cells to respond to harmful chemicals without saturation even at high concentration.

Characterization of novel thermostable dextranase from Thermotoga lettingae TMO

Biochemical properties of a putative thermostable dextranase gene from Thermotoga lettingae TMO were determined in a recombinant protein (TLDex) expressed in Escherichia coli and purified to sevenfold apparent homogeneity. The 64-kDa protein displayed maximum activity at pH 4.3, and enzyme activity was stable from pH 4.3-10. The optimal temperature was 55-60 degrees C during 15 min incubation, and the half-life of the enzyme was 1.5 h at 65 degrees C. The enzyme showed higher activity against alpha-(1 --> 6) glucan and released isomaltose and isomaltotriose as main products from dextran T2000. An unusual kinetic feature of TLDex was the negative cooperative behavior on the reaction of dextran T2000 cleavage. Enzyme activity was not significantly affected by the presence of metal ions, except for the strong inhibited by 1 mM Fe(2+) and Ag(2+). TLDex may prove useful as an enzyme for high temperature sugar milling processes.

Negative Cooperativity in a Protein Ion Channel Revealed by Current Noise, Conductance and Selectivity Experiments

Cooperativity is a phenomenon of universal importance in biophysics and has been extensively reported in many systems including enzymes, protein receptors and protein ion channels, among others. The concept of positive cooperativity appeared in the study of oxygen uptake by hemoglobin to explain that binding of a molecule of oxygen makes it easier the subsequent binding of other molecules. In contrast, negative cooperativity is found when the presence of the first molecule makes the binding of the second molecule more difficult. In particular, we study here the pH titration of the OmpF channel through measurement of current noise amplitude, conductance and ion selectivity. The steep pH dependence found both in channel conductance and Reversal Potential, together with the wide peak found in current noise amplitude are analyzed in terms of the Hill formalism. In all cases, Hill coefficients lower than unity are found, suggesting a negative cooperative behavior. Although OmpF porin is a trimer, previous studies demonstrate that each monomer is identical and both structural and functionally independent. The origin of cooperativity in each monomeric is subtle and does not necessarily demand the existence of different binding domains or subunits. Finally, experiments performed at different electrolyte concentrations evidence that salt cations play a major role in the observed channel features.

Purification and Characterization of α-Glucosidase I from Japanese Honeybee (Apis cerana japonica) and Molecular Cloning of Its cDNA

alpha-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.

On the mechanism and specificity of soluble, quinoprotein glucose dehydrogenase in the oxidation of aldose sugars.

Kinetic and optical studies were performed on the reductive half-reaction of soluble, quinoprotein glucose dehydrogenase (sGDH), i.e., on the conversion of sGDHox plus aldose sugar into sGDHred plus corresponding aldonolactone. It appears that the nature and stereochemical configuration of the substituents at certain positions in the aldose molecule determine the substrate specificity pattern: absolute specificity exists with respect to the C1-position (only sugars being oxidized which have the same configuration of the H/OH substituents at this site as the beta-anomer of glucose, not those with the opposite one) and with respect to the overall conformation of the sugar molecule (sugars with a 4C1 chair conformation are substrates, those with a 1C4 one are not); the nature and configuration of the substituents at the 3-position are hardly relevant for activity, and an equatorial pyranose group at the 4-position exhibits only aspecific hindering of the binding of the aldose moiety of a disaccharide. The pH optimum determined for glucose oxidation appeared to be 7.0, implying that reoxidation of sGDHred is rate-limiting with those electron acceptors displaying a different value under steady-state conditions. The kinetic mechanism of sGDH consists of (a) step(s) in which a fluorescing intermediate is formed, and a subsequent, irreversible step, determining the overall rate of the reductive half-reaction. The consequences of this for the likeliness of chemical mechanisms where glucose is oxidized by covalent catalysis in which a C5-adduct of glucose and PQQ are involved, or by hydride transfer from glucose to PQQ, followed by tautomerization of C5-reduced PQQ to PQQH2, are discussed. The negative cooperative behavior of sGDH seems to be due to substrate-occupation-dependent subunit interaction in the dimeric enzyme molecule, leading to a large increase of the turnover rate under saturating conditions.

Alterations in AMP deaminase activity and kinetics in skeletal muscle of creatine kinase-deficient mice.

Alterations in the competency of the creatine kinase system elicit numerous structural and metabolic compensations, including changes in purine nucleotide metabolism. We evaluated molecular and kinetic changes in AMP deaminase from skeletal muscles of mice deficient in either cytosolic creatine kinase alone (M-CK-/-) or also deficient in mitochondrial creatine kinase (CK-/-) compared with wild type. We found that predominantly fast-twitch muscle, but not slow-twitch muscle, from both M-CK-/- and CK-/- mice had much lower AMP deaminase; the quantity of AMP deaminase detected by Western blot was correspondingly lower, whereas AMP deaminase-1 (AMPD1) gene expression was unchanged. Kinetic analysis of AMP deaminase from mixed muscle revealed negative cooperativity that was significantly greater in creatine kinase deficiencies. Treatment of AMP deaminase with acid phosphatase abolished negative cooperative behavior, indicating that a phosphorylation-dephosphorylation cycle may be important in the regulation of AMP deaminase.

A Triangular Copper(I) Complex Displaying Allosteric Cooperativity in Its Electrochemical Behavior and a Mixed-Valence Cu(I)-Cu(I)-Cu(II) State with Unusual Temperature-Dependent Behavior.

Reaction of the tris-chelating hexadentate podand ligand tris[3-(2-pyridyl)pyrazol-1-yl]hydroborate (Tp(Py)) with [Cu(MeCN)(4)][PF(6)] affords [Cu(I)(3)(Tp(Py))(2)][PF(6)] (1), which was crystallographically characterized. 1.(MeCN)(2): C(52)H(44)B(2)Cu(3)F(6)N(20)P, orthorhombic, Pna2(1); a = 24.592(7), b = 16.392(5), c = 13.365(5) Å; Z = 4. Each Cu(I) ion is four coordinated by one N,N '-bidentate arm from each ligand; each ligand therefore donates each bidentate arm to a different Cu(I) ion. The isosceles triangular arrangement of Cu(I) ions with N-donor ligands is reminiscent of the tricopper(I) site of ascorbate oxidase. One-electron oxidation of 1 affords the Cu(I)(2)Cu(II) complex [Cu(3)(Tp(Py))(2)][PF(6)](2) (2). The potentials of the Cu(I)/Cu(II) redox couples are affected by the ease with which the accompanying geometric rearrangement can occur. Thus, the first oxidation of 1 is facile (-0.52 V vs the ferrocene/ferrocenium couple, Fc/Fc(+)), but as a result of the concomitant structural rearrangement the second oxidation is rendered much more difficult (+0.12 V vsFc/Fc(+)) and results in slow decomposition of the product. A third oxidation does not occur at accessible potentials. This complex therefore exhibits negative cooperative behavior, in which the geometric change accompanying one metal-based redox change hinders further redox changes at other sites via an allosteric effect. EPR studies on the mixed-valence complex 2 show that in frozen glasses below 120 K the unpaired electron is delocalized over two metal centers (7-line spectrum), but above 160 K the electron becomes localized and gives a simple axial spectrum. The electronic spectrum of 2 in solution shows an intense band at 910 nm (epsilon 2100 dm(3) mol(-)(1) cm(-)(1)) which we believe to be an IVCT band. The combination of EPR and electronic spectral studies show that 2 is class III (fully delocalized over 2 centers) below 120 K but class II (localized but strongly interacting) at higher temperatures.

Effects of NAD+ binding on the luminescence of tryptophans 84 and 310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

The individual fluorescence and phosphorescence properties of W84 and W310 in Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase were identified through the construction of a single tryptophan mutant (W84F) and by comparison of the emission between mutant and wild-type enzymes. The results show that the luminescence of W310 is red-shifted and substantially quenched relative to that of W84. It displays an average subnanosecond fluorescence lifetime (tau F) and a very short, 50 microseconds, room-temperature phosphorescence (RTP) lifetime (tau P). The perturbation of W310 luminescence is believed to arise from a stacking interaction with Y283. In contrast, W84 exhibits a fluorescence lifetime tau F of several nanoseconds and a long-lived phosphorescence lifetime tau P, typical of buried, unperturbed TrP residues. NAD+ binding to the tetrameric enzyme causes a 55% reduction of W310 fluorescence intensity together with a nearly complete quenching of its low-temperature phosphorescence. W84, which is located far from the nicotinamide moiety of NAD+, is much less affected by the binding of the coenzyme; the reduction in fluorescence intensity is 35%, and its phosphorescence intensity is unchanged. Another consequence of NAD+ binding is a significant decrease of the RTP lifetime tau P of W84, manifesting thereby a conformational change in the region of the coenzyme-binding domain. However, no change is observed in the RTP lifetime tau P of W310 located in the catalytic domain. These findings and those obtained at partial coenzyme saturation support the conclusions derived from high-resolution crystallographic structures [Skarzynski, T., & Wonacott, A. J., (1988) J. Mol. Biol. 203, 1097-1118] that the NAD(+)-induced conformational change is sequential and that subtle rearrangement in the structure of unligated subunits might be responsible for the negative cooperative behavior of NAD+ binding.

Structural Asymmetry and Half-Site Reactivity in the T to R Allosteric Transition of the Insulin Hexamer

The zinc-insulin hexamer, the storage form of insulin in the pancreas, is an allosteric protein capable of undergoing transitions between three distinct conformational states, designated T6, T3R3, and R6, on the basis of their ligand binding properties, allosteric behavior, and pseudo point symmetries [Kaarsholm, N. C., Ko, H.-C., & Dunn, M. F. (1989) Biochemistry 28, 4427-4435]. The transition from the T-state to the R-state involves a coil-to-helix transition in residues 1-8 of the B-chain wherein the ring of PheB1 is displaced by approximately 30 A. This motion also is accompanied by small changes in the positions of A-chain residues and other B-chain residues. In this paper, one- and two-dimensional (COSY and NOESY) 1H NMR are used to characterize the ligand-induced T to R transitions of wild-type and EB13Q mutant human zinc-insulin hexamers and to make sequence-specific assignments of all resonances in the aromatic region of the R6 complex with resorcinol. The changes in the 1H NMR spectrum (at 500 and 600 MHz) that occur during the T to R transition provide specific signatures of the conformation change. Analysis of the dependence of these spectral changes for the phenol-induced transition as a function of the concentration of phenol establish (1) that the interconversion of T6 and R6 occurs via a third species assigned as T3R3 and (2) that the system shows both negative and positive cooperative allosteric behavior. One- and two-dimensional COSY and NOESY studies show that, in the absence of phenolic compounds, anions act as heterotropic effectors that shift the distribution of hexamer conformations in favor of the R-state with the order of effectiveness, SCN- > N3- >> I- >> Cl-. Analysis of one- and two-dimensional spectra indicate that with wild-type insulin, SCN- and N3- give T3R3 species, whereas the EB13Q mutant gives an R6 species. An allosteric model for the insulin T to R transition based on the structural asymmetry model [Seydoux, F., Malhotra, O. P., & Bernhard, S. A. (1974) CRC Crit. Rev. Biochem. 2, 227-257] is proposed that explains the negative and positive allosteric properties of the system, including the role of T3R3 and the action of homotropic and heterotropic effectors.

Enzyme kinetic and inhibition analyses of cytochrome P450XVII, a protein with a bifunctional catalytic site. Quantification of effective substrate concentrations at the active site and their significance for intrinsic control of the hydroxylase/lyase reaction sequence.

An enzyme kinetic analysis of the cytochrome P450XVII-dependent testicular steroid-17 alpha-hydroxylase/17,20-lyase system was performed which catalyzes simultaneously, at one active site, the formation of androstenedione from progesterone (productive pathway, coordinated performance of both hydroxylase and lyase activities) and the formation of 17 alpha-hydroxyprogesterone as the intermediate (abortive pathway, isolated performance of hydroxylase activity only). Progesterone was used as the substrate and promegestone (17 alpha, 21-dimethyl-4,9(10)-pregnadiene-3,20-dione) or estradiol as competitive inhibitors, and description of the data was based on the discrimination of those alternative routes of progesterone metabolism. The overall catalytic activity of P450XVII obeyed Michaelis-Menten kinetics (control incubations: V = 440 nM/min with 80 nM P450XVII, Km = 140 nM), and KI values amounted to 6.8 microM for promegestone and to 23.5 microM for estradiol. In contrast, analysis of the rates of both abortive and productive events yielded curvilinear Eadie-Hofstee plots. The former presented an apparent negative cooperative behavior with nh = 0.78 in the region of Kh, while the latter presented an apparent substrate inhibition behavior with the maximal rate occurring at that progesterone concentration where nh for the abortive path reached its minimum. Consequently, the ratio of productive versus abortive catalytic events decreased with increasing substrate supply, but did not reach zero. Effective substrate concentrations [S]eff with respect to either the productive or the abortive pathway were derived from Hill plots. Linearization of Eadie-Hofstee plots and parallel calculation methods using these [S]eff instead of [S] yielded V = 236 nM/min and [S]eff(0.5) = 131 nM for the abortive events and V = 217 nM/min and [S]eff(0.5) = 38 nM for the productive events. Both promegestone and estradiol were identified as competitive inhibitors of either reaction after consideration of effective progesterone concentrations. With the inhibitors, ratios of productive versus abortive events were constantly higher at a given substrate concentration than in control incubations, although neither promegestone nor estradiol affected progesterone or 17 alpha-hydroxyprogesterone accumulation in the endoplasmic reticulum membrane compartment. Rather, a linear correlation between ratios of productive versus abortive events and the overall catalytic rate as a measure of E.S complex concentration was obtained irrespective of the absence or presence of inhibitor. It was therefore concluded that promegestone and estradiol inhibit access or binding of progesterone to P450XVII, whereas the relative efficiency of androgen formation is solely dictated by the local substrate concentration being effective with respect to E.S formation. A model of P450XVII function is presented which proposes that excess substrate at the active site of the P450XVII enzyme protein hinders a certain fraction of a putative transient intermediate from being retained at the bifunctional catalytic site with the consequence that it cannot be further processed to androgen.

Applicability of the induced-fit model to glyceraldehyde-3-phosphate dehydrogenase from sturgeon muscle. Study of the binding of oxidized nicotinamide adenine dinucleotide and nicotinamide 8-bromoadenine dinucleotide.

A new method of calculation, based on a direct fitting of the protein fluorescence intensity observed upon coenzyme binding (H.-P. Lutz, unpublished results), is used to study the negative cooperative behavior of glyceraldehyde-3-phosphate dehydrogenase from sturgeon muscle. The calculation procedure simultaneously elaborates data obtained for four different protein concentrations, and it is able to compare different models by computing the minimal and critical sum of squares. Using this approach, it is shown that the induced-fit model [Koshland, D. E., Jr., Nemethy, G., & Filmer, D. (1966) Biochemistry 5,365] and the dimer of dimer model [Malhotra, O. P., & Bernhard, S. A. (1968) J. Biol. Chem. 243, 1243-1252] can both be applied for explaining the negative cooperativity observed upon coenzyme binding to sturgeon glyceraldehyde-3-phosphate dehydrogenase. In addition to the progressive modification of the binding affinity during ligand binding, different maximal fluorescence quenchings for the binding steps must be postulated; and furthermore, the binding capability decreases by decreasing the protein concentration. The fact that the induced-fit model can also be applied is rather in contradiction with the view generally accepted of a dimer of dimer structure of sturgeon glyceraldehyde-3-phosphate dehydrogenase. By use of the same approach, nicotinamide 8-bromoadenine dinucleotide is shown to bind to glyceraldehyde-3-phosphate dehydrogenase from sturgeon in a negative cooperative manner.

Insulin-dependent and insulin-independent low Km cyclic AMP phosphodiesterase from rat adipose tissue.

Chromatographic analysis of a soluble extract of rat adipose tissue on DEAE-Sephacel resolves four distinct peaks of 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) activity. Kinetic investigation indicates that two of these fractions have a high affinity for cyclic AMP and show negative cooperative kinetic behavior at high substrate concentration. They differ in the degree of inhibition by cyclic GMP and in their response to insulin. If rat epididymal fat pads are incubated with insulin prior to homogenization, only one of the low Km cyclic AMP phosphodiesterase forms is stimulated.

Characterization of particulate and soluble guanylate cyclases from rat lung

Rat lung homogenates contained significant amounts of guanylate cyclase activity in both 100,000 times g (60 min) particulate and supernatant fractions. In the presence of detergent, the particulate fraction contained 40% as much activity as did the supernatant fraction. Detergent-dispersed particulate and partially purified soluble guanylate cyclase preparations were characterized with respect to divalent cation requirements, divalent cation interactions, kinetic behavior, and gel filtration profiles. Both soluble and particulate guanylate cyclases required divalent cation for activity. The soluble preparation was 10 times more active in the presence of Mn-2plus than in the presence of Mg-2plus or Ca-2plus and no detectable activity was seen with Ba-2plus or Sr-2plus. Particulate guanylate cyclase activity was detectable only in the presence of Mn-2plus. Both enzyme preparations required Mn-2plus in excess of GTP for optimal activity at subsaturating amounts of GTP. At near-saturating GTP, the soluble enzyme required excess Mn-2plus, but the particulate enzyme did not. For kinetic analyses the enzymes were considered to require two substrates: metal-GTP and Me-2plus. Apparent negative cooperative behavior was seen with the soluble enzyme when excess Mn-2plus (in excess of GTP) was varied from 0.01 to 0.2 mM; above 0.2 mM excess Mn-2plus classical kinetic behavior was seen with an apparent KMn-2plus of 0.2 mM at near-saturating MnGTP. Similar studies using the particulate preparation yielded only classical kinetic behavior, but the apparent KMn-2plus decreased to near zero when MnGTP was near-saturating. Kinetic patterns for the particulate and soluble enzymes also differed when reciprocal initial velocities were plotted as a function of reciprocal MnGTP concentrations; classical kinetic behavior was seen with the soluble enzyme with an apparent KMnGTP of about 12 muM (at near-saturating excess Mn-2plus), whereas apparent positive cooperative behavior was seen with the particulate preparation (Hill coefficient equals 1.6, S0.5 EQUALS 70 MUM. Ca-2plus "activation" of soluble guanylate cyclase was related to the Mn-2plus:GTP ratio. Activation was most apparent when saturating amounts of Mn-2plus and MnGTP. At relatively high concentrations of Ca-2plus (0.1 to 4 mM), the addition of 10 muM Mn-2plus resulted in a 3- to 5-fold increase in soluble guanylate cyclase activity. In contrast, Ca-2plus sharply inhibited particulate guanylate cyclase activity. Gel filtration profiles of particulate and soluble preparations indicated differences in physical properties of the enzymes. As estimated by gel filtration, particulate (detergent-dispersed)evels. Here, removal of renal tissue is contraindicated. In all renal hy