Effects of Temperature on Allosteric and Catalytic Properties of the cGMP-stimulated Cyclic Nucleotide Phosphodiesterase from Calf Liver*

We have investigated effects of temperature on the oatalytic and allosteric properties of the cGMP-stimu- lated cyclic nucleotide phosphodiesterase from calf liver. V,, for cAMP and cGMP increased as assay temperature increased from 5 to 45 "C. At substrate concentrations below KZp, however, hydrolysis in- creased as temperature decreased from 45 to 5 "C and was much greater at 5 OC than at 45 OC. As assay temperature decreased, K Z p for cAMP and cGMP de- creased. Hill coefficients for cAMP and cGMP were -1.9 at 45 "C and 1.2-1.0 at 5 "C. cGMP stimulated hydrolysis of 0.5 NM ['HICAMP at all assay temperatures. Although maximal activity stimulated by cGMP, like V,,., was lowest at 5 "C, presumably because of the effect of temperature on catalytic activity, the apparent activation constant (Kim) for cGMP stimulation was lower at 5 "C than at 45 OC. Thus, affinity for both substrate and effector was increased at 5 "C, suggest- ing that low temperature promotes transitions of the cGMP-stimulated phosphodiesterase to a "high affin-ity'' state. That cGMP stimulated cAMP hydrolysis at 5 "C suggests that temperature-induced transitions are incomplete and/or readily reversible. In assays at 30 "C competitive inhibitors, like sub-


Effects of Temperature on Allosteric and Catalytic Properties of the cGMP-stimulated Cyclic Nucleotide Phosphodiesterase from Calf Liver*
(Received for publication, February 18,1986) Hide0 Wadal, James C. Osborne, Jr.$ll, and Vincent C. Manganielloll We have investigated effects of temperature on the oatalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. V , , for cAMP and cGMP increased as assay temperature increased from 5 to 45 "C. At substrate concentrations below KZp, however, hydrolysis increased as temperature decreased from 45 to 5 "C and was much greater at 5 OC than at 45 O C . As assay temperature decreased, K Z p for cAMP and cGMP decreased. Hill coefficients for cAMP and cGMP were -1.9 at 45 "C and 1.2-1.0 at 5 "C. cGMP stimulated hydrolysis of 0.5 NM ['HICAMP at all assay temperatures. Although maximal activity stimulated by cGMP, like V , , . , was lowest at 5 "C, presumably because of the effect of temperature on catalytic activity, the apparent activation constant (Kim) for cGMP stimulation was lower at 5 "C than at 45 OC. Thus, affinity for both substrate and effector was increased at 5 "C, suggesting that low temperature promotes transitions of the cGMP-stimulated phosphodiesterase to a "high affinity'' state. That cGMP stimulated cAMP hydrolysis at 5 "C suggests that temperature-induced transitions are incomplete and/or readily reversible.
In assays at 30 "C competitive inhibitors, like substrates, induce allosteric transitions which result in enhanced hydrolysis of low substrate (1.0 PM ['HI CAMP) concentrations. At higher substrate concentrations (50 PM ['HICAMP), with the enzyme in the "activated'' state, inhibitors compete with substrate at catalytic sites and reduce hydrolysis. At 45 "C, as at 30 "C, 1-methyl-3-isobutylxanthine (IBMX) and papaverine increased hydrolysis of 1.0 PM ['HICAMP and reduced hydrolysis of 50 PM ['HICAMP. At 5 "C, however, IBMX and papaverine inhibited hydrolysis of both 1.0 and 50 PM ['HICAMP. Enzyme activity was relatively more sensitive to inhibition by IBMX at 5 "C than at 45 OC.
Taken together, these observations support the notion that low temperature induces incomplete or readily reversible transitions to the high affinity state for substrates, effectors, and inhibitors. These observed effects of temperature also point out that enzyme determinants and topographical features responsible for transitions to the high affinity state and expression of catalytic activity can be regulated independently.
The cGMP-stimulated cyclic nucleotide phosphodiesterase, purified from bovine heart, adrenal, and liver (1)(2)(3), hydrolyzes both cAMP and cGMP with positively cooperative kinetics (nap, for CAMP -1.6-1.8; napp for cGMP -1.3-1.5). As might be expected, at substrate concentrations well below KF, hydrolysis of one cyclic nucleotide is increased by appropriate concentrations of the other; cGMP is preferred as substrate and effector (1)(2)(3). In one simple model for the allosteric activation of the phosphodies~ra~, binding of substrate or effector to "ligand-free" enzyme ("low affinity state") induces allosteric transitions to an active or high affinity state (4), and hydrolysis of substrate at low concentrations is enhanced by virtue of an alteration in K,,, and not Vm,,. Certain competitive inhibitors, like substrates, bind to the low affinity form of the phosphodiesterase, induce allosteric transitions, and increase hydrolysis of low concentrations of substrate; at higher substrate concentrations (ie. with the activated enzyme), these compounds compete with substrate at catalytic sites (5, 6).
In the experiments reported here, effects of temperature on allosteric transitions and catalytic activity were investigated. Whereas Vmax for cAMP and cGMP hydrolysis increases as assay temperature increases from 5 to 45 "C, hydrolysis of low substrate concentrations is highest at 5 "C. Hill coefficients (napp) for cAMP and cGMP hydrolysis approach 2 at 45 "C and 1 at 5 "C. From these and other observations and from effects of competitive inhibitors on cAMP hydrolysis in assays at different temperatures, we suggest that low temperature promotes transitions of the c G M P -s t i m u l a~ phosphodiesterase to the high affinity state and higher temperatures to the low affinity state. In light of these and other properties of the cGMP-stimulated phosphodiesterase, it is conceivable that transitions between low and high affinity states are important in regulation of phosphodiesterase activity in vivo and that a physiological mechanism for cGMP action might involve allosteric activation of the phosphodiesterase and regulation of intracellular cAMP degradation.

Effect of Temperature on c~M P -s t~m u~~e d P~s p h o d i e s t e r~e
Behring. Sources of all other materials have been published (2,6).

P r e~r a t w~ ami Assay of ~G~P -s t i r n~~d
Cyclic Nucleotide Pho3phodiesterase-The enzyme was purified to apparent homogeneity from calf liver (2). Stock enzyme was diluted in Buffer A (50 mM HEPES, pH 7.5, 3 mg/ml fatty acid-free bovine serum albumin, 0.1 mM EDTA, 1 mM NaN3, 0.4 mM phenylmethylsulfonyl fluoride, 1 HM pepstatin, 2 p~ leupeptin). Activity was assayed as described (2) at the indicated temperature in a final volume of 0.3 ml containing 15 pmol of HEPES buffer, 2.5 @mol of MgCI,, 15 nmol of EGTA, 150 pg of ovalbumin, -20,000-30,OOO cpm [3H]cAMP or cGMP, indicated concentrations of cAMP and cGMP, at pH 7.5. The reaction was terminated and *H-labeled nucleosides isolated for radioassay as described (2). Initial rates of hydrolysis of high and low substrate concentrations were established at every assay temperature (cf. Fig.  1) before measuring hydrolysis of the wide range of substrate concentrations in kinetic experiments at different assay temperatures. Data reported are means of values for duplicate assays in representative experiments. Protein was assayed using Coomassie Brilliant Blue G-250 (Bio-Rad) with bovine serum albumin as a standard (7).

RESULTS
Effect of Temperature on V, , , , KZp, and nnPp-With 250 p~ [3H]cAMP as substrate (a saturating concentration sufficient to induce allosteric transitions and activate the phosphodiesterase), the rate of cAMP hydrolysis increased with assay temperature between 5 and 45 "C ( Fig. L4). In contrast, initial rates of hydrolysis of 1.0 p~ [3H]cAMP, a concentration well below KZP, were highest at 5 "C and lowest at 45 "C ( Fig. 1s). Increasing concentrations of cGMP, up to 5-10 pM, stimulated hydrolysis of 0.5 p~ [3H]cAMP and higher concentrations decreased it ( Fig. 2.4). -Fold stimulation was highest at 45 "C and lowest at 5 "C ( Fig. 2B). Apparent activation constants (KZpp) for cGMP were calculated from graphs of (1/ V-V,) (where Vo is activity in absence of cGMP at each temperature) uersus l/cGMP (Fig, 3). K:PP is a function of substrate concentration; in this case, substrate concentration was 0.5 p~ [3H]cAMP, a value well below P $ P for cAMP (1-3). K : P P decreased from -5 p~ at 45 "C to -0.2 p~ at 5 "C ( Fig. 3), indicating that affinity for effector increased as temperature decreased.
Vmax and KZp for cAMP and cGMP hydrolysis increased with temperature between 5 and 45 "C ( Fig. 4). As seen in Fig. 4 (inserts), napp for cAMP and cCMP hydrolysis approached 1.9 at 45 "C and 1.0-1.2 at 5 "C; K,"P (estimated as that concentration of substrate where log(v/V,,,v) = 0) for cAMP and cGMP was lowest at 5 "C. In Fig. 5, v/V,,,,, is plotted as a function of substrate concentration. At 5 "C, the concentration of cAMP and cGMP required to produce 50% V, , , was much lower than at 45 "C. From a plot of log VmnX versus 1/T, the activation energy (Ea) for cAMP and cGMP hydrolysis was estimated. At 30 "C, AH (AH = Ea + RT) was approximately 11,000 cal/mol for both cAMP and cGMP (data not shown). These results are consistent with the idea that at 5 "C, the phosphodiesterase undergoes transitions to a high affinity state (increased hydrolysis of low substrate concentrations, reduced napp and K,"P). Although catalytic activity is markedly reduced at 5 "C (decreased V, , , , decreased cGMP-stimulated activity), hydrolysis at low substrate Concentrations is increased because of the increased affinity.
Effects of Competitive Inhibitors ut 5 and 45 T-In assays at 30 "C, certain competitive inhibitors mimic substrate, bind to the low-affinity state of the phosphodiesterase, induce allosteric transitions to the high affinity or activated state, and increase hydrolysis of low substrate concentrations (4,5). In the activated state, i.e. in assays performed in the presence of 1 p~ cGMP or at high substrate concentrations, inhibitors compete with substrate at catalytic sites according to classical competitive kinetics (4,5). IBMX and papaverine stimulated hydrolysis of 1 p~ ['HICAMP at 30 "C, as previously reported (5), and at 45 "C, but inhibited at 5 "C ( Fig. 6), observations consistent with the idea that high and low temperatures induce transitions to the low and high affinity states, respectively. Higher concentrations of IBMX were required to produce maximal stimulation of cAMP hydrolysis at 45 "C than at 30 "C (data not shown). In assays carried out at higher substrate concentrations, i.e. 50 p~ [3H]cAMP, hydrolysis was inhibited by both IBMX and papaverine at 5, 30, and 45 "C, the IC5, being highest at 45 "C (Fig. 6).
IBMX was relatively more effective in inhibiting cAMP hydrolysis at 5 "C than at 45 "C, as seen from plots of velocity versus log S (Fig. 7). Lineweaver-Burk plots of cAMP hydrolysis at 5 "C were linear (Kkpp -20 pM CAMP; KFp -22 pM IBMX) but plots at 45 "C were curvilinear, consistent with an napp of -1.9 (data not shown). At 45 "C, in the presence of IBMX and high substrate concentrations, double-reciprocal plots were linear; graphical analysis indicated a KJKI of -1. 4 and a KTpp of -45 pM.
Effects of Solvent Polarity on cAMP Hydrolysis-In assays at 30 "C, hydrolysis of 0.5 p~ [3H]cAMP was stimulated by increasing concentration of short chain alcohols, especially isopropyl and butyl alcohols, as well as dimethyl sulfoxide and N,N-dimethylformamide (Fig. 8). Methanol, up to 3% by volume, had little or no effect on enzyme activity (data not shown). Ethanol and isopropyl alcohol slightly enhanced stimulation of 0.5 p~ [3H]cAMP hydrolysis by cGMP and had little or no effect on hydrolysis of 250 p~ [3H]cAMP; higher concentrations of butanol and isobutanol inhibited hydrolysis (Fig. 8). Increasing concentrations of isopropyl alcohol reduced both KzP and nap, for CAMP, with no change in Vmax (Fig. 9). Whether these effects are related to changes in overall solvent polarity or specific interactions of the alcohols with hydrophobic enzyme domains is not known. Specific hydrophobic domains may be important for enzyme activity, since of a number of xanthine derivatives tested, IBMX and 1-methyl-3-isopropylxanthine were much more effective inhibitors of the cGMP-stimulated phosphodiesterase than theophylline (1,3-dimethyl~anthine).~

DISCUSSION
According to a common model for allosteric activation of the cGMP-stimulated phosphodiesterase, the interaction of substrates, effectors, and even competitive inhibitors with the ligand-free or low affinity state induces transitions to the high affinity or activated state (1)(2)(3)(4)(5)(6). In the activated state, n, , for cAMP is reduced, and hydrolysis of low concentrations of cAMP is increased by virtue of a decrease in for cAMP (1)(2)(3). Taken together, all of the observations reported herein suggest that at high temperature (ie. >30 "C), the cGMPstimulated phosphodiesterase is in the low affinity state, with * H. Wada, J. C. Osborne, Jr., and V. C. Manganiello, unpublished observations.  Fig. 4, V,, was estimated at each assay temperature. u/Vmax ratio at each substrate concentration is plotted ver.su.s log S at 5 "C (0), 20 "C (e), 30 "C (A), 40 "C (A), and 45 "C (El) for cGMP @anel A ) and CAMP (punel B). PDE, phosphodiesterase. reduced affinity for substrate (K,"" at 45 "C > 5 "C), effector ( E p p for cGMP stimulation of cAMP hydrolysis at 45 "C > 5 "C), and inhibitors (KIaw for IBMX and for IBMX and papaverine at 45 "C > 5 "C). In assays at 45 "C, nepp for cAMP hydrolysis was -1.9, and papaverine and IBMX stimulated hydrolysis of low cAMP concentrations, indicating that at 45 "C, as at 30 "C (4,5), substrates and competitive inhibitors bind to the low affinity form and induce transitions to the activated state. In the low affinity state, however, at physiological temperatures the phosphodiesterase, by virtue of its sigmoidal velocity-substrate responses, would be very sensitive to changes in concentrations of cAMP or to stimulation by cGMP (at concentrations within the presumed physiological range for both cyclic nucleotides (16-21)). Conversely, at low (5 "C) temperature, hydrolysis of low cAMP concentrations was increased, r a p p for cGMP stimulation of cAMP hydrolysis was reduced, and KZP and n , , for cAMP and cGMP were also decreased. In addition, at 5 "C, competitive inhibitors did not stimulate hydrolysis of low CAMP concentrations, and IBMX was more effective in inhibiting cAMP hydrolysis at 5 "C than at 45 "C. These findings are consistent with the view that low temperature (5 "C) induces transitions to the high affinity or activated state. Basal hydrolysis of low substrate concentrations was increased at 5 "C, presumably due to the increased affinity for substrate at this temperature, despite apparent effects of temperature on catalytic activity ( Vmax for hydrolysis of cAMP and cGMP and stimulation of cAMP hydrolysis by cGMP was lowest at 5 "C).
Exposure of the phosphodiesterase to high pH (9-10.5) activates the enzyme, without altering VmeX? Activation (i.e. transitions to the high affinity state) induced by high pH is complete in the sense that after exposure to high pH hydrolysis of low cAMP concentrations is increased to such an extent that it is not further stimulated by cGMP.3 Certain Wada, H., Osborne, J. C., Jr., and Manganiello, V. C. (1987) B~~~~t~, submitted for publication. short chain alcohols also activate, i.e. increase hydrolysis of low cAMP concentrations with reduced KZp and napp, without change in Vmax. Temperature, on the other hand, seems to affect both cooperative interactions (aapp) and catalytic activity ( Vmax). In the presence of short chain alcohols and at all temperatures tested, however, cGMP stimulated cAMP hydrolysis. Thus, although low temperature does increase affinity for substrates, effectors, and inhibitors, the apparent activation is incomplete since at 5 "C hydrolysis of cAMP is stimulated by cGMP. Transitions to the high affinity or activated state induced by low temperature or short chain alcohols are either incomplete, readily reversible, or proceed via different mechanisms than the pH-induced changes. In any case, these and earlier findings by us and Erneux and coworkers (3,5,(8)(9)(10) are consistent with the idea that the molecular interactions important in promoting allosteric transitions can be regulated independently of those important for catalysis, and that high and low affinity states possess binding domains with distinct topographical features. Whether temperature or short chain alcohols alter subunit interactions or the conformation of these high and low affinity binding domains, or both, is not known. Along these lines, Ross and Subramanian (11) have suggested that the initial steps in protein-protein subunit interactions and protein-ligand interactions are driven by hydrophobic forces and the second steps are stabilized by short range interactions such as electrostatic forces and hydrogen bonding. The ability to control the dis-tribution of low and high affinity states in uitro by use of effectors, inhibitors, and various reaction conditions, including temperature, expands greatly the experimental approaches to study and understand regulation of this phosphodiesterase.
Of the major classes of phosphodiesterase, the cGMPstimulated form is unique in exhibiting positively cooperative homotropic kinetics for hydrolysis of both cAMP and cGMP (12)(13)(14)(15). As a result of the cooperative responses to cAMP and cGMP, one cyclic nucleotide can increase hydrolysis of the other. cGMP is, however, preferred as substrate and effector (3,8). A number of other characteristics of the enzyme (1)(2)(3)5,6,(8)(9)(10)(11), including effects of temperature on allosteric regulation, strongly support the concept that under physiologic conditions cGMP, functioning as an allosteric activator of the phosphodiesterase, could regulate cAMP degradation. At physiological temperatures, the cGMP-stimulated phosphodiesterase is in the low affinity state, with KZp well above presumed tissue concentrations of cAMP ( p~) (16-21) and cGMP (10 or 100 times less than CAMP) (16)(17)(18)(19)(20)(21). At substrate concentrations below saturation, sigmoidal responses in substrate-velocity relationships exhibited by the cGMP-stimulated phosphodiesterase provide a sensitive, "on-off" type, regulation of velocity. In addition, and perhaps more importantly, aIthough the KZp for cGMP is about 2 orders of magnitude above tissue cGMP, M cGMP (within the physiological range) (16-21) can increase hydrolysis of micromolar concentrations of CAMP, strongly suggesting that under physiological conditions, elevations in tissue cGMP content could increase cAMP degradation. Activation of this phosphodiesterase by cGMP could thus provide a molecular basis for the opposing actions of cAMP and cGMP described in some biological systems (20). In fact, Hartzell and Fischmeister (22) have recently concluded from patch clamp studies with frog ventricle cells that decreases in CAMP-induced slow inward Ca" current were brought about via degradation of cAMP by the cGMP-stimulated phosphodiesterase.
Activity of the cGMP-stimulated phosphodiesterase can be influenced by a number of conditions that alter the macroand microenvironment of the cell, e.g. hormonal balance (14), cellular differentiation (15), pH: temperature, as well as by potential physiological and therapeutic effectors, substrates (1)(2)(3), fatty acids (23), short chain alcohols, sulfhydryl reagents (24), and competitive inhibitors (4,5). Subcellular localization may also be an important element in enzyme function, since the cGMP-stimulated phosphodiesterase has been found in both particulate and soluble fractions of various tissues and cells (12), including cultured Madin-Darby canine kidney cells (15), and in association with bovine brain coated vesicles (25). The integration of these factors in regulation of this phosphodiesterase in the intact cell and the role this enzyme plays in metabolism of cyclic nucleotides in general or in control or functional compartmentalization of discrete processes regulated by cAMP remain to be established.