Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments.

Frog (Rana catesbiana) rod outer segment disc membranes contain a cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which is activated by light in the presence of ATP. This enzyme is firmly bound to the disc membrane, but can be eluted from the membrane with 10 mM Tris-HCl buffer, pH 7.4 and 2 mM EDTA. The eluted phosphodiesterase has reduced activity, but can be activated approximately 10-fold by polycations such as protamine and polylysine. The eluted phosphodiesterase can no longer be activated by light in the presence of ATP, that is, activation by light apparently depends on the native orientation of phosphodiesterase in relationship to other disc membrane components. The eluted phosphodiesterase was purified to homogeneity as judged by analytical polyacrylamide gel electrophoresis and polyacrylamide gel isoelectric focusing. The over-all purification from intact retina was approximately 925-fold. The purification of phosphodiesterase from the isolated rod outer segment preparation was about 185-fold with a 28% yield. Phosphodiesterase accounts for approximately 0.5% of the disc membrane protein. The eluted phosphodiesterase (inactive form) has a sedimentation coefficient of 12.4 S corresponding to an approximate molecular weight of 240,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates the purified phosphodiesterase into two subunits of 120,000 and 110,000 daltons. With cyclic 3':5'-GMP (cGMP) as substrate the Km for the purified phosphodiesterase is 70 muM. Protamine increases the Vmax without changing the Km for cGMP. The isoelectric point (pI) of the native dimer is 5.7. Limited exposure of the eluted phosphodiesterase (inactive form) to trypsin produces a somewhat greater activation than is obtained with 0.5 mg/ml of protamine. The trypsin-activated phosphodiesterase has a sedimentation coefficient of 7.8 S corresponding to an approximate molecular weight of 170,000. The 110,000-dalton subunit is much less sensitive to trypsin hydrolysis and the 120,000-dalton subunit is rapidly replaced by smaller fragments. On the basis of the molecular weight of the purified phosphodiesterase (240,000) and the concentrations of phosphodiesterase and rhodopsin in the rod outer segment, it is estimated that the molar ratio ophosphodiesterase to rhodopsin in the rod outer segment is approximately 1:900. Since all of the disc phosphodiesterase molecules are activated when 0.1% of the rhodopsins are bleached, we conclude that in the presence of ATP 1 molecule of bleached rhodopsin can activate 1 molecule of phosphodiesterase.     

Inhibition of cyclic nucleotide phosphodiesterase from the rod outer segments of the frog retina by 3-hydroxypyridine

The influence of 8 analogues of 3-hydroxypyridine upon the phosphodiesterase of rod outer segments of frog retinae has been investigated. It has been shown that the analogues of 3-hydroxypyridine inhibit the enzeme reversely and noncompetitively in case of hydrolysis towards the cAMP and cGMP. The natural analogues of 3-hydroxypyridine (pyridoxol, pyridoxale, pyridoxale-phosphate) do not exert the inhibiting effect. It is suggested that the inhibition of phosphodiesterase from rod outer segments of retinae is caused by the interaction of 3-hydroxypyridines with the hydrophobic microenvironment of the active site of the enzyme.     

Light activation of phosphodiesterase activity in retinal rod outer segments

Light “activates” phosphodiesterase activity of bovine rod outer segments in the presence of 0.1 mM ATP. In contrast, no difference in phosphodiesterase activity can be observed between dark-adapted and light-bleached outer segments in the absence of ATP.     

The effect of rhodopsin phosphorylation on the light-dependent activation of phosphodiesterase from bovine rod outer segments

ATP quenches light-dependent phosphodiesterase (PDE) activation in rod outer segments presumably due to rhodopsin phosphorylation. Here we compared the efficiency of phosphorylated and non-phosphorylated rhodopsins as PDE activators in a reconstituted cell-free system. It is shown that the ability of phosphorylated membranes to activate this enzyme is suppressed compared with non-phosphorylated ones.     

Purification of guanylyl cyclase from rod outer segments.

The particulate form of guanylyl cyclase from bovine rod outer segments has been solubilized and purified to near homogeneity by a combination of liquid chromatography and native gel electrophoresis. The procedure enriches enzyme activity 6700-fold from rod outer segment extracts to a final specific activity of 17.5 mumol/min per mg (when assayed with Mn-GTP as substrate). Purified preparations of guanylyl cyclase contain a single glycoprotein with an apparent molecular mass of 60,000 Da and a native isoelectric point of 7.6. Although crude or partially purified enzyme activity is modulated by sub-micromolar concentrations of Ca2+, the fully purified enzyme is insensitive to this cation. However, the purified enzyme remains sensitive to nitrovasodilators, being stimulated over 10-fold by sodium nitroprusside. These data suggest that retinal rods contain a unique isoform of guanylyl cyclase.     

Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.     

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

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

On a soluble system for studying light activation of rod outer segment cyclic GMP phosphodiesterase

Bovine rod outer segment (ROS) cyclic GMP phosphodiesterase (PDE) could be activated about 6-fold by light, an effect that could be simulated by isolated bleached rhodopsin. About 90% of PDE activity in ROS could be extracted with 10 mM Tris-HCl, pH 7.5, but light is ineffective in activating the soluble enzyme. However, bleached rhodopsin could activate it in the presence of a very low concentration of ATP, strongly suggesting the mediation of rhodopsin in the light activation of the enzyme in ROS. Direct evidence is presented to suggest that the phosphorylation of opsin (bleached rhodopsin) is unrelated to the activation of PDE by bleached rhodopsin and ATP. The reconstitution of the light activation of PDE in a soluble system presented here opens up a new direction to future investigations on the mechanism of light regulation of cyclic GMP levels in retina and its implication in the photoreceptor function.     

Size changes of phosphodiesterase in bovine rod outer segments on illumination

Light activates a 3',5'-cyclic GMP phosphodiesterase (PDE) in bovine retinal rod outer segments. The light is absorbed by rhodopsin situated in the disk membranes. PDE is a three-subunit peripheral protein on the disks and appears to be activated via a guanine nucleotide binding protein (G) in the presence of activated rhodopsin and GTP. Does the activation occur by collision coupling of G and PDE? We have studied the protein-protein interactions of PDE in situ in disk membranes by radiation inactivation. Irradiation of a protein with high-energy electrons leads to loss of activity in proportion to radiation dose and the molecular weight of the protein. We see no change in the size of PDE upon activation by light and 100 microM guanosine 5'-(beta, gamma-imidotriphosphate) (Gpp[NH]p) compared with PDE in dark with 260 microM GTP. Application of statistics to our data shows that a 27 000 change in molecular weight would be significant at the 95% level but that smaller changes would go undetected. The apparent molecular weight is 176 000 +/- 27 000 (mean +/- 95% confidence limit), in agreement with the size determined by polyacrylamide gel electrophoresis. Thus there appears to be either (i) no permanent change in PDE size on activation or (ii) a small change, undetectable by the technique, or (iii) an exchange of subunits such that no net change in molecular weight is seen.     

Properties and content of cyclic nucleotide phosphodiesterase in photoreceptor outer segments of ground squirrel retina

Cyclic GMP-specific phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 1.3.4.17) (PDE) is thought to be a key enzyme of the retinal-rod phototransduction cyclic nucleotide pathway. We attempted to investigate the properties and content of PDE in retinal-cone photoreceptors. The fractions obtained from cone-dominant ground squirrel retinas were analyzed for cone visual pigment content and PDE activity. The cone visual pigment content was estimated to be approx. 65 pmol per retina. The distribution of cone visual pigment coincided with that of the PDE activity through several steps of photoreceptor membrane purification by sucrose density gradient centrifugation. The ground squirrel retinal PDE was similar to the retinal-rod PDE by its kinetic properties, thermostability, sensitivity to tryptic activation, Stokes radius and pI values. The cone visual pigment enriched fractions contained the heat-stable trypsin-inactivated PDE inhibitor. Its functional properties seem to be similar to those of the retinal-rod PDE inhibitory subunit. The PDE content in ground squirrel retina was roughly estimated to be about five copies of enzyme per 100 cone visual pigment molecules. The obtained results indicated that the major portion of ground squirrel retinal cyclic GMP-specific PDE is the endogenous cone photoreceptor membrane enzyme and strongly supported the conception about the key role of PDE in cone phototransduction. The existence of essential differences between rod and cone systems rapidly returning cyclic GMP-specific amplification cascade components to the dark (or inactivated) states after photon absorption was suggested. If this suggestion is true, the well-known distinctions between response kinetics and light sensitivity of these two kinds of photoreceptor can be explained.     

cGMP phosphodiesterase in rod and cone outer segments of the retina

Immunochemical, chromatographic, and sodium dodecyl sulfate gel electrophoresis studies suggest that immunologically related but distinct cyclic GMP phosphodiesterases are present in rod and cone outer segments of the retina. Immunocytochemical studies demonstrated that one monoclonal antibody (ROS-1) recognized a determinant present in both rod and cone outer segments, while another monoclonal antibody (ROS-2) only recognized rod outer segments. At least two peaks of phosphodiesterase activity could be separated by high-performance anion-exchange chromatography of retinal extracts. Both peaks were recognized by ROS-1. None of the first peak and only 80% of the second broad peak of activity were recognized by ROS-2. High-performance liquid chromatography profiles from human fovea and several other types of cone-enriched retina showed that most of the activity was contained in the first peak, suggesting that this activity was derived from cone outer segments. Conversely, the phosphodiesterase in rod-enriched preparations migrated predominately in the second peak. Sodium dodecyl sulfate-gel electrophoresis indicated that this first peak contained a single large immunoreactive polypeptide (alpha') that migrated with the same mobility as a phosphorylase b standard and was distinct from the more rapidly migrating large immunoreactive polypeptides (alpha and beta) present in a broad second peak. The second peak could be further separated into a first part that contained a doublet of two immunoreactive polypeptides (alpha and beta) that migrated faster than phosphorylase b and a later part that contained only the most rapidly migrating polypeptide (beta). All of the peaks could be activated by histone or transducin:GTP, implying that all contained a small 11-kDa inhibitory subunit (gamma) of the enzyme. Since the larger (alpha') and smaller (beta) immunoreactive polypeptides could be completely separated from the alpha polypeptide and from each other, yet still retain the ability to be activated by histone or transducin, the data suggest that only a single species of polypeptide-inhibitor complex (e.g. alpha' gamma, alpha gamma, or beta gamma) was required for histone or transducin:GTP activation.     

Involvement of ATP in activation and inactivation sequence of phosphodiesterase in frog rod outer segments

Cyclic GMP phosphodiesterase in frog rod outer segments is activated after flash illumination and is inactivated when left in the dark. ATP reduces the initial peak activity caused by dim flashes (with 50 microM ATP being required for a half-maximal effect) and also accelerates inactivation (with 2 microM ATP being required for a half-maximal effect). An acceleration of inactivation caused by ATP addition is 3-7-fold, depending on the preparation, and ATP effect can be observed even 1 min after a dim flash is given. The accelerated inactivation is also flash intensity-dependent. A low intensity of light causes more rapid inactivation than does a high intensity of light. ATP appears to control phosphodiesterase activity in various ways.     

Isolation of cyclic AMP and cyclic GMP by thin-layer chromatography. Application to assay of adenylate cyclase, guanylate cyclase, and cyclic nucleotide phosphodiesterase

A procedure is described for isolation of cAMP and cGMP by thin-layer chromatography on polyethylenimine cellulose. Chromatographs are developed (descending) twice in the same direction with two different solvents. This procedure separates cAMP and cGMP from other radioactive metatolites of [³H] or [¹⁴C] ATP or GTP. Application of this isolation method to assay of adenylate cyclase, (EC 4.6.1.1), guanylate cyclase (EC 4.6.1.2), and cyclic nucleotide phosphodiesterase (EC 3.1.4.17) has proven convenient and provides results of unusual quality.     

Proteolytic activation of calmodulin-dependent cyclic nucleotide phosphodiesterase

Purified calmodulin-stimulated cyclic nucleotide phosphodiesterase from brain, a homodimer of 59-kDa subunits, was activated by limited proteolysis with trypsin, alpha-chymotrypsin, Pronase, or papain and could not be further stimulated by addition of Ca2+ and calmodulin. Proteolysis increased Vmax and had little effect on the Km for cGMP. Treatment with alpha-chymotrypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) produced, sequentially, 57- and 45-kDa peptides from the bovine and 55-, 53-, and 38-kDa peptides from the ovine enzyme. This protease-treated phosphodiesterase exhibited a Stokes radius of 3.9 nm and an S20,w value of 4.55; comparison with the hydrodynamic properties observed for native enzyme (4.3 nm, 5.95 S) strongly suggests a dimeric protein of Mr approximately 80,000-90,000. The proteolyzed species does not interact significantly with calmodulin immobilized on agarose, nor does it show complex formation with 2-dimethylaminonaphthalene-1-sulfonyl-calmodulin even at micromolar concentrations of protein. Proteolysis, in the presence of calmodulin plus Ca2+, fully activated phosphodiesterase, producing the same intermediate peptides; however, final peptides from the bovine and ovine enzymes were 47 and 42 kDa, respectively, indicating a new, specific conformation of the enzyme. When EGTA was added to such incubations, these peptides were cleaved to those of the size seen when proteolysis was carried out entirely in the presence of EGTA. The initial rate of activation was increased by the presence of Ca2+ and calmodulin, suggesting that, in complex, phosphodiesterase exhibits a site with increased susceptibility to proteolysis. Since calmodulin can still interact with a fully activated form of the enzyme, it appears that retention of calmodulin binding can occur concomitantly with damage to that portion of the phosphodiesterase molecule responsible for suppression of its basal catalytic activity.     

Characterization of guanylate cyclase activity in single retinal rod outer segments

cGMP mediates vertebrate phototransduction by directly gating cationic channels on the plasma membrane of the photoreceptor outer segment. This second messenger is produced by a guanylate cyclase and hydrolyzed by a light-activated cGMP-phosphodiesterase. Both of these enzyme activities are Ca2+ sensitive, the guanylate cyclase activity being inhibited and the light-activated phosphodiesterase being enhanced by Ca2+. Changes in these activities due to a light-induced decrease in intracellular Ca2+ are involved in the adaptation of photoreceptors to background light. We describe here experiments to characterize the guanylate cyclase activity and its modulation by Ca2+ using a truncated rod outer segment preparation, in order to evaluate the enzyme's role in light adaptation. The outer segment of a tiger salamander rod was drawn into a suction pipette to allow recording of membrane current, and the remainder of the cell was sheared off with a probe to allow internal dialysis. The cGMP-gated channels on the surface membrane were used to monitor conversion of GTP, supplied from the bath, into cGMP by the guanylate cyclase in the outer segment. At nominal 0 Ca2+, the cyclase activity had a Km of 250 microM MgGTP and a Vmax of 25 microM cGMP s-1 in the presence of 1.6 mM free Mg2+; in the presence of 0.5 mM free Mg2+, the Km was 310 microM MgGTP and the Vmax was 17 microM cGMP s-1. The stimulation by Mg2+ had an EC50 of 0.2 mM Mg2+ for MgGTP at 0.5 mM. Ca2+ inhibited the cyclase activity. In a K+ intracellular solution, with 0.5 mM free Mg2+ and 2.0 mM GTP, the cyclase activity was 13 microM cGMP s-1 at nominal 0 Ca2+; Ca2+ decreased this activity with a IC50 of approximately 90 nM and a Hill coefficient of approximately 2.0.     

Stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments

The stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments was investigated using phosphorothioate analogs of GTP as chiral probes. (Sp)-Guanosine 5'-O-(1-thiotriphosphate) (Sp-GTP alpha S) is a substrate, whereas (Rp)-GTP alpha S is a competitive inhibitor (K1 = 0.1 mM), but not a substrate. (Sp)-GTP alpha S is converted into (Rp)-guanosine 3':5'-monophosphorothioate, showing that the reaction proceeds with inversion of configuration at the alpha-phosphorus atom. Km and Vmax for (Sp)-GTP alpha S (at low [Ca2+], 20 nM) are 3.7 mM and 1.1 nmol/min/mg of rhodopsin, respectively, compared with 1.1 mM and 23.1 nmol/min/mg of rhodopsin for GTP. Vmax for the cyclization of (Sp)-GTP alpha S, as for GTP, increases 10-20-fold when the calcium level is lowered. This activity change is centered at approximately 90 nM and has a Hill coefficient of 4.8. The configuration of the metal-substrate complex was determined by measuring the effectiveness of the Sp and Rp isomers of GTP alpha S and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) in the presence of Mg2+ or Mn2+. (Sp)-GTP alpha S is a substrate with either Mg2+ or Mn2+, whereas (Rp)-GTP beta S is a substrate with only Mn2+. These findings suggest that the substrate is a metal-beta, gamma-bidentate complex with delta screwsense. We also found that the cyclization reaction catalyzed by the membrane-bound guanylate cyclase from sea urchin sperm proceeds with inversion of configuration at the alpha-phosphorus atom. The stereochemical course of the reactions catalyzed by all prokaryotic and eukaryotic adenylate cyclases and guanylate cyclases studied thus far is the same.