Binding of the delta subunit to rod phosphodiesterase catalytic subunits requires methylated, prenylated C-termini of the catalytic subunits

PDE6 (type 6 phosphodiesterase) from rod outer segments consists of two types of catalytic subunits, alpha and beta; two inhibitory gamma subunits; and one or more delta subunits found only on the soluble form of the enzyme. About 70% of the phosphodiesterase activity found in rod outer segments is membrane-bound, and is thought to be anchored to the membrane through C-terminal prenyl groups. The recombinant delta subunit has been shown to solubilize the membrane-bound form of the enzyme. This paper describes the site and mechanism of this interaction in more detail. In isolated rod outer segments, the delta subunit was found exclusively in the soluble fraction, and about 30% of it did not coimmunoprecipitate with the catalytic subunits. The delta subunit that was bound to the catalytic subunits dissociated slowly, with a half-life of about 3.5 h. To determine whether the site of this strong binding was the C-termini of the phosphodiesterase catalytic subunits, peptides corresponding to the C-terminal ends of the alpha and beta subunits were synthesized. Micromolar concentrations of these peptides blocked the phosphodiesterase/delta subunit interaction. Interestingly, this blockade only occurred if the peptides were both prenylated and methylated. These results suggested that a major site of interaction of the delta subunit is the methylated, prenylated C-terminus of the phosphodiesterase catalytic subunits. To determine whether the catalytic subunits of the full-length enzyme are methylated in situ when bound to the delta subunit, we labeled rod outer segments with a tritiated methyl donor. Soluble phosphodiesterase from these rod outer segments was more highly methylated (4.5 +/- 0.3-fold) than the membrane-bound phosphodiesterase, suggesting that the delta subunit bound preferentially to the methylated enzyme in the outer segment. Together these results suggest that the delta subunit/phosphodiesterase catalytic subunit interaction may be regulated by the C-terminal methylation of the catalytic subunits.     

Comparative immunochemical characteristics of different phosphodiesterases of cyclic nucleotides

Precipitating monospecific antibodies against purified bovine retinal rod outer segment phosphodiesterase (EC 3.1.4.17) were obtained from rabbit blood serum. These antibodies do not form precipitating complexes with phosphodiesterase isolated from rat or ox brain tissues or from the heart, lung, liver, kidney, testes and uterus of the rat. The antibodies inhibit the activity of retinal rod outer segment phosphodiesterase or that of rat brain, liver, heart and uterus enzyme (despite the lack of precipitation) but have no effect on the phosphodiesterase activity of preparations obtained from rat lungs, kidney or testes. The same effect on the phosphodiesterase activity of all these tissues is exerted by monovalent fragments of the antibodies. Using partially purified preparations of phosphodiesterase from retinal rod outer segments and brain of the ox and from human myometrium, the mechanisms of inhibition of the enzyme catalytic activity by the antibodies was studied. In the presence of the antibodies, the Km and V values appeared to be different, depending on the preparation. It was assumed that a certain site in the phosphodiesterase molecule is characterized by great structural rigidity. Taking into account the shifts in the Km values induced by the antibodies, the differences in the localization of the antigenic determinant in relation to the enzyme active center are discussed.     

Distribution of enzyme activities in subcellular fractions of bovine retina.

Centrifugation of homogenates of bovine retinas to isopycnic equilibrium in sucrose density gradients yielded three partially overlapping bands of particles which were, in the order of increasing density: (a) photoreceptor cell (rod) outer segments; (b) plasma membranes, lysosomes, and large fragments of endoplasmic reticulum; and (c) mitochondria. The only enzyme activity investigated which had a peak coinciding only with outer segment fractions was guanylate cyclase. Enzyme activities with peaks in both the outer segment and denser fractions included 5'-nucleotidase and cyclic GMP phosphodiesterase. Enzyme activities with peaks only in the denser fractions included sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase), adenylate cyclase, cyclic AMP phosphodiesterase, beta-glucosidase, beta-galactosidase, and succinate-dependent cytochrome c reductase. These results suggest that some of the activities once thought to be present in rod outer segments are actually present in particles from elsewhere in the retina which contaminate rod outer segment preparations.     

A Ca2+-Binding Protein that Regulates the Lifetime of the Transducin Active State

Biophysics - Abstract—It has been previously shown in the study of the kinetic behavior of cGMP-specific phosphodiesterase in preparations of the outer segments of bovine retinal rods, which...     

Role of cyclic nucleotide phosphodiesterase in the effect of estradiol on uterine tissue

It is shown that 17 beta-estradiol (10(-7)--10(-5) M) inhibited phosphodiesterase activity of the preparations (supernatant 100000 epsilon, 1 h) obtained from uterine tissue of sexually mature rats and did not affect adenylate cyclase activity of crude membrane fraction of this tissue. The hormone did not change phosphodiesterase activity of the preparations obtained from the brain, heart and outer segments of the retinal rods. Cytosol preparations from uterine tissue were demonstrated to be able to specific hormone binding. The antiestrogen clomifen completely blocked the binding. In the presence of clomifen estradiol had no effect on phosphodiesterase activity. It is suggested that estrogen receptors are necessary for the effect of 17 beta-estradiol on phosphodiesterase to be realized in uterine tissue.     

Interleukin-1α (IL-1α) production by alveolar macrophages in patients with acute lung diseases: the influence of zinc supplementation

In vertebrate retina, rod outer segment is the site of visual transduction. The inward cationic current in the dark-adapted outer segment is regulated by cyclic GMP. A light flash on the outer segment activates a cyclic GMP phosphodiesterase resulting in rapid hydrolysis of the cyclic nucleotide which in turn causes a decrease in the dark current. Restoration of the dark current requires inactivation of the phosphodiesterase and synthesis of cyclic GMP. The latter is accomplished by the enzyme guanylate cyclase which catalyzes the formation of cyclic GMP from GTP. Therefore, factors regulating the cyclase activity play a critcal role in visual transduction. But regulation of the cyclase by some of these factors — phosphodiesterase, ATP, the soluble proteins and metal cofactors (Mg and Mn) — is controversial. The availability of different types of cyclase preparations, dark-adapted rod outer segments with fully inhibited phosphodiesterase activity, partially purified cyclase without PDE contamination, cloned rod outer segment cyclase free of other rod outer segment proteins, permitted us to address these controversial issues. The results show that ATP inhibits the basal cyclase activity but enhances the stimulation of the enzyme by soluble activator, that cyclase can be activated in the dark at low calcium concentrations under conditions where phosphodiesterase activity is fully suppressed, and that greater activity is observed with manganese as cofactor than magnesium. These results provide a better understanding of the controls on cyclase activity in rod outer segments and suggest how regulation of this cyclase by ATP differs from that of other known membrane guanylate cyclases.This work was supported by the grants from the National Institutes of Health (EY07158, EY 05230, EY 10828, NS 23744) and the equipment grant from Pennsylvania Lions Eye Research Foundation.     

Phosphorylation reactions in bovine rod outer segments studied by 32P-labelling of intact retina

The protein phosphorylation pattern in the intact bovine retina has been investigated by labelling with 32P-phosphate under incubation conditions that preserve the electrical photoresponse of the photoreceptor cells. The phosphorylation of rod outer segment proteins was analysed after isolation of outer segments from the labelled retina. The global influence of light, Ca2+ and the phosphodiesterase inhibitor, isobutylmethylxanthine, on protein phosphorylation in rod outer segments was analysed. A 12 kDa protein is the most prominent phosphorylated species in the intact bovine retina. Its phosphorylation is increased by light and/or Ca2+. Evidence is presented that this strongly phosphorylated protein is not located in the outer segment, and we suggest that it may be a synaptic protein. Retinal rod outer segment membrane proteins with apparent molecular weights of 245, 226, 125, 110, 50, 46, 38 and 20 all show light-stimulated phosphorylation. Lowering the extracellular Ca2+ levels results in a decrease of the phosphorylation level of some of these proteins, viz. at 125, 50, 38 and probably at 20 kDa. Such proteins, whose phosphorylation level is influenced both by light and by elevated Ca2+, are candidates for mediators of phototransduction. The phosphorylated species at 245, 226, 110, 50 and 20 kDa are enriched in rod outer segment plasma membrane preparations. These protein species could participate in the light-regulated modulation of the Na+-conductance of the plasma membrane.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF THE BOVINE RETINA: ACTIVITY, SUBCELLULAR DISTRIBUTION AND KINETIC PARAMETERS

Abstract— High phosphodiesterase activity for cyclic AMP and cyclic GMP was found in subcellular fractions of the bovine retina with more rapid hydrolysis of cyclic GMP than cyclic AMP in each fraction. Rod outer segments (ROS) and the supernatant fraction had highest activity. High enzyme activity remained associated with ROS membranes through several steps of purification by gradient centrifugation. A complex kinetic pattern was observed for cyclic AMP hydrolysis by the supernatant fraction yielding two values for K _m; a simple kinetic pattern was observed with cyclic GMP hydrolysis in supernatant and for both cyclic nucleotides in preparations of purified outer segments. Phosphodiesterase activity of outer segments was enhanced by Mg²⁺. Mn²⁺ and inhibited by EDTA. Cyclic AMP had relatively little effect on the hydrolysis of cyclic GMP in supernatant or ROS while cyclic GMP inhibited hydrolysis of cyclic AMP in both fractions.     

Biosynthetic studies of A2E, a major fluorophore of retinal pigment epithelial lipofuscin.

We have examined questions related to the biosynthesis of A2E, a fluorophore that accumulates in retinal pigment epithelial cells with aging and in some retinal disorders. The use of in vitro preparations revealed that detectable levels of A2-PE, the A2E precursor, are formed within photoreceptor outer segments following light-induced release of endogenous all-trans-retinal. Moreover, experiments in vivo demonstrated that the formation of A2-PE in photoreceptor outer segment membrane was augmented by exposing rats to bright light. Whereas the generation of A2E from A2-PE by acid hydrolysis was found to occur very slowly, the detection in outer segments of a phosphodiesterase activity that can convert A2-PE to A2E may indicate that some portion of the A2-PE that forms in the outer segment membrane may undergo hydrolytic cleavage before internalization by the retinal pigment epithelial cell. The identities of additional minor components of retinal pigment epithelium lipofuscin, A2E isomers with cis olefins at positions other than the C13-C14 double bond, are also described.     

Influence of effectors of hormone-sensitive adenylate cyclase on the activation system of photostimulated cyclic nucleotide phosphodiesterase from outer rod segments

The effect of preincubation of preparations of the outer segments of optic rods with the nonhydrolyzed analog GTP-guanilyl-5'-imidodiphosphate (Gpp(NH)p) and NaF, the combined effect of these agents as well as the action of (NH4)2SO4 (10-800 mM), MgSO4 (2-50 mM) and induction of peroxide oxidation of lipids are studied as applied to the catalytic activity of phosphodiesterase of cyclic nucleotides. Gpp(NH)p and NaF are shown to be tightly bound to GTP-binding proteins (G-proteins) of outer segments of optic rods, additional activation of phosphodiesterase in the presence of Gpp(NH)p being observed after preincubation with NaF and subsequent washing of the membrane. A problem on different binding sites of the ion F and Gpp(NH)p on G-proteins is discussed. It is found that (NH4)2SO4 does not affect the basal activity of phosphodiesterase but inhibits the activating effect of Gpp(NH)p and NaF on the enzyme. Induction of peroxide oxidation of lipids prevented by the addition of ionol (antioxidant) in a dose of 5.10(-4) M has the same effect. Changes in the concentration of Mg2+ in the medium influence insignificantly the basal activity of phosphodiesterase but are necessary for manifestation of the activating effect of Gpp(NH)p and NaF.     

Protein inhibitor of the retinal cyclic nucleotide phosphodiesterase: its localization in the outer segment of a photoreceptor]

The content of a protein inhibitor of the cyclic nucleotides phosphodiesterase (PDE) in different retinal preparations as well as its distribution in the subfractions of rod outer segments (ROS) was studied. The content of protein inhibitor of PDE in different preparations of the retina was found to correlate with the rhodopsin content. The distribution of this protein over different ROS subfractions appeared to be exactly the same as that of rhodopsin, the content of protein inhibitor of PDE being more than a half of its content in the native ROS. The protein inhibitor of PDE could be easily washed out from the ROS fractions. It is concluded that the cattle protein inhibitor of PDE is localized in ROS, and is absent in the other retinal layers.     

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.     

Isolation of an inhibitory protein for the cyclic guanosine 3','5'-monophosphate phosphodiesterase of bovine rod outer segments

Cyclic guanosine 3',5'-monophosphate phosphodiesterase in crude extracts from bovine rod outer segments can be activated by the addition of bleached rod outer segment membranes and GTP. In the absence of rhodopsin-containing membranes, the phosphodiesterase specific activity decreases with increasing concentration. A trypsin-sensitive inhibitor believed to be responsible for this phenomenon can be separated from the phosphodiesterase by DEAE-cellulose chromatography of the crude extract. Phosphodiesterase eluted from the DEAE-cellulose column shows considerably less concentration-dependence than in the crude extract. This partially purified phosphodiesterase was used as the substrate to assay for inhibitor. A GTPase which is active only in the presence of bleached rod outer segment membranes coelutes with the phosphodiesterase and is distinct from the phosphodiesterase inhibitor we have isolated.     

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.     

Cyclic nucleotide phosphodiesterases associated with bovine retinal outer-segment fragments.

ATP-dependent cyclic GMP phosphodiesterase activity (EC 3.1.4.16) associated with bovine retinal outer-segment fragment preparations was stimulated an order of magnitude by light, confirming the results of Miki et al. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3820-3824 at Yale for the frog system. In contrast to the results of the Yale group, however, light stimulation was not observed for cyclic AMP as substrate. A direct relationship of bovine rhodopsin bleaching to phosphodiesterase activation differs from a previous report by the Yale group that full activation of the frog enzyme was achieved by bleaching of a maximum of 2% rhodopsin. Phosphodiesterase activity could be qualitatively removed from the fresh outer-segment preparations with isotonic sucrose which apparently did not disrupt the plasmalemma or discs. Activity recovered from the washing was not light sensitive. Two Km values were determined for cyclic AMP, 5 and 0.05 mM; for cyclic GMP a Km of 0.22 mM was found. All Km values were determined in the presence of 1 mM ATP in the dark. Sonication of fresh outer segments or storing at -20 degrees C abolished the light response. However, storage at -76 degrees C fully preserved it.     

Immunologic characterization of the photoreceptor outer segment cyclic GMP phosphodiesterase

High affinity (KD approximately 1 X 10(-9) M) monoclonal antibodies (ROS-1 and ROS-2) were prepared to bovine photoreceptor outer segment cGMP phosphodiesterase. ROS-1 immunoadsorbed greater than 95% of the cGMP phosphodiesterase activity from a detergent-solubilized bovine retina extract while ROS-2 immunoadsorbed only a subfraction of the same activity. Sodium dodecyl sulfate gel analysis of these immunoadsorbates demonstrated that ROS-1 and ROS-2 specifically adsorbed only peptides that comigrated with purified rod outer segment phosphodiesterase. Limited trypsin digestion of purified rod outer segment phosphodiesterase greatly reduced its affinity for ROS-1 but not ROS-2. When a crude heat-stable inhibitor fraction was added back to the activated enzyme, the affinity for ROS-1 was restored, suggesting that the inhibitor was necessary for ROS-1 binding. ROS-1 but not ROS-2 was found to inhibit cGMP phosphodiesterase which had been activated either by dilution or guanyl nucleotide. The inhibitory property of ROS-1 may provide a useful probe for directly studying the effects of this phosphodiesterase on the phototransduction response in the retina. Sodium dodecyl sulfate gel analysis demonstrated that the ROS-1 immunoadsorbates from mammals, fish, and amphibia contained peptides of similar mobility. Immunocytochemistry performed with ROS-1 and fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG localized the antigenic determinant to both rod and cone outer segments suggesting the presence of an antigenically similar phosphodiesterase in both types of photoreceptors.     

Characterization of the light-induced increase in the Michaelis constant of the cGMP phosphodiesterase in frog rod outer segments

Activation of cGMP phosphodiesterase in rod disk membrane in the light is thought to be an intermediary process of phototransduction. In various preparations of frog rod outer segments, the Michaelis constant (Km) of the phosphodiesterase was measured with pH assay method. On illumination, the Km increased from the value of the dark (130 microM) by about 8-fold (1 mM) in crude preparations, but did not change appreciably in purified disk membranes, confirming the previous finding by Robinson et al. (Robinson, P.R., Kawamura, S., Abramson, B. and Bownds, M.D. (1980) J. Gen. Physiol. 76, 631-645). The present work further showed that the light-induced Km increase is labile to various experimental manipulations such as sonication, freeze-thawing, etc. However, the Km in the light was relatively high in a crude disk membrane preparation and in a lyzed preparation. In addition, reconstitution experiments revealed that the Km increase does not require soluble components. Both proteolytic digestion and phospholipase treatment reduced the light Km of the phosphodiesterase in crude disk membranes. The above results suggest that there is a labile factor(s) responsible for the light-induced Km increase and that the factor is a membrane-bound protein and requires structural integrity of the disk membrane to exert its function. The latency of the Km increase after light stimulation was less than 2 s.     

Isolation and recombination of bovine rod outer segment cGMP phosphodiesterase and its regulators

cGMP phosphodiesterase extracted from rod outer segments can be activated by GTP in the presence of phospholipid vesicles containing bleached rhodopsin. I have separated the phosphodiesterase from a phosphodiesterase inhibitory protein and a GTPase also present in the crude extracts from rods. The GTPase can be activated by bleached rhodopsin. However, in the absence of the GTPase and inhibitor, the phosphodiesterase was not activated by GTP in the presence of bleached rhodopsin. Recombination with these proteins partially restored the activation by GTP and bleached rhodopsin.     

Localization of several rhodopsin regeneration enzymes in retinal rods]

The distribution of NAD kinase and glucose-6-phosphate dehydrogenase within membranes of both outer and inner retina rod segments was studied by the sucrose gradient centrifugation of crude outer segment preparations. Rhodopsin and retinoldehydrogenase served as markers for outer segment membranes, whereas succinate dehydrogenase was a marker for inner ones. It is shown that NAD kinase and glucose-6-phosphate dehydrogenase are localized within inner segment membranes of the photoreception cell and that the activity of these enzymes in the crude preparations is due to contamination of the inner segments.     

Light-stimulated protein movement in rod photoreceptor cells of the rat retina

We examined the intracellular distribution of three proteins involved in the cyclic GMP cascade of visual transduction; cGMP phosphodiesterase, the alpha-subunit of G-protein and arrestin. In adult rats, light-induced changes in the amounts of G and arrestin in the photoreceptor cell outer segments were observed both by polyacrylamide gel analysis of purified ROS and by immunocytochemical localization on retinal sections. In dark conditions, G was concentrated in the outer segments of photoreceptor cells while in the light G alpha was seen in the inner segments and the outer nuclear layer. Arrestin had the opposite distribution, appearing in the inner segments and outer nuclear layer under dark conditions and in the ROS under light conditions. In contrast, PDE, the enzyme which is activated by G and inhibited by arrestin showed no light-stimulated movement. In both light- and dark-adapted retinas, PDE was localized primarily in the outer segments of the photoreceptor cells.