Search PubMed⌕ Search

Biomedical subjects

J A Beavo

Publications and source records attributed to J A Beavo.

At least 73 records · Page 4Linked to original sources
PubMed1992

Regulation and function of cyclic nucleotides.

Recent work has greatly expanded our knowledge of the structure, regulation and diversity of enzymes involved in the synthesis and degradation of cyclic nucleotides. This review focuses on recent work that provides insight into the structure and function of the cyclases and phosphodiesterases that regulate cyclic nucleotide metabolism. Particular emphasis is given to the roles played by multiple isoforms of each enzyme system.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1991

Structure and function studies of the cGMP-stimulated phosphodiesterase.

Studies of cGMP binding to both the native cyclic GMP-stimulated phosphodiesterase and to two unique isolated chymotryptic fragments lacking the catalytic domain suggest that the enzyme contains two noncatalytic cGMP-binding sites/homodimer. In the presence of high concentrations of ammonium sulfate, 2 mol of cGMP are bound/mol of cGMP-stimulated phosphodiesterase homodimer. Under these conditions, linear Scatchard plots of binding are obtained that give an apparent Kd of approximately 2 microM. The inclusion of 3-isobutyl-1-methylxanthine produces a curvilinear plot. In the absence of ammonium sulfate, the dissociation of cGMP from the holoenzyme is rapid, having a t1/2 of less than 10 s, and addition of ammonium sulfate to the incubation greatly decreases this rate of dissociation. The native enzyme is resistant to degradation by chymotrypsin in the absence of cGMP; however, in its presence, chymotrypsin treatment produces several discrete fragments. Similarly, in the presence but not in the absence of cGMP, dicyclohexylcarbodiimide causes an irreversible activation of the enzyme without cross-linking the nucleotide to the phosphodiesterase. Both observations provide evidence that a different conformation in the enzyme results from cGMP binding. Only the conformation formed upon cGMP binding is easily attacked by chymotrypsin or permanently activated by treatment with dicyclohexylcarbodiimide. One major chymotryptic cleavage site exposed by cGMP binding is at tyrosine 553, implying that this region takes part in the conformational change. Limited proteolysis experiments indicate that these noncatalytic binding sites are located within a region of internal sequence homology previously proposed to include the cGMP-binding site(s) and that they retain a high affinity and specificity for cGMP independent of the catalytic domain of the enzyme. The products formed by partial proteolysis can be separated into individual catalytically active and cGMP-binding fractions by anion exchange chromatography. Gel filtration and electrophoresis analysis of the isolated fractions suggest that the cGMP-binding peak has a dimeric structure. Moreover, it can be further resolved by polyethyleneimine high performance liquid chromatography into two peaks (Peaks IIIA and IIIB). Peak IIIA binds 2 mol of cGMP/mol of dimer with an apparent Kd of 0.2 microM. Peak IIIB, however, has greatly reduced cGMP binding. Further digestion of these fragments with cyanogen bromide show that the differences between Peaks IIIA and IIIB are due to one or more additional proteolytic nicks in IIIB that remove a few residues near its C terminus, most probably residues 523-550 or 534-550. This in turn suggests that this region is essential for cGMP-binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1991

Molecular cloning of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase cDNA. Identification and distribution of isozyme variants.

We have cloned a 4.2-kilobase pair (kb) cDNA that encodes the cyclic GMP-stimulated phosphodiesterase (cGS PDE) from a bovine adrenal cortex library. The 921-residue polypeptide deduced from the cDNA nucleotide sequence is nearly identical with the complete amino acid sequence of the cGS PDE purified from a soluble bovine heart extract. Moreover, PPD-S49 cells transfected with the cGS PDE cDNA express a soluble cAMP hydrolytic activity that is enhanced by cGMP. Total RNA isolated from several bovine tissues were screened for cGS PDE transcript by Northern blot analysis. The cGS PDE cDNA appears to hybridize to a single 4.5-4.6-kb mRNA species. Although the cGS PDE mRNA is most abundant in the adrenal cortex, it is also concentrated in the adrenal medulla and heart and in anatomically distinct regions of the brain and kidney. A mRNA species encoding a putative variant cGS PDE isoform was detected by RNase protection. Total RNA isolated from adrenal cortex, adrenal medulla, liver, kidney, trachea, lung, spleen, and T-lymphocytes completely protected a 452-base riboprobe encoding 100 residues of the adrenal cortex cGS PDE amino terminus. In contrast, RNAs isolated from brain (cerebral cortex, hippocampus, and basal ganglia) protected only 268 bases of this riboprobe. The RNase protection pattern of this same probe using heart RNA showed major bands at both 268 and 452 bases, suggesting that two different cGS PDE mRNA species are expressed. These results indicate that the cGS PDE is widely expressed in a variety of tissues. Moreover, these studies suggest that at least one different cGS PDE isoform having a structurally distinct amino-terminal domain is expressed in brain and heart.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

Evidence for domain organization within the 61-kDa calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain.

The complete amino acid sequence of the 61-kDa calmodulin-dependent, cyclic nucleotide phosphodiesterase (CaM-PDE) from bovine brain has been determined. The native protein is a homodimer of N alpha-acetylated, 529-residue polypeptide chains, each of which has a calculated molecular weight of 60,755. The structural organization of this CaM-PDE has been investigated with use of limited proteolysis and synthetic peptide analogues. A site capable of interacting with CaM has been identified, and the position of the catalytic domain has been mapped. A fully active, CaM-independent fragment (Mr = 36,000), produced by limited tryptic cleavage in the absence of CaM, represents a functional catalytic domain. N-Terminal sequence and size indicate that this 36-kDa fragment is comprised of residues 136 to approximately 450 of the CaM-PDE. This catalytic domain encompasses a approximately 250 residue sequence that is conserved among PDE isozymes of diverse size, phylogeny, and function. CaM-PDE and its PDE homologues comprise a unique family of proteins, each having a catalytic domain that evolved from a common progenitor. A search of the sequence for potential CaM-binding sites revealed only one 15-residue segment with both a net positive charge and the ability to form an amphiphilic alpha-helix. Peptide analogues that include this amphiphilic segment were synthesized. Each was found to inhibit the CaM-dependent activation of the enzyme and to bind directly to CaM with high affinity in a calcium-dependent manner. This site is among the sequences cleaved from a 45-kDa chymotryptic fragment that has the complete catalytic domain but no longer binds CaM. These results indicate that residues located between position 23 and 41 of the native enzyme contribute significantly to the binding of CaM although the involvement of residues from additional sites is not excluded.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

Sequence comparison of the 63-, 61-, and 59-kDa calmodulin-dependent cyclic nucleotide phosphodiesterases.

Partial protein sequences from the 59-kDa bovine heart and the 63-kDa bovine brain calmodulin-dependent phosphodiesterases (CaM-PDEs) were determined and compared to the sequence of the 61-kDa isozyme reported by Charbonneau et al. [Charbonneau, H., Kumar, S., Novack, J. P., Blumenthal, D. K., Griffin, P. R., Shabanowitz, J., Hunt, D. F., Beavo, J. A. & Walsh, K. A. (1991) Biochemistry (preceding paper in this issue)]. Only a single segment (34 residues) at the N-terminus of the 59-kDa isozyme lacks identity with the 61-kDa isozyme; all other assigned sequence is identical in the two isozymes. Peptides from the 59-kDa isozyme that correspond to residues 23-41 of the 61-kDa protein bind calmodulin with high affinity. The C-terminal halves of these calmodulin-binding peptides are identical to the corresponding 59-kDa sequence; the N-terminal halves differ. The localization of sequence differences within this single segment suggests that the 61- and 59-kDa isozymes are generated from a single gene by tissue-specific alternative RNA splicing. In contrast, partial sequence from the 63-kDa bovine brain CaM-PDE isozyme displays only 67% identity with the 61-kDa isozyme. The differences are dispersed throughout the sequence, suggesting that the 63- and 61-kDa isozymes are encoded by separate but homologous genes.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

High concentrations of a cGMP-stimulated phosphodiesterase mediate ANP-induced decreases in cAMP and steroidogenesis in adrenal glomerulosa cells.

An adrenal cGMP-stimulated phosphodiesterase (cGS-PDE) has been shown to mediate atrial natriuretic peptide (ANP)-induced reductions in aldosterone secretion and cAMP levels in primary bovine glomerulosa cells. High concentrations of cGS-PDE have been localized to the zona glomerulosa cell layer of the adrenal cortex using biochemical and immunological techniques. Immunoblot analysis using an affinity-purified, isozyme-specific antiserum revealed a single band that comigrated with a purified cGS-PDE (105 kDa) (1) and that was most highly concentrated in the outermost 1-2 mm of the cortex, representing the capsule and zona glomerulosa regions. Greater than 90% of the overall phosphodiesterase activity present in tissue extracts prepared from these regions was immunoprecipitated using a solid-phase monoclonal antibody reagent, indicating the cGS-PDE as the predominant phosphodiesterase isozyme. Immunohistochemical staining experiments of frozen thin sections of intact adrenal tissue revealed that the cGS-PDE present in this region was localized in the glomerulosa cells themselves. The role of this isozyme as a mediator of ANP-induced decreases in intracellular cAMP concentrations and aldosterone production was tested in primary cultures of bovine adrenal glomerulosa cells. In cells stimulated by ACTH, ANP treatment produced dose-dependent reductions in aldosterone secretion and cellular cAMP content over the same concentration range. Increases in aldosterone production elicited by three cell-permeable cAMP derivatives (8-bromo-cAMP, 8-p-chlorophenylthio-cAMP, and N6-2'-O-dibutyryl-cAMP) were antagonized by ANP, indicating a site of action distal to adenylate cyclase for this hormone. Because the relative magnitude of the ANP effect differed depending upon the derivative used, the three derivatives were compared with respect to their relative rates of in vitro hydrolysis by adrenal cGS-PDE. A positive correlation between their rates of hydrolysis and the degree to which the steroidogenic response produced by these derivatives was antagonized by ANP was demonstrated, further suggesting an ANP-induced activation of the cGS-PDE as being responsible for this effect. The possible contribution of an additional pathway mediated by an inhibitory guanine nucleotide binding regulatory protein (Gi) acting on adenylate cyclase was tested by pretreatment of primary glomerulosa cells with pertussis toxin. Levels of pertussis toxin sufficient to inhibit subsequent in vitro ribosylation did not significantly alter the ANP effect on aldosterone production, although a partial reduction in the ANP effect on cAMP levels was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1990

Amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase from bovine heart.

The complete amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase (cGS-PDE) of bovine heart has been determined by analysis of five digests of the protein; placement of the C-terminal 330 residues has been confirmed by interpretation of the corresponding partial cDNA clone. The holoenzyme is a homodimer of two identical N alpha-acetylated polypeptide chains of 921 residues, each with a calculated molecular weight of 103,244. The C-terminal region, residues 613-871, of the cGS-PDE comprises a catalytic domain that is conserved in all phosphodiesterase sequences except those of PDE 1 from Saccharomyces cerevisiae and a secreted PDE from Dictyostelium. A second conserved region, residues 209-567, is homologous to corresponding regions of the alpha and alpha' subunits of the photoreceptor phosphodiesterases. This conserved domain specifically binds cGMP and is involved in the allosteric regulation of the cGS-PDE. This regulatory domain contains two tandem, internal repeats, suggesting that it evolved from an ancestral gene duplication. Common cyclic nucleotide binding properties and a distant structural relationship provide evidence that the catalytic and regulatory domains within the cGS- and photoreceptor PDEs are also related by an ancient internal gene duplication.

Amino Acid Sequence↗
PubMed1990

Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors.

Primary sequence information has been reported for more than 15 different mammalian cyclic nucleotide phosphodiesterases. Moreover, recent observations suggest that many of these isozymes are selectively expressed in a limited number of cell types. The fact that nearly all these different phosphodiesterases have unique primary sequences in their catalytic or regulatory domains and that they are often selectively expressed implies that it may be possible to modulate individual isozymes using specific drugs. Joe Beavo and David Reifsnyder summarize much of the evidence that has led to our current understanding of multiple isozymes of phosphodiesterase, with emphasis on aspects that may be relevant to drug design. They also discuss why many previous attempts to isolate isozyme-selective inhibitors may have failed.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1990

Identification of a noncatalytic cGMP-binding domain conserved in both the cGMP-stimulated and photoreceptor cyclic nucleotide phosphodiesterases.

Partial amino acid sequence has been determined for the cone, alpha' subunit of the bovine photoreceptor cyclic nucleotide phosphodiesterase (PDE) and deduced from nucleotide sequences of a partial cDNA clone. These sequences identify the alpha' subunit as the product of a gene that is distinct from those encoding the alpha or beta subunits of the membrane-associated rod photoreceptor PDE. Comparisons between the recently determined cGMP-stimulated-PDE sequence and those of the alpha and alpha' photoreceptor PDE subunits reveal an unexpected sequence similarity. In addition to the catalytic domain conserved in eukaryotic PDEs, all three PDEs possess a second conserved segment of approximately 340 residues that contains two internally homologous repeats. Limited proteolysis and direct photolabeling studies indicate that the noncatalytic, cGMP-binding site(s) in the cGMP-stimulated PDE is located within this conserved domain, suggesting that it also may serve this function in the photoreceptor PDEs. Moreover, other PDEs that do not bind cGMP at noncatalytic sites do not contain this conserved domain. The function of the conserved segment in the photoreceptor PDEs is not known, but the homology to allosteric sites of the cGMP-stimulated PDE suggests a role in cGMP binding and modulation of enzyme activity.

Amino Acid Sequence↗
PubMed1989

Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase.

cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with [32P]cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments. The labeled site not blocked by cAMP is also contained in a Mr = 28,000 cyanogen bromide fragment of the enzyme that does not overlap with the Mr = 36,000 proteolytic fragment. Limited chymotryptic proteolysis also increases basal activity and eliminates cGMP stimulation of cAMP hydrolysis. The chymotryptic fragments can be separated by either ion exchange high performance liquid chromatography (HPLC) or solid-phase monoclonal antibody treatment. A solid-phase monoclonal antibody against the cGMP-stimulated PDE removes the Mr = 60,000 and 57,000 labeled fragments and any intact, unproteolyzed protein but does not remove the Mr = 36,000 fragment or the majority of activity. Ion exchange HPLC separates the fragments into three peaks (I, II, and III). Peaks I and II contain activity of approximately 40 and 100 units/mg, respectively. Peak II is the undigested or slightly nicked native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

A soluble form of bovine rod photoreceptor phosphodiesterase has a novel 15-kDa subunit.

A substantial fraction (20-30%) of the bovine rod outer segment phosphodiesterase (PDE) activity is not associated with outer segment membranes prepared with buffers of moderate ionic strength; this PDE activity appears to represent a distinct, soluble isozyme. Although this PDE isozyme can be demonstrated to be present in sealed rod outer segments, it is discarded from most standard rod outer segment preparations. A method was developed that allowed the rapid purification of the soluble rod PDE by 2600-fold, to apparent homogeneity, using a monoclonal antibody column (ROS-1a). The soluble rod PDE isozyme has a novel Mr = 15,000 subunit (delta) in addition to subunits of Mr = 88,000 (alpha sol), 84,000 (beta sol), and 11,000 (gamma sol). The delta subunit comigrates with and may be identical to the cone PDE 15-kDa subunit. The small subunits of the soluble rod PDE and the membrane-associated rod PDE were isolated by reverse-phase chromatography. The gamma sol subunit was a potent inhibitor of trypsin-activated rod PDE, inhibiting 50% of 1 pM PDE activity at a concentration of 11 pM. This concentration was similar to that observed for the gamma subunit of the membrane-associated rod PDE. The purified delta subunit did not appear to affect PDE activity; this subunit was, however, unusually difficult to keep in solution. All of the kinetic and physical properties of the soluble rod PDE tested thus far are similar to those of the membrane-associated form, except for the presence of the delta subunit, suggesting that this unique subunit could mediate the solubility of the soluble rod PDE and the cone PDE in the intact photoreceptor.

Animals↗
PubMed1989

cGMP is tightly bound to bovine retinal rod phosphodiesterase.

Although the total concentration of cGMP in rod outer segments is thought to be substantially greater than the free concentration, no quantitatively relevant site for the bound cGMP has been described in mammalian photoreceptors. We have found that preparations of purified bovine rod photoreceptor cyclic nucleotide phosphodiesterase (PDE) contain 1.8 +/- 0.3 mol of tightly bound cGMP per mol of PDE. When subunits of the purified PDE were separated by reverse-phase HPLC in 0.1% trifluoroacetic acid and acetonitrile, a peak of material having spectral properties characteristic of a guanine ring was seen. This material was identified as cGMP by comigration with authentic cGMP on HPLC, conversion to 5-GMP by trypsin-activated rod PDE, and conversion to guanosine by a combination of trypsin-activated PDE and 5'-nucleotidase-containing snake venom. When incubated with 1 microM [3H]cGMP, only 0.1 mol of [3H]cGMP bound per mol of purified PDE, presumably because nearly all binding sites were occupied by tightly bound endogenous cGMP carried through the purification. Scatchard plots of [3H]cGMP binding have indicated that two classes of binding sites are present on the rod PDE. The off-rate of cGMP from the slowly dissociating site is extremely slow; it has a t1/2 of approximately 4 hr at 37 degrees C. At lower temperatures, very little cGMP dissociates; the amount of [3H]cGMP bound to rod PDE after 2 hr at 4 degrees C was essentially the same as at the beginning of the incubation. The observation that stoichiometric amounts of cGMP are tightly bound to PDE accounts for the inability to purify the bovine rod PDE on cGMP affinity columns or to demonstrate stoichiometric high-affinity binding sites with [3H]cGMP. More significantly, the tightly bound cGMP may resolve the apparent discrepancy between the free and total cGMP concentrations of photoreceptor outer segments.

Animals↗
PubMed1989

Inhibition and stimulation of photoreceptor phosphodiesterases by dipyridamole and M&B 22,948.

Few high affinity inhibitors of the photoreceptor phosphodiesterases have been identified. We show here that dipyridamole and M&B 22,948 (Zaprinast), potent inhibitors of the cGMP-binding, cGMP-specific phosphodiesterase (PDE), also inhibit trypsin- or transducin-activated bovine rod and cone photoreceptor phosphodiesterases at submicromolar concentrations. Dixon plots demonstrated that the inhibition of trypsin-activated rod PDE was competitive, with Ki values of 140 nM for M&B 22,948 and 380 nM for dipyridamole. Both of these drugs were much more potent than other PDE inhibitors, including isobutylmethylxanthine (IBMX). These results reinforce the suggestion that the photoreceptor and the cGMP-binding, cGMP-specific PDE are closely related. In addition, the high affinity and selectivity of these agents should make them useful for probing the regulation and function of PDE in the photoreceptor. At low substrate concentrations, the effects of these drugs on basal unactivated PDE activity were similar to those seen with trypsin- or transducin-activated PDE. At millimolar substrate concentrations, however, the effects of the drugs were biphasic; PDE activity was stimulated at drug concentrations from 1 to 10 microM and inhibited at higher concentrations. Stimulation was not observed with IBMX. This stimulation of activity apparently was not an allosteric effect caused by direct binding of the dipyridamole and M&B 22,948 to the high affinity noncatalytic cGMP binding sites on the PDEs; whereas no cooperativity of cGMP binding to this site has been demonstrated, the drugs actually stimulated the binding of low concentrations of cGMP to this site. In addition, whereas preincubation with cGMP and cGMP analogs blocked the stimulation exerted by the drugs, they did so only at much higher concentrations than those necessary for saturation of the high affinity noncatalytic cGMP site. Because the stimulation can only be seen at higher substrate levels than are thought to exist in the photoreceptor, only the inhibitory effects of the drugs are likely to be pharmacologically relevant. However, the stimulation exerted by these drugs may point to a hitherto unknown allosteric interaction between the catalytic and regulatory sites on the PDE or to a previously unrecognized regulatory site.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1988

Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulin-dependent enzymes.

Nanomolar concentrations of synthetic peptides corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase were found to inhibit calmodulin activation of seven well-characterized calmodulin-dependent enzymes: brain 61 kDa cyclic nucleotide phosphodiesterase, brain adenylate cyclase, Bordetella pertussis adenylate cyclase, red blood cell membrane Ca++-pump ATPase, brain calmodulin-dependent protein phosphatase (calcineurin), skeletal muscle phosphorylase b kinase, and brain multifunctional Ca++ (calmodulin)-dependent protein kinase. Inhibition could be entirely overcome by the addition of excess calmodulin. Thus, the myosin light chain kinase peptides used in this study may be useful antagonists for studying calmodulin-dependent enzymes and processes.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Phosphorylation results in activation of a cAMP phosphodiesterase in human platelets.

Agents such as prostaglandins E1 and I2 which elevate cAMP levels in platelets also increase cAMP phosphodiesterase activity. Since much of the cAMP phosphodiesterase activity in human platelets is due to the cGMP-inhibited isozyme (Macphee, C. H., Harrison, S. A., and Beavo, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6600-6663), we examined the regulation of this isozyme by prostaglandins E1 and I2 in intact platelets. Because this isozyme is a minor component of platelet protein, normally requiring several thousand-fold purification to achieve homogeneity, a specific monoclonal antibody (CGI-5) was utilized to identify and isolate the cGMP-inhibited phosphodiesterase activity. Treatment of intact platelets with the prostaglandins promoted an increase in the phosphorylation state of the cGMP-inhibited phosphodiesterase and a corresponding increase in phosphodiesterase activity. The effect on activity and phosphorylation of the cGMP-inhibited phosphodiesterase was observed within 2 min after intact platelets were exposed to the prostaglandins. The half-maximal effective dose for prostaglandin I2 (10 nM) was approximately 10-fold lower than that for prostaglandin E1. The phosphorylated, cGMP-inhibited isozyme migrated as a 110-kDa peptide following sodium dodecyl sulfate gel electrophoresis. Direct in vitro phosphorylation of the platelet cGMP-inhibited phosphodiesterase by the catalytic subunit of cAMP-dependent protein kinase caused a similar increase in phosphodiesterase activity. Treatment with PKI peptide, a specific inhibitor of cAMP-dependent protein kinase, blocked the phosphorylation and the effect on activity. Taken together, the data strongly suggest that the effects of prostaglandins E1 and I2 on platelet phosphodiesterase activity are mediated by a direct cAMP-dependent protein kinase-catalyzed phosphorylation of the cGMP-inhibited phosphodiesterase isozyme.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Characterization of a bovine cone photoreceptor phosphodiesterase purified by cyclic GMP-sepharose chromatography.

The biochemical bases for the differences in cone and rod photoreceptor physiology have not been thoroughly examined because of the difficulty in obtaining cone photoreceptor components. We report here the purification and preliminary characterization of a bovine cyclic GMP phosphodiesterase (PDE) which is enriched in cone photoreceptors. The cone PDE was purified at least 15,000-fold to apparent homogeneity from bovine retinas by DEAE-cellulose and cGMP-Sepharose affinity chromatography. The trypsin-activated cone PDE hydrolyzed cGMP with efficiency similar to that of the rod PDE. However, a number of characteristics distinguished the cone PDE from the rod isozyme including the subunit structure. As previously reported, the apparent molecular weight of the cone PDE large subunit (alpha') was slightly larger than either of the large subunits of the rod PDE (93,500 versus 88,000 and 84,000). Three other smaller polypeptides were associated with the alpha' subunit (Mr = 11,000, 13,000, and 15,000), one of which (11,000) may be identical to the rod PDE gamma subunit. Cone phosphodiesterase binds at least 10-fold more cyclic GMP/mol of PDE than the rod photoreceptor isozyme. Cyclic GMP binds to this noncatalytic site with high affinity (Kd = 11 nM) and dissociates very slowly (t1/2 = 10-20 min at 37 degrees C). Purified rod transducin activated the cone PDE in solution to at least 90% of the trypsin-activated level. The concentration of rod transducin required for half-maximal activation of cone PDE (15 nM) was 50-fold lower than that necessary for half-maximal activation of rod PDE. Thus several properties of the cone phosphodiesterase clearly distinguish it from the rod isozyme and could account for some differences in cone and rod physiology.

3',5'-Cyclic-GMP Phosphodiesterases↗