Purification and Chemical Characterization of Peptide G1, an Invertebrate Neuropeptide That Stimulates Cyclic GMP Metabolism

Bioassay analysis of extracts of the major neurosecretory structures of the American lobster have revealed several different agents with stimulatory effects on the cyclic GMP metabolism of various lobster tissues. The most potent of these is a peptide extracted from the sinus gland, a neurohemal organ found in the animal's eyestalk. This molecule, called peptide G1 (for its effects on cyclic GMP metabolism), can increase the cyclic GMP content of every lobster tissue tested, sometimes by as much as 200-fold. In this article, we describe the purification and some of the chemical properties of peptide G1. Purification was accomplished by sequential anion exchange and reverse-phase HPLC. The purified peptide is a large, extremely hydrophobic molecule. Its apparent molecular mass on a reducing sodium dodecyl sulfate-containing gel is 6.4 kDa, and its calculated molecular mass (based on an amino acid analysis of the purified material) is 8.2 kDa. Amino acid analysis reveals a high proportion of leucine and valine residues. The amino terminus of the molecule is not susceptible to Edman degradation, but sequencing studies were successfully carried out on tryptic fragments. Based on the estimated size of the molecule, these studies provide approximately 60% of the total sequence. No homologies with any previously sequenced peptide were observed, but biochemical similarities to as yet unsequenced peptides found in extracts of sinus glands from other crustaceans (hyperglycemic hormone and moult-inhibiting hormone) are described.     

Isolation from porcine intestinal extracts of a cholecystokinin-like peptide and a peptide with homology to cytochrome oxidase polypeptide VII and chymodenin

A number of different forms of cholecystokinin (CCK) exist in the brain and intestine. Gel permeation and ion exchange chromatography and high performance liquid chromatography have been used to isolate a peptide from partially purified porcine intestinal extracts with N-terminal homology to porcine brain CCK-58. This peptide contracted both the guinea pig ileum longitudinal muscle and gallbladder muscle and these responses were inhibited by dibutyryl cyclic GMP or proglumide. The potency was approximately 1/100 of that of CCK-8. The reason for this low potency is unclear, but it is possible that a critical part of the biologically active region is modified or that it is a truncated form of CCK-58. A further peptide was isolated with a sequence homologous to cytochrome oxidase polypeptide VII and chymodenin.     

Activation of membrane guanylate cyclase by an invertebrate peptide hormone.

Peptide hormones can stimulate cyclic GMP synthesis through either of two general mechanisms: some peptides activate the cytoplasmic form of guanylate cyclase via a coupling factor called EDRF (endothelium-derived relaxation factor), while others activate the membrane form by interacting directly with an extracellular binding domain of the cyclase molecule itself. We have investigated the mechanism(s) by which crustacean hyperglycemic hormone (CHH), a neuropeptide that regulates energy metabolism in crustaceans, elevates cyclic GMP levels in lobster muscle. Phosphodiesterase inhibitors potentiate the response in intact tissue. This indicates that the primary effect of the peptide is to activate a cyclase rather than inhibit a phosphodiesterase. Methylene blue, a specific inhibitor of the EDRF pathway, does not block the actions of CHH. In addition, nitroprusside, an agent that directly activates the EDRF pathway in vertebrate animals, does not activate guanylate cyclase either in intact or homogenized lobster muscle. This indicates that the EDRF pathway, although prominent in vertebrate muscle, is not found in crustaceans and further suggests that the membrane cyclase is the most likely target of CHH. Membrane and soluble cyclases can be isolated from homogenates of lobster muscle (in a 3.5:1 ratio), and both are stimulated by Mn2+ and inhibited by Ca2+. CHH has no effect on the soluble enzyme. Coupling of CHH receptors to the particulate cyclase, however, remains intact in isolated membranes, thus providing a new model system for the study of receptor/cyclase interactions.     

Effect of andrenalectomy on cyclic 3',5'-guanosine monophosphate metabolism of rat liver and other tissues

Adrenalectomy increased guanyl cyclase and cyclic GMP phosphodiesterase activities in liver and other rat tissues. Liver guanyl cyclase activities from adrenalectomized rats were increased above those of normal controls according to kinetic analysis, gel filtration, ion-exchange chromatography, discontinuous sucrose gradient fractionation, sulfhydryl inhibition, and secretin activation. The effects of adrenal insufficiency on hepatic guanyl cyclase and cyclic GMP phosphodiesterase were prevented by cortisone acetate administration. Immunoassay of liver and skeletal muscle cyclic GMP after adrenalectomy showed markedly decreased levels in liver, but increased levels in skeletal muscle. In liver and other tissues, basal adenyl cyclase and cyclic AMP phosphodiesterase activities were unaffected by adrenalectomy. Hepatic levels of cyclic AMP were also unchanged by adrenalectomy. Hypophysectomy raised guanyl cyclase activity in liver but had no effect on liver cyclic GMP phosphodiesterase activity. These alterations are discussed in relation to possible glucocorticoid regulation of cyclic GMP metabolism.     

Speract. Purification and characterization of a peptide associated with eggs that activates spermatozoa

A low molecular weight peptide (speract) associated with sea urchin eggs has been purified to apparent homogeneity by charcoal adsorption, DEAE-Sephacel chromatography, Bio-Gel P-2 filtration, and Dowex AG 50W-X4 chromatography. Gametes from 5000 female sea urchins were required for the isolation of approximately 9 mg of the peptide. The isolated peptide is homogenous based on [3H]acetic anhydride labeling, gel filtration, and reverse phase high pressure liquid chromatography. Speract is composed entirely of neutral and acidic amino acids with glycine as the major component, and it appears to have a blocked NH2 terminus based on its insensitivity to leucine aminopeptidase, its failure to react with dansyl chloride, and its chromatographic behavior on strong cation exchange resins. Speract is a potent stimulator of sea urchin sperm oxygen consumption, causing significant increases of sperm respiration rates at concentrations as low as 10(-12) M and producing 20-fold increases of oxygen consumption at maximal concentrations of 10(-8) M. Sperm cyclic GMP and cyclic AMP concentrations are also increased by speract, but concentrations of at least 10(-10) M and 10(-9) M are required for half-maximal elevations, respectively. The peptide, purified from Strongylocentrotus purpuratus eggs, also cross-reacts with spermatozoa from Lytechnis pictus sea urchins, suggesting that speract does not show species specificity. These results represent the first report of the purification of a peptide associated with eggs that may affect spermatozoa under natural conditions.     

Truncated atrial natriuretic peptide analogs. Comparison between receptor binding and stimulation of cyclic GMP accumulation in cultured vascular smooth muscle cells

A series of truncated atrial natriuretic peptide analogs were examined as a means of defining the structural requirements for receptor occupancy and stimulation of cyclic GMP accumulation in bovine aortic smooth muscle cells. It was determined that deletion of amino acids from the carboxyl and/or amino termini of the peptides diminished their ability to increase cyclic GMP levels. Deletion of amino acids from the carboxyl terminus had the greatest effect, and atrial natriuretic peptide analogs lacking the carboxyl-terminal phenylalanyl-arginyl-tyrosine tripeptide were 100-1000-fold less active than parent compounds in stimulating intracellular cyclic GMP accumulation. In marked contrast to the cyclic GMP effects, deletion of amino- and/or carboxyl-terminal amino acids had only minor effects on the affinity of the peptides for specific smooth muscle cell-associated receptors. Peptide analogs lacking the phenylalanyl-arginyl-tyrosine tripeptide bound to receptors with an affinity only 1.1-5-fold weaker than the parent compounds. Thus, there was no correlation between apparent receptor binding affinity of atrial natriuretic peptide analogs and potency of these same peptides for stimulating intracellular cyclic GMP accumulation. Furthermore, analogs that bound to receptors and failed to elicit significant cyclic GMP responses did not antagonize or modulate increases in cyclic GMP induced by parent compounds. These data are most consistent with the existence of multiple subpopulations of atrial natriuretic peptide receptors on aortic smooth muscle cells.     

Cyclic nucleotide phosphodiesterases from rat anterior pituitary. Characterization of multiple forms and regulation by protein activator and Ca+.

Phosphodiesterase activities for adenosine and guanosine 3':5'-monophosphates (cyclic AMP and cyclic GMP) were demonstrated in particulate and soluble fractions of rat anterior pituitary gland. Both fractions contained higher activity for cyclic GMP hydrolysis than that for cyclic AMP hydrolysis when these activities were assayed at subsaturating substrate concentrations. Addition of protein activator and CaCl2 to either whole homogenate, particulate or supernatant fraction stimulated both cyclic AMP and cyclic GMP phosphadiesterase activities. Almost 80% of cyclic AMP and 90% of cyclic GMP hydrolyzing activities were localized in soluble fraction. Particulate-bound cyclic nucleotide phosphodiesterase activity was completely solubilized with 1% Triton X-100. Detergent-dispersed particulate and soluble enzymes were compared with respect to Ca2+ and activator requirements and gel filtration profiles. Particulate, soluble and partially purified phosphodiesterase activities were also characterized in relation to divalent cation requirements, kinetic behavior and effects of Ca2+, activator and ethyleneglycol-bis-(2-aminoethyl)-N,N'-tetraacetic acid. Gel filtration of either sonicated whole homogenate or the 10500 X g supernatant fraction showed a single peak of activity, which hydrolyzed both cyclic AMP and cyclic GMP and was dependent upon Ca2+ and activator for maximum activity. Partially purified enzyme was inhibited by 1-methyl-3-isobutylxanthine and papaverine with the concentration of inhibitor giving 50% inhibition at 0.4 muM substrate being 20 muM and 24 muM for cyclic AMP and 7 muM and 10 muM for cyclic GMP, respectively. Theophylline, caffeine and theobromine were less effective. The rat anterior pituitary also contained a protein activator which stimulated both pituitary cyclic nucleotide phosphodiesterase(s) as well as activator-deficient brain cyclic GMP and cyclic AMP phosphodiesterases. Chromatography of the sonicated pituitary extract on DEAE-cellulose column chromatography resolved the phosphodiesterase into two fractions. Both enzyme fractions hydrolyzed cyclic AMP and cyclic GMP and had comparable apparent Km values for the two nucleotides. Hydrolysis of cyclic GMP and cyclic AMP by fraction II enzyme was stimulated 6--7-fold by both pituitary and brain activator in the presence of micromolar concentrations of Ca2+.     

Cyclic nucleotide phosphodiesterases in the cricket, Acheta domesticus.

Exceptionally high levels of guanosine 3'-5'-cyclic monophosphate (cyclic GMP) in the accessory reproductive gland of the male house cricket, Acheta domesticus, led to an investigation of cyclic nucleotide phosphodiesterase (EC 3.1.4.--) as a possible regulatory enzyme. Cricket cyclic nucleotide phosphodiesterase activity with cyclic GMP or cyclic AMP as substrate had a pH optimum around 9.0, required Mg2+ or Mn2+ for maximal activity, and was inhibited by EDTA and methylxanthines. Cyclic GMP phosphodiesterase occurred mainly in the soluble fraction of homogenates of accessory glands or whole crickets, but cyclic AMP phosphodiesterase in the accessory gland was primarily particulate. Kinetic analysis indicated three forms of cyclic GMP phosphodiesterase, with Km values at 2.9 muM, 71 muM and 1.5 mM. Chromatography of whole cricket or accessory gland extracts on DEAE cellulose gave an initial peak having comparable activity with either cyclic GMP or cyclic AMP, and a second peak specific for cyclic AMP. There were no appreciable changes in the specific activity or kinetic properties of accessory gland cyclic GMP phosphodiesterase during a developmental period over which cyclic GMP levels rise more than 500-fold. Thus, the accumulation of cyclic GMP in the accessory gland is probably not associated with concomitant developmental modulation of phosphodiesterase activity.     

Isolation and characterization of a fibrogenic factor from CCl4-damaged rat liver

A fibrogenic factor which stimulates collagen production without cell proliferation of rat skin fibroblast cultures was isolated from CCl₄-damaged rat liver. (1) The factor was isolated from saline extracts of CCl₄-induced fibrotic rat liver and fractionated by Sephadex G-50S gel filtration and DEAE-cellulose chromatography. The original extract produced a 6-fold increase in collagen synthesis and the active factor eluted from gel filtration columns in a region corresponding to 5000 daltons. (2) The active factor was destroyed by heat (57°C, 30 min), phospholipase C digestion, but was insensitive to proteolytic enzymes or phospholipase A. Chemical analysis of the partially purified factor revealed relatively high quantities of phosphorus (3%) and low quantities of protein (13.3%), neutral sugar (1.9%) and uronic acid (4.9%). The possibility of this component being a complex phospholipid containing polypeptide is suggested. (3) Fibrogenic properties of the isolated factor was enhanced by apparent oxidation in air, to a more active, yet insoluble complex. Attempts to solubilize the oxidized product completely destroyed its biological activity.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

A low molecular weight factor from sea urchin eggs elevates sperm cyclic nucleotide concentrations and respiration rates.

A factor associated with sea urchin eggs that increases sperm cyclic nucleotide concentrations and respiration rates was identified as having a low molecular weight. The factor was more potent at elevating cyclic GMP concentrations than cyclic AMP concentrations, and represents the first demonstration of a factor associated with eggs that is capable of causing elevations of sperm cyclic GMP. Concentration-response curves of the crude mixture of egg factors to increase sperm cyclic AMP and cyclic GMP concentrations and respiratory rates were very similar, and comparable losses of these three activities were observed after extensive dialysis and heat treatment of the crude egg factors. The factor was partly purified by ethanol precipitation of a large molecular weight egg jelly component, and by charcoal adsorption and LH-20 chromatography of the resultant ethanol-soluble material. The factor was not extracted into a variety of organic solvents and had an apparent molecular weight of between 1000 and 2000, as estimated by gel filtration.     

Cyclic nucleotide phosphodiesterase activities in Neurospora crassa.

Cyclic nucleotide phosphodiesterase activities in soluble Neurospora crassa mycelial extracts were resolved into two peaks, phosphodiesterase I and II, by chromatography on DEAE-cellulose columns. Phosphodiesterase I hydrolysed cyclic AMP and cyclic GMP equally well. Phosphodiesterase II was active on cyclic GMP but scarcely active on cyclic AMP. Phosphodiesterase I was resolved by gel filtration and sucrose-density-gradient centrifugation into three peaks having molecular weights of about 57 000, 125 000 and 225 000. This suggests that this enzyme activity has at least three aggregation forms, tentatively defined as monomeric, dimeric and tetrameric. Similarly, phosphodiesterase II was resolved into two forms, having molecular weights of about 170 000 and 320 000. Evidence on the interconversion between phosphodiesterase I forms was obtained.     

Purification and succinylation of cyclic GMP from large volume samples and radioimmunoassay of succinyl cyclic GMP.

Improved procedures for isolation of cyclic GMP and cyclic AMP and radioimmunoassay of cyclic GMP with succinylation are described. Procedures involved include modified chromatography on alumina and succinylation of cyclic GMP followed by purification of succinyl cyclic GMP on a Dowex AG 1×8 column. These procedures are convenient and applicable to any volume up to 50 ml of tissue extracts and especially for isotonic incubation mixtures. This assay system is sensitive to 6 femtomoles of cyclic GMP/tube. On radioimmunoassay, free and antibody bound [¹²⁵I]-labeled cyclic GMP are separated by Millipore filtration. Cyclic GMP levels in several tissue samples were determined in order to show the applicability of the procedures.     

The purification of luteinizing-hormone-releasing factor with some observations on its properties

Bullock median-eminence tissue was used as a source of luteinizing-hormone-releasing factor for small-scale experiments to explore methods for its isolation. The presence of luteinizing-hormone-releasing factor was detected by the ovulation response in rabbits after intrapituitary infusion of the extract. Gel filtration was found to be suitable for the purification of these extracts. The releasing factor appeared to be a basic peptide of molecular weight in the range 1200-2500. On a larger scale, an extract of hypothalamic tissue from sheep was used to establish a multi-stage isolation procedure that resulted in a 200000-fold purification of luteinizing-hormone-releasing factor. After the initial extraction the isolation process consisted of: (1) two cycles of gel filtration; (2) anion-exchange chromatography; (3) gel filtration in a partially organic medium; (4) thin-layer chromatography on cellulose. Stage (3) separated two zones of activity each containing peptides. One of these was purified further by stage (4) to give a preparation that was active at a dose of 6mug. of peptide/animal, although activity diminished seriously during storage. This preparation contained only five or six components, but the small amounts of peptides obtained at this stage of purity were insufficient for full characterization.     

Lobster muscle and synapse respond to cortisol, a vertebrate hormone

Cortisol (0.28 mumol X L-1) applied to lobster (Homarus americanus) neuromuscular preparations produces a hyperpolarization in muscle fibers and an increase in amplitude of excitatory postsynaptic potentials. The effect appears to be surface-mediated, because of its rapid onset (within seconds). It is also Na+-K+ ATPase dependent, because ouabain blocks the effects. The effects are relatively short-lasting, and gradually subside within 15 min. The increase in excitatory postsynaptic potentials is attributed in part to increased quantal output of transmitter, and not to changes in muscle fiber membrane resistance. The effects of cortisol on neuromuscular transmission and membrane potential indicate that cortisol may have a physiological role in crustaceans.     

Partial purification and properties of guanosine 3':5'-monophosphate-dependent protein kinase from pig lung.

Guanosine 3':5'-monophosphate(cyclic GMP)-dependent protein kinase which catalyzes the phosphorylation of histone was purified about 200-fold from the soluble fraction of pig lung by pH 5.5 precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. The apparent Ka values for guanosine 3':5'-monophosphate and adenosine 3':5'-monophosphate were determined to be about 17 and 360 nM, respectively. Mg2+ was essential for the activity exhibiting biphasic stimulation behavior and neither Mn2+ nor Ca2+ could substitute for Mg2+. However, these divalent ions markedly inhibited the protein kinase activity stimulated by cyclic GMP in the presence of Mg2+.     

Renal and systemic effects of synthetic atrial natriuretic factor

A synthetic peptide corresponding to a sequence of 26 amino acids contained in endogenous rat atrial natriuretic factor (ANF), was infused into one renal artery of anesthetized dogs for a comprehensive in vivo evaluation of the renal and systemic effects of pure ANF. The results proved conclusively that ANF acted directly on the kidney since urine volume and fractional excretion of sodium, potassium, chloride and calcium were elevated in a dose-related manner in the ANF-treated kidney, but were not significantly affected in the contralateral saline-infused organ. The maximum effects achieved with the synthetic ANF were higher than any reported following intravenous administration of crude extracts of rat atria and were similar to those produced by thiazide diuretics. In four of the five dogs studied, renal vascular resistance fell progressively as doses of ANF were increased. Glomerular filtration rate was not significantly elevated during ANF infusion, but was correlated with sodium excretion rates. Even though mean arterial pressure was progressively reduced, there was no significant change in heart rate and no stimulation of renin secretion. Arterial cyclic GMP concentration was higher in the basal state and rose more rapidly than did renal venous levels, indicating that increases in circulating concentrations of arterial cyclic GMP originated from an extrarenal source. Dose-related elevations in urinary cyclic GMP excretion could be explained by increased cyclic GMP filtration, by enhanced production in tubular cells, or by renal tubular secretion. Especially in the saline-infused kidney, there was a clear dissociation between excretion of cyclic GMP and fractional sodium excretion. We conclude that the synthetic ANF increased electrolyte excretion via a direct renal action which was not solely dependent upon changes in renal vasculature, renin secretion or cyclic GMP levels.     

Amino acid sequence of a peptide with both molt-inhibiting and hyperglycemic activities in the lobster, Homarus americanus

A hydrophobic peptide of 71 residues was isolated from lobster sinus gland extracts that prolonged intermolt periods and lowered ecdysteroid titers in juvenile lobsters. Removal of the N-terminal pyroglutamyl residue allowed sequencing of 30 of the first 36 residues. Additional data were obtained from HPLC-purified fragments from endoproteinase cleavages (Lys-C, Glu-C, Arg-C, Asp-N), and carboxypeptidase Y digestion. This is the first reported amino acid sequence of a crustacean molt-inhibiting hormone. This peptide also has significant hyperglycemic activity.     

Identification of Nitric Oxide-Sensitive and -Insensitive Forms of Cytoplasmic Guanylate Cyclase

Abstract: Cytoplasmic, nitric oxide-activated guanylate cyclases are expressed in many regions of the mammalian brain and are thought to participate in functions as diverse as synaptogenesis and long-term potentiation. In this study, we have characterized cytoplasmic guanylate cyclases in the nervous system of an invertebrate, the American lobster. Cytoplasmic cyclase specific activity is higher in lobster nerve cord than in any other lobster tissue tested, and considerably higher than in typical rat tissues (cerebellum, lung, and liver). However, nitric oxide donors have minimal effects on lobster nerve cord cyclic GMP production, when applied either to intact tissue or to cytoplasmic extracts. Parallel immunocytochemical studies, using an anti-cyclic GMP antibody, reveal that only a small subset of lobster neurons responds to nitric oxide with a significant elevation of cyclic GMP levels. HPLC analysis of nerve cord cytoplasm reveals two chromatographically separable cyclases, a minor nitric oxide-sensitive form whose retention time is identical to that of the conventional mammalian enzyme and a more abundant nitric oxide-insensitive form that appears to be novel. The physiological function and phylogenetic distribution of this nitric oxide-insensitive enzyme, and the signaling mechanisms that regulate its activity, are not known.     

Purification, characterization and production of rabbit antibodies to rat liver particulate, high-affinity, cyclic AMP phosphodiesterase

The cyclic nucleotide phosphodiesterase (EC 3.4.16) activities of a rat liver particulate fraction were analyzed after solubilization by detergent or by freeze-thawing. Analysis of the two extracts by DEAE-cellulose chromatography revealed that they contain different complements of phosphodiesterase activities. The detergent-solubilized extract contained a cyclic GMP phosphodiesterase, a low affinity cyclic nucleotide phosphodiesterase whose hydrolysis of cyclic AMP was activated by cyclic GMP and a high affinity cyclic AMP phosphodiesterase. The freeze-thaw extract contained a cyclic GMP phosphodiesterase and two high affinity cyclic AMP phosphodiesterase, but no low affinity cyclic nucleotide phosphodiesterase. The cyclic AMP phosphodiesterase activities from the freeze-thaw extract and from the detergent extract all had negatively cooperative kinetics. One of the cyclic AMP phosphodiesterases from the freeze-thaw extract (form A) was insensitive to inhibition by cyclic GMP; the other freeze-thaw solubilized cyclic AMP phosphodiesterase (form B) and the detergent-solubilized cyclic AMP phosphodiesterase were strongly inhibited by cyclic GMP. The B enzyme appeared to be converted into the A enzyme when the particulate fraction was stored for prolonged periods at -20 degrees C. The B form was purified extensively, using DEAE-cellulose, a guanine-Sepharose column and gel filtration. The enzyme retained its negatively cooperative kinetics and high affinity for both cyclic AMP and cyclic GMP throughout the purification, although catalytic activity was always much greater for cyclic AMP. Rabbit antiserum was raised against the purified B enzyme and tested via a precipitin reaction against other forms of phosphodiesterase. The antiserum cross-reacted with the A enzyme and the detergent-solubilized cyclic AMP phosphodiesterase from rat liver. It did not react with the calmodulin-activated cyclic GMP phosphodiesterase of rat brain, the soluble low affinity cyclic nucleotide phosphodiesterase of rat liver or a commercial phosphodiesterase preparation from bovine heart. These results suggest a possible interrelationship between the high affinity cyclic nucleotide phosphodiesterase of rat liver.