Association of cyclic nucleotide phosphodiesterase of buffalo spermatozoa with sperm chromatin

Buffalo sperm heads contain more than 50% of the total cyclic AMP-phosphodiesterase activity (EC 3.1.4.17) present in spermatozoa. Its distribution in sperm heads revealed no activity in acrosome and other membrane structures present in the head. All the cyclic AMP-phosphodiesterase activity was found firmly bound to sperm chromatin which could not be solubilized. In addition to cyclic AMP, cyclic GMP was also hydrolysed by chromatin preparation. The rate of hydrolysis was 2.5-times more rapid with cyclic AMP than with cyclic GMP at their optimum pH of 7.5 and 8.0, respectively. The pH and heat stability profiles, inhibition studies and the effect of divalent metal ions indicated that the two activities are not associated with the same protein. Mixed substrate analysis showed two sites at which the hydrolysis of cyclic AMP and cyclic GMP is catalysed. Chromatin cyclic nucleotide phosphodiesterases exhibited kinetics typical of one enzyme species both for cyclic AMP (K m = 100 microM; V = 1.0 nmol/min per mg protein) and cyclic GMP (Km = 23 microM; V = 0.4 nmol/min per mg protein). Each cyclic nucleotide was found to be a competitive inhibitor of the hydrolysis of the other with a Ki value of 30.18 microM for cyclic AMP hydrolysis and 256 microM for cyclic GMP hydrolysis. Hill coefficients of 1.0 obtained in the presence of cyclic AMP for cyclic GMP hydrolysis and vice-versa indicated no allosteric interactions. It is suggested that chromatin cyclic nucleotide phosphodiesterase may have a role post fertilization in cell growth and differentiation with no role in sperm motility which is regulated by similar enzymes present in sperm flagella.     

Cyclic nucleotide phosphodiesterase in silkworm. Separation and characterization of multiple forms of the enzyme.

The existence of two forms of cyclic AMP phosphodiesterase (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) was demonstrated in silkworm larvae by kinetic analysis and DEAE-cellulose column chromatography. The two forms of the enzyme (phosphodiesterase II and III) differ apparently in their characteristics from the previously reported cyclic nucleotide phosphodiesterase (phosphodiesterase I) of silkworm. The higher K-m form (phosphodiesterase II) has a molecular weight of approx. 50 000 and optimum pH of 7.8, and requires Mn-2-+ for maximum activity. The lower K-m form (phosphodiesterase III) has a molecular weight of approx. 97 000 and optimum pH of 7.2, and requires Mg-2-+ for maximum activity. Phosphodiesterase II and probably phosphodiesterase III are specific enzymes for the hydrolysis of cyclic AMP.     

Multiple forms of cyclic nucleotide phosphodiesterase in a murine adrenal cortex cell line (Y-1)

Murine adrenal cortex tumor Y-1 cells contained both soluble and particulate forms of cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotide hydrolase, EC 3.1.4.17). The soluble forms of the enzyme comprised 80% of total cellular phosphodiesterase activity. The soluble enzyme(s) hydrolyzed both cyclic AMP and cyclic GMP, with apparent Km values of 125 and 30 microM, respectively. Soluble cyclic AMP phosphodiesterase showed marked inhibition by the calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and the anticalmodulin drugs, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium. No alteration in soluble cyclic GMP phosphodiesterase activity was observed when cyclic AMP was added to the assay. Resolution of the soluble enzymatic activity by DEAE-cellulose chromatography in the presence of calcium showed two peaks of phosphodiesterase activity. Further purification of one of these peaks on DEAE-cellulose in the presence of EGTA yielded a phosphodiesterase activity peak that was stimulated fivefold by calmodulin. The particulate form of the enzyme hydrolyzed both cyclic AMP anc cyclic GMP; the apparent Km values for these substrates were similar (90 and 100 microM, respectively). Hydrolysis of cyclic GMP by the particulate enzyme was inhibited by cyclic AMP in a concentration-dependent manner with an apparent half-maximal inhibitory concentration of 100 microM. The particulate form of phosphodiesterase was not inhibited by EGTA or anticalmodulin drugs.     

Cyclic nucleotide phosphodiesterases and thyroid hormones.

Evidence is presented that modulation of the maximum velocity of a particulate low K-m cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase by thyroid hormones is one mechanism for the regulation of the responsiveness of rat epididymal adipocytes to lipolytic agents such as epinephrine and glucagon. Fat cells of propylthiouracil-induced hypothyroid rats are unresponsive to lipolytic agents and the V-max of particulate low K-m cyclic AMP phosphodiesterase of these cells is elevated above normal. In vivo treatment of hypothyroid rats with triiodothyronine restores to control values both the lipolytic response of the fat cells to epinephrine and the V-max of the particulate bound low K-m cyclic AMP phosphodiesterase. No similar correlation is found with the soluble high K-m cyclic AMP phosphodiesterase. The phosphodiesterases of fat cells from normal and hypothyroid rats respond identically in vitro to propylthiouracil, triiodothyronine, methylisobutylxanthine, or theophylline, although the particulate low K-m cyclic AMP phosphodiesterase is inhibited to a greater extent than soluble cyclic guanosine 3':5'-monophosphate phosphodiesterase activity. Protein kinase of fat cells from hypothyroid rats can be stimulated by cyclic AMP to the same total activity as observed in fat cells of normal rats. However, less of the protein kinase in fat cells from hypothyroid rats was in the cyclic AMP-independent form. This shift in the equilibrium of protein kinase forms is consistent with an increased activity of low K-m cyclic AMP phosphodiesterase and probably results from a lowering of the lipolytically significant pool of cyclic AMP.     

Characterisation of cyclic adenosine 3':5'-monophosphate phospodiesterase from Walker carcinoma sensitive and resistant to bifunctional alkylating agents.

Walker carcinoma cell lines sensitive or resistant to bifunctional alkylating agents have been found to contain multiple forms of cyclic AMP phosphodiesterase (3':5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). These activities have been resolved using Sepharose 6B gel filtration and their apparent molecular weights have been estimated. The enzyme appears to occur in four active forms of apparent mol. wts of greater than 1 000 000, 430 000, 350 000 and 225 000, when assayed at low substrate concentrations. Evidence has been obtained which suggests that all four forms of the enzyme are composed of subunits of mol. wt of approximately 15 000 and are interconvertible. While the ionic strength of the buffer affected the predominance of the different forms, the presence of cyclic AMP at 10(-6) M had no effect on aggregation or dissociation of the enzyme. An activity shift from high molecular weight forms of the enzyme to low molecular weight forms has been found in the resistant tumour at low substrate concentration. No change in elution profile between sensitive and resistant tumours was observed for the low affinity form of the enzyme. The pH optima of the enzymes with both high and low affinity for the substrate was found to be pH 8.0 in the sensitive line. In the resistant tumour the pH optima of the high affinity form is shifted to pH 8.4 while the low affinity form remains at pH 8.0. The high affinity forms of the phosphodiesterase in the sensitive and resistant tumour also differed in their inhibition by theophylline. In both cases inhibition was of the competitive type with Ki values for the sensitive and resistant lines being 2.35 and 0.32 mM, respectively. There was no significant difference in the inhibition of the low affinity form between the sensitive and resistant tumour.     

Cyclic nucleotide phosphodiesterase in rat neostriatum: multiple isoelectric forms with similar kinetic properties

Separation of multiple forms of cyclic nucleotide phosphodiesterase from the soluble supernatant fraction of rat neostriatum by isoelectric focusing yielded five separate peaks of cyclic nucleotide hydrolysing activity. Each separated enzyme form displayed a complex kinetic pattern for the hydrolysis of both cyclic AMP and cyclic GMP, and there were two apparent Km's for each nucleotide. At 1 microM substrate concentration, four enzyme forms exhibited higher activity with cyclic AMP than with cyclic GMP, while one form yielded higher activity with cyclic GMP than with cyclic AMP. Cyclic AMP and cyclic GMP were both capable of almost complete inhibition of the hydrolysis of the other nucleotide in all the peaks separated by isoelectric focusing; the IC50's for this interaction correlated well with the relative rates of hydrolysis of each nucleotide in each peak. The ratio of activity at 1 microM substrate concentration for the five enzyme forms separated by isoelectric focusing was 10:10:5:15:1 for cyclic AMP hydrolysis; and 6:6:4:8:2 for cyclic GMP hydrolysis; and the isoelectric points of the five peaks were 4.3, 4.45, 4.7, 4.85, and 5.5, respectively. Known phosphodiesterase inhibitors did not preferentially inhibit any of the separated forms of activity for either cyclic AMP or cyclic GMP hydrolysis, at either high (100 microM) or low (1 microM) substrate concentrations. Preliminary examination of the subcellular distribution of the different forms of enzyme activity indicated a different degree of attachment of the various forms to particulate tissue components. Isoelectric focusing of the soluble supernatant of rat cerebellum gave rise to a slightly different pattern of isoelectric forms from the neostriatum, indicating a different cellular distribution of the isoelectric forms of PDE in rat brain. Polyacrylamide disc gel electrophoresis of the soluble supernatant of rat neostriatum also generated a characteristic pattern of five separate peaks of cyclic nucleotide phosphodiesterase activity, each of which hydrolysed both cyclic AMP and cyclic GMP. Polyacrylamide gel electrophoresis of single enzyme forms previously separated by isoelectric focusing gave single peaks, with a marked correspondence between the enzyme forms produced by isoelectric focusing and those produced by gel electrophoresis, suggesting that both protein separation procedures were isolating the same enzyme forms. The results indicate the existence of multiple isoelectric forms of cyclic nucleotide phosphodiesterase in the soluble supernatant fraction of rat neostriatum, all of which exhibit similar properties. In this tissue a single kinetic form of this enzyme appears to exist displaying complex kinetic behaviour indicative of negative cooperativity and hydrolysing both cyclic AMP and cyclic GMP, with varying affinities.     

Studies on the soluble phosphodiesterases of chicken gizzard smooth muscle.

In this study we describe the identification of four soluble forms of cyclic nucleotide phosphodiesterase from chicken gizzard smooth muscle. These isoenzymes were separated from one another by ion-exchange chromatography on DEAE-cellulose and by calmodulin-Sepharose affinity chromatography. Each form migrates as a single discrete band when it is electrophoresed on non-denaturing polyacrylamide gels and stained for phosphodiesterase activity. Each form is also eluted as a single peak on gel-permeation chromatography, giving apparent Mr values of 114 000, 116 000, 122 000 and 59 000. All four enzymes have apparent Km values in the 0-20 microM range, although their relative specificities for cyclic AMP and cyclic GMP differ. Two of the forms bind to calmodulin in a Ca2+-dependent manner; however, only one is activated by calmodulin. The interaction of the second calmodulin-binding form with calmodulin is disrupted by the papaverine derivative verapamil without significantly altering the hydrolytic activity of the enzyme.     

Evidence for convertible forms of soluble uterine cyclic nucleotide phosphodiesterase

The cyclic nucleotide phosphodiesterase (3':5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) systems of many tissues show multiple physical and kinetic forms. In contrast, the soluble rat uterine phosphodiesterase exists as a single enzyme form with non-linear Lineweaver-Burk kinetics for cyclic AMP (app. Km of approx. 3 and 20 microM) and linear kinetics for cyclic GMP (app. Km of approx. 3 microM) since the two hydrolytic activities are not separated by a variety of techniques. In uterine cytosolic fractions, cyclic AMP is a non-competitive inhibitor of cyclic GMP hydrolysis (Ki approx. 32 microM). Also, cyclic GMP is a non-competitive inhibitor of cyclic AMP hydrolysis (Ki approx 16 microM) at low cyclic GMP/cyclic AMP substrate ratios. However, cyclic GMP acts as a competitive inhibitor of cyclic AMP phosphodiesterase (Ki approx 34 microM) at high cyclic GMP/cyclic AMP substrate ratios. When a single hydrolytic form of uterine phosphodiesterase, separated initially by DEAE anion-exchange chromatography, is treated with trypsin (0.5 microgram/ml for 2 min) and rechromatographed on DEAE-Sephacel, two major forms of phosphodiesterase are revealed. One form elutes at 0.3 M NaOAc- and displays anomalous kinetics for cyclic AMP hydrolysis (app. Km of 2 and 20 microM) and linear kinetics for cyclic GMP (app. Km approx. 5 microM), kinetic profiles which are similar to those of the uterine cytosolic preparations. A second form of phosphodiesterase elutes at 0.6 M NaOAc- and displays a higher apparent affinity for cyclic AMP (app. Km approx. 1.5 mu) without appreciable cyclic GMP hydrolytic activity. These data provide kinetic and structural evidence that uterine phosphodiesterase contains distinct catalytic sites for cyclic AMP and cyclic GMP. Moreover, they provide further documentation that the multiple forms of cyclic nucleotide phosphodiesterase in mammalian tissues may be conversions from a single enzyme species.     

Identification and characterization of a third form (D3) of cyclic 3'5'-nucleotide phosphodiesterase from rhizobwm fredii

• 1.1. A third form (D3) of cyclic nucleotide phosphodiesterase from Rhizobiumfrediiv/as detected and characterized for the first time.
• 2.2. The enzyme could hydrolyse both cyclic AMP and cyclic GMP with apparent K_m for cyclic AMP of approx. 0.2 μM.
• 3.3. D3 cyclic nucleotide phosphodiesterase had a pH optimum of about 6.0 when hydrolysing cyclic AMP.
• 4.4. The enzyme lost almost all its activity when heated to 60°C for 20 min.
• 5.5. Gel filtration with Sephadex G-100 gave a mol. wt of approx. 42.5 kD for the native enzyme.

     

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.     

Characterization of an adenosine 3'':5''-cyclic monophosphate phosphodiesterase from baker''s yeast. Its binding to subcellular particles, catalytic properties and gel-filtration behaviour.

1. The 3':5'-cyclic AMP phosphodiesterase in the microsomal fraction of baker's yeast is highly specific for cyclic AMP, and not inhibited by cyclic GMP, cyclic IMP or cyclic UMP. Catalytic activity is abolished by 30 micrometer-EDTA. At 30 degrees C and pH8.1, the Km is 0.17 micrometer, and theophylline is a simple competitive inhibitor with Ki 0.7 micrometer. The pH optimum is about 7.8 at 0.25 micrometer-cyclic AMP, so that over the physiological range of pH in yeast the activity changes in the opposite direction to that of adenylate cyclase [PH optimum about 6.2; Londesborough & Nurminen (1972) Acta Chem. Scand. 26, 3396-3398].2. At pH 7.2, dissociation of the enzyme from dilute microsomal suspensions increased with ionic strength and was almost complete at 0.3 M-KCl. MgCl2 caused more dissociation than did KCl or NaCl at the same ionic strength, but at low KCl concentrations binding required small amounts of free bivalent metal ions. In 0.1 M-KCl the binding decreased between pH 4.7 and 9.3. At pH 7.2 the binding was independent of temperature between 5 and 20 degrees C. These observations suggest that the binding is electrostatic rather than hydrophobic. 3. The proportion of bound activity increased with the concentration of the microsomal fraction, and at 22 mg of protein/ml and pH 7.2 was 70% at I0.18, and 35% at I0.26. Presumably a substantial amount of the enzyme is particle-bound in vivo. 4. At 5 degrees C in 10 mM-potassium phosphate, pH 7.2, the apparent molecular weight of KCl-solubilized enzyme decreased with enzyme concentration from about 200 000 to 40 000. In the presence of 0.5M-KCl, a constant mol.wt. of about 55 000 was observed over a 20-fold range of enzyme concentrations.     

Properties and prenatal ontogeny of beta-D-mannosidase in selected goat tissues.

beta-D-Mannosidase activity in selected normal adult, neonatal and foetal goat tissues and in tissues from animals affected with caprine beta-mannosidosis was examined with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. The enzyme in normal adult thyroid, kidney and brain exhibited a sharp unimodal pH optimum at pH 5.0, whereas the enzyme in both normal adult and mutant liver exhibited broad pH ranges of activity (pH 4.5-8.0). No residual enzyme was detectable in mutant kidney or brain; in contrast, residual activity in mutant liver was 52% of that in a neonatal control. Concanavalin A-Sepharose 4B (Con A-Sepharose) fractionation of normal adult liver beta-D-mannosidase resolved the enzyme into an unbound (non-lysosomal) from (52%) with a broad pH range of activity (pH 4.5-8.0) and a bound (lysosomal) form (48%) with a sharp pH optimum of 5.5. The enzyme in mutant liver consisted entirely of the unbound (non-lysosomal) form. Beta-D-Mannosidase activity in normal adult thyroid, kidney and brain was resolved by chromatofocusing into two major isoenzymes, with pI 5.5 and 5.9, and traces of a minor isoenzyme, with pI 5.0. In normal adult liver the enzyme was also resolved into three isoenzymes with similar pI values; however, that with pI 5.0 predominated. The predominant form of the enzyme in 60-day-foetal liver was bound by Con A, exhibited a unimodal pH optimum (5.0) and was resolved into two isoenzymes, with pI 5.4 and 5.8; only traces of an isoenzyme with pI 5.0 were detectable. Total hepatic beta-D-mannosidase activity increased progressively towards adult values during the last 90 days of gestation as a result of increasing non-lysosomal isoenzyme activity (pI 5.0). Lysosomal beta-D-mannosidase was shown to occur in all normal goat tissues studied as multiple isoenzymes, which are genetically and developmentally distinct from the non-lysosomal isoenzyme occurring predominantly, if not exclusively, in liver.     

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.     

Characterization of the soluble cyclic nucleotide phosphodiesterases in Xenopus laevis oocytes. Evidence for a calmodulin-dependent enzyme

Cyclic nucleotide phosphodiesterase activities (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the 40,000 X g supernatant fraction of homogenates of Xenopus laevis oocytes. In the supernatant, the ratio of the specific activity of cyclic AMP phosphodiesterase to that of cyclic GMP phosphodiesterase was 1.1 at the 1 micro substrate level. Two phosphodiesterase forms were isolated by centrifugation on sucrose gradient: a 3-4 S form hydrolyzing specificity cyclic AMP and a 6-7 S form hydrolyzing both cyclic nucleotides (cyclic AMP and cyclic GMP). The activity of the 6-7 S phosphodiesterase was characterized by its activation by 0.1 micro M calmodulin purified from beef pancreas in the presence of 50 micro M CA2+. The calmodulin dependence of this form was completely abolished in the presence of 1 mM ethyleneglycobis(beta-aminoethyl ether)-N-N,N',N'-tetraacetic acid (EGTA). Trifluoperazine at 0.1 mM inhibited both the freshly prepared crude enzyme and the partially purified 6-7 S form. On the other hand, no effect of cyclic GMP at 3 micro M was observed on cyclic AMP hydrolysis in the case of the supernatant or that of the partially purified phosphodiesterases. These data show the presence of a calmodulin-dependent phosphodiesterase in the soluble fraction of X. laevis oocytes.     

The pH and temperature dependence of the activity of the high Km cyclic nucleotide phosphodiesterase of bakers' yeast

The hydrolysis of adenosine 3':5'-monophosphate by the high Km cyclic nucleotide phosphodiesterase of bakers' yeast was studied over a range of temperature and pH at I = 0.17. The effects of ionic strength and MgCl2 concentration were studied at pH 7.7 and 30 degrees C. Km and Vmax were insensitive to changes in the MgCl2 concentration between 1 and 30 mM, implying that this enzyme (which does not require free divalent metal ions) does not discriminate between free cyclic AMP- and the Mg-cyclic AMP+ complex. Vmax decreased below pH 6.8 because of protonation of a group required in the basic form in the enzyme x substrate complex. On the basis of its pK (5.46 at 30 degrees C) and delta H (23 kJ/mol) this group was tentatively identified as imidazole. Vmax/Km decreased above pH 6.8 because of ionization of a group required in the acid form in the free enzyme, with a pK of 7.88 at 30 degrees C and a delta H of about 13 kJ/mol. Several possibilities exist for the identity of this group, the most likely being a second imidazole, sulfhydryl, or a water molecule bonded to tightly bound zinc. At pH 7.90, log Vmax and log Km both changed linearly with 1/T (between 12 degrees C and 37 degrees C) with enthalpies of 47 and 55 kJ/mol, respectively. Consequently, at low enough cyclic AMP concentration, the rate of reaction at pH 7.90 decreases slightly when the temperature is increased. This is also true at higher pH, but in the physiological pH range (6.4 to 7.5) Vmax/Km and, therefore, the rate of reaction at very low cyclic AMP concentration were nearly independent of temperature. Under physiological conditions, the Km approaches the upper limit of in vivo cyclic AMP concentrations in yeast, and at normal in vivo cyclic AMP concentrations the pH optimum is within or below the physiological range of pH in yeast.     

Cardiac adenosine 3':5'-monophosphate. Free and bound forms in the isolated rat atrium.

Adenosine 3':5'-monophosphate (cyclic AMP), a mediator of hormone action in a variety of tissues, has been measured in its free and bound forms in intact cardiac tissue. We have used a rapid high dilution technique which involves tissue homogenization, subcellular fractionation, and separation of bound from free cyclic AMP by Millopore filtration. The precision of this method is dependent upon minimization of binding and dissociation of cyclic AMP that occur during the preparation and handling of tissue homogenates. In each experiment, a tracer of cyclic [3H]AMP prebound to isolated cardiac binding protein was freed of unbound cyclic [3H]AMP by Sephadex gel filtration and added to the tissue just prior to homogenization in cold EDTA buffer. This tracer was therefore treated identically to the sample through all subsequent dilution, fractionation, and filtration procedures, and provided an acurate internal monitor for total cyclic AMP dissociation during the course of the free-bound determination. Each tissue sample was then individually corrected for dissociation. Rapid dilution to produce a 1:1000 homogenate was found to lower endogenous cyclic AMP levels sufficiently to make binding (or rebinding) during the procedure negligible (less than 5%). Spontaneously beating rat right atria (controls) contained 5.96 +/- 0.28 pmol of cyclic AMP/mg of protein (n = 19) of which 41 and 14% were bound to soluble and particulate proteins, respectively. The remaining cyclic AMP was free. Pretreatment of the tissue with 1 muM isoproterenol (30 s at 30 degrees) increased both the bound and free forms of cyclic AMP (n = 8). While free cyclic AMP increased 420% with the catecholamine, the bound forms increased 240% (soluble) and 60% (particulate). Similar results were obtained when atria (n = 6) were treated with the phosphodiesterase inhibitor, methylisobutylxanthine (0.5 mM, 10 min at 30 degrees). When both agents were used together, cyclic AMP bound to soluble proteins was elevated 4-fold over control while free cyclic AMP increased 27-fold (n = 7), indicating saturation of the soluble sites. It could be calculated that less than one-third of these sites are occupied in the unstimulated cell. These sites may represent the R subunit of cyclic AMP-dependent protein kinase. The data suggest that half-maximal binding in vivo occurs at an intracellular free cyclic AMP concentration of about 1 muM.     

Roles of pH and cyclic adenosine monophosphate in the acquisition of potential for sperm motility during migration from the sea to the river in chum salmon

In the natural process of the migration of chum salmon from the sea to the river, spermatozoa moved from the testis to the sperm duct, and the pH value of seminal plasma, concentration of cyclic adenosine monophosphate (AMP) in the sperm cells, and potential for sperm motility increased. Cyclic AMP levels and the potential for motility gradually increased when testis spermatozoa with no capacity for movement were incubated in the artificial seminal plasma of which the pH was much the same as, or higher than, the pH of natural seminal plasma from the sperm duct. Such correlation in motility, pH, and cyclic AMP suggests that the increases in seminal pH and intracellular cyclic AMP level during passage of spermatozoa from the testis to the sperm duct cause the acquisition of potential for motility. Motility of testicular spermatozoa demembranated with Triton X-100 was very low in fish caught in the sea, while motility of spermatozoa from the posterior portion of the sperm duct was much higher in fish caught in the river. Furthermore, nondemembranated, intact spermatozoa showed a lag in the timing of the acquisition of potential for motility vs. demembranated spermatozoa: The demembranated sperm exhibited the potential earlier than the nondemembranated sperm. These data suggest that increase in activity of the motile apparatus, the axoneme, is a prerequisite, in part, for the acquisition of sperm motility, whereas the development of some function of the plasma membrane also contributes to this phenomenon. © 1993 Wiley-Liss, Inc.     

Analysis of cyclic nucleotide phosphodiesterase(s) by radioimmunoassay

A high-affinity form of cyclic AMP phosphodiesterase, purified to apparent homogeneity from dog kidney, was labeled with ¹²⁵I using a solid-state lactoperoxidaseglucose oxidase system and its purity confirmed by acrylamide gel electrophoresis and isoelectric focusing. Sheep anti-cyclic AMP phosphodiesterase immunoglobulin fraction was analyzed for ¹²⁵I-enzyme binding and covalently bound to agarose A 1.5m for isotopically labeled antigen displacement. Anti-phosphodiesterase antiserum was purified by Sepharose 4B-cAPDE affinity chromatography and used for a radioimmunoassay employing second-antibody precipitation. The specificity of the anti-cyclic AMP phosphodiesterase antibody was established by its use as a covalently bound affinity ligand for cyclic AMP phosphodiesterase purification and analysis of sodium dodecyl sulfate-gel extracts of partially purified and purified dog kidney supernatants. Radioimmunoassay using a monospecific antibody preparation demonstrated the similarity of high-affinity cyclic AMP phosphodiesterase forms of different tissues and species that had been separated by DEAE-cellulose chromatography. Various purified preparations of calmodulin, as well as brain calcineurin, did not cross-react in the high-affinity cyclic AMP phosphodiesterase radioimmunoassay. However, higher molecular weight cyclic GMP/lower affinity cyclic AMP phosphodiesterase enzyme forms, partially purified by anion-exchange chromatography, gel filtration, and Cibacron blue adsorption, were shown to cross-react in the high-affinity cAMP phosphodiesterase radioimmunoassay. These studies suggest immunological similarities between the major forms of this enzyme system and the possibility of higher molecular weight complexes containing both cyclic GMP and cyclic AMP hydrolytic sites.     

Cyclic adenosine 3':5'-monophosphate phosphodiesterase. Distinct forms in human lymphocytes and monocytes.

Adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase activity of normal human peripheral blood leukocyte suspensions containing 90% lymphocytes and 10% monocytes showed anomalous kinetic behavior indicative of multiple enzyme forms. Kinetic analyses of purified lymphocyte (99%) or monocyte preparations (95%) indicated that only one type of phosphodiesterase was present in each cell type. None of the preparations contained any detectable guanosine 3':5'-monophosphate (cyclic GMP) hydrolytic activity. The lymphocyte enzyme had an apparent Km congruent to 0.4 muM for cyclic AMP and Vmax congruent to 0.5 picomoles/min/10(6) cells. These kinetic parameters were confirmed by several cell purification techniques used alone and sequentially. Sedimentation velocity analyses indicated that the higher Km monocyte enzyme had a molecular weight near 45,000 and that the lower Km lymphocyte enzyme most likely had a molecular weight near 98,000. A variety of procedures led to a loss of the higher molecular weight, high affinity enzyme leaving only the enzyme of 45,000 daltons with a much lower substrate affinity. A long term, stable human lymphoblastoid cell line had cyclic AMP phosphodiesterase activity that was similar to the lymphocyte enzyme by both physical and kinetic criteria. Lymphocyte cyclic AMP phosphodiesterase appears to be a soluble enzyme whose pH and temperature optima and cationic requirements are similar to those of other mammalian phosphodiesterases. The distinct cyclic AMP phosphodiesterase forms of these cells may possibly represent the basic, active subunit of mammalian cyclic nucleotide phosphodiesterases. We hypothesize that the extremely high affinity cyclic AMP phosphodiesterase of normal lymphocytes plays an important role in the regulation of normal function in these cells, and also in the rapid proliferative responses characteristic of the stimulated lymphocyte.     

Adenosine 3'':5''-cyclic monophosphate-binding proteins in bovine and rat tissues.

1. At least two classes of high-affinity cyclic AMP-binding proteins have been identified: those derived from cyclic AMP-dependent protein kinases (regulatory subunits) and those that bind a wide range of adenine analogues (adenine analogue-binding proteins). 2. In fresh-tissue extracts, regulatory subunits could be further subdivided into 'type I or 'type II' depending on whether they were derived from 'type I' or 'type II' protein kinase [see Corbin et al. (1975) J. Biol. Chem. 250, 218-225]. 3. The adenine analogue-binding protein was detected in crude tissue supernatant fractions of bovine and rat liver. It differed from the regulatory subunit of cyclic AMP-dependent protein kinase in many of its properties. Under the conditions of assay used, the protein accounted for about 45% of the binding of cyclic AMP to bovine liver supernatants. 4. The adenine analogue-binding protein from bovine liver was partially purified by DEAE-cellulose and Sepharose 6B chromatography. It had mol.wt. 185000 and was trypsin-sensitive. As shown by competition and direct binding experiments, it bound adenosine and AMP in addition to cyclic AMP. At intracellular concentrations of adenine nucleotides, binding of cyclic AMP was essentially completely inhibited in vitro. Adenosine binding was inhibited by only 30% under similar conditions. 5. Rat tissues were examined for the presence of the adenine analogue-binding protein, and, of those examined (adipose tissue, heart, brain, testis, kidney and liver), significant amounts were only found in the liver. The possible physiological role of the adenine analogue-binding protein is discussed. 6. Because the adenine analogue-binding protein or other cyclic AMP-binding proteins in tissues may be products of partial proteolysis of the regulatory subunit of cyclic AMP-dependent protein kinase, the effects of trypsin and aging on partially purified protein kinase and its regulatory subunit from bovine liver were investigated. In all studies, the effects of trypsin and aging were similar. 7. In fresh preparations, the cyclic AMP-dependent protein kinase had mol.wt. 150000. Trypsin treatment converted it into a form of mol.wt 79500. 8. The regulatory subunit of the protein kinase had mol.wt. 87000. It would reassociate with and inhibit the catalytic subunit of the enzyme. Trypsin treatment of the regulatory subunit produced a species of mol.wt. 35500 which bound cyclic AMP but did not reassociate with the catalytic subunit. Trypsin treatment of the protein kinase and dissociation of the product by cyclic AMP produced a regulatory subunit of mol.wt. 46500 which reassociated with the catalytic subunit. 9. These results may be explained by at least two trypsin-sensitive sites on the regulatory subunit. A model for the effects of trypsin is described.