Proteolytic activation and solubilization of endoplasmic-reticulum cyclic AMP phosphodiesterase activity.

Dithiothreitol led to the activation and solubilization of the cyclic nucleotide phosphodiesterase activities associated with the smooth and various rough subfractions of rat liver endoplasmic reticulum. The activity in each of the subfractions exhibited somewhat different time courses, and sensitivities to dithiothreitol concentration, in respect of their solubilization and activation. Both activation and solubilization by dithiothreitol could be blocked by either thiol proteinase inhibitors or excess bovine serum albumin. Freeze-thaw solubilization was not blocked by the thiol proteinase inhibitor antipain and did not lead to the activation of the enzyme. After dithiothreitol-induced solubilization, all of the enzymes exhibited non-linear Lineweaver-Burk plots indicative of apparent negative co-operativity. In contrast, after freeze-thaw solubilization the enzyme in the smooth-endoplasmic-reticulum-plus-Golgi fraction still obeys Michaelis kinetics, as does the membrane-bound enzyme. It is possible to mimic the action of dithiothreitol in solubilizing and activating the enzyme by limited proteolysis with trypsin. Triton X-100 is highly efficient at solubilizing these enzymes, yet has little effect on their activities. Charged detergents exhibit highly selective effects on the enzymes as regards their solubilization and activity expressed.     

Subcellular localization and hormone sensitivity of adipocyte cyclic AMP phosphodiesterase.

Treatment of intact adipocytes with either or both insulin and adrenaline stimulated membrane cyclic AMP phosphodiesterase activity only in the endoplasmic reticulum subfraction. The cyclic GMP-inhibited cyclic AMP phosphodiesterase activity was also found in this fraction. Quantitative Western blotting using a specific polyclonal antibody, raised against the homogeneous 'dense-vesicle' cyclic AMP phosphodiesterase from rat liver, identified a single 63 kDa species which was localized in the adipocyte endoplasmic reticulum fraction. The ability of adrenaline to stimulate adipocyte membrane cyclic AMP phosphodiesterase was shown to be mediated via beta-adrenoceptors and not alpha 1-adrenoceptors. Membrane cyclic AMP phosphodiesterase was stimulated by glucagon but not by vasopressin, A23187 or 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment of adipocytes with either chloroquine or dansyl cadaverine failed to affect the ability of insulin to stimulate cyclic AMP phosphodiesterase activity. Treatment of an isolated adipocyte endoplasmic reticulum membrane fraction with purified protein kinase A increased its cyclic AMP phosphodiesterase activity some 2-fold. When this fraction was treated with purified protein kinase A and [32P]ATP, label was incorporated into a 63 kDa protein which was specifically immunoprecipitated with the antiserum against the liver 'dense-vesicle' cyclic AMP phosphodiesterase.     

Distribution of adenylate cyclase among membrane fractions of rat liver.

Adenylate cyclase activity was detected in plasma membranes, Golgi apparatus, and endoplasmic reticulum from rat liver. Adenylate cyclase activities of purified membranes were determined biochemically by two methods. In one, the synthesis of radioactive cyclic AMP from ATalpha32P was monitored. In the other, the synthesis of cyclic AMP was quantitiated using a protein which specifically binds cyclic AMP. The enzyme activity was responsive to activation by both glucagon and sodium fluoride although differences in degree of activation were noted comparing plasma membrane, Golgi apparatus, and endoplasmic reticulum. Cytochemical studies, using both whole tissue and purified cell fractions and conducted in parallel, confirmed the biochemical results. Deposition of lead phosphate, enhanced by glucagon and NaF with samples incubated with appropriate substrates, was not restricted to plasma membranes of hepatocytes but was present in intracellular membranes as well. Adenylate cyclase of rat hepatocytes appears more widely distributed among internal membranes than previously recognized.     

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+.     

Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity. Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied. The Golgi complex fraction contained most of the galactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions. The dolichol- dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex. Although the dolichol-independent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction. Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A1 suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed.     

Cyclic AMP-dependent protein kinase in canine pancreatic rough endoplasmic reticulum

A canine pancreas homogenate was subfractionated by several differential centrifugation steps. The distribution of cAMP-dependent protein kinase in the various fractions was monitored by assaying [3H]cAMP binding and photo-cross-linking of the regulatory subunits of the enzyme (RI and RII) with radiolabeled 8-azido-cAMP. The distribution of the kinase was also compared to that of markers for the plasma membrane, the endoplasmic reticulum and the cytosol. While our results confirm previous studies suggesting the presence of cyclic AMP-dependent protein kinase in the cytosol and Golgi, a significant amount of the total [3H] cAMP binding and photolabeled R subunits (both RI and RII) were found in rough microsomes (RM). The association is relatively resistant to extraction with EDTA, low and high ionic strength solutions. These extractions unmasked several new phosphorylation substrates in the "stripped" RM that were inaccessible in the RM, possibly because they were covered by ribosomes or peripheral membrane proteins. RII with a molecular mass of 52 kDa (RII-52 kDa) was the predominant RII found in the cytosolic fraction, whereas RII-52 kDa and RII with a molecular mass of 54 kDa (RII-54 kDa) were approximately equally enriched in the RM fraction. The mobility of the RII-52 kDa-photolabeled band could be shifted to the mobility of the RII-54 kDa band by phosphorylation with purified catalytic subunit and ATP, indicating that they represent "dephospho" and "phospho" forms of RII, respectively. A more precise localization to the rough endoplasmic reticulum was accomplished by isopycnic floatation in sucrose gradients. The enzyme cobanded at the density of rough microsomes and shifted to the lower density of "stripped" microsomes after treatment with puromycin/high salt, which specifically removes ribosomes.     

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.     

Characterization of the protein kinase activities of human platelet supernatant and particulate fractions.

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.     

Ultrastructural immunocytochemical localization of cyclic AMP-dependent protein kinase regulatory subunits in rat parotid acinar cells

Polyclonal antibodies to types I and II regulatory (R) subunits of cyclic AMP-dependent protein kinase (cA-PK) were utilized in a post-embedding immunogold-labeling procedure to localize these proteins in rat parotid acinar cells. Both RI and RII were present in the nuclei, cytoplasm, rough endoplasmic reticulum (RER), Golgi apparatus, and secretory granules. In the nuclei, gold particles were mainly associated with the heterochromatin. In the cytoplasm, the label was principally found in areas of RER. Most gold particles were located between adjacent RER cisternae or over their membranes and attached ribosomes; occasional particles were also present over the cisternal spaces. Labeling of the Golgi apparatus was significantly greater than background, although it was slightly lower than that over the RER cisternae. In secretory granules, gold particles were present over the granule content; no preferential localization to the granule membrane was observed. Morphometric analysis revealed equivalent labeling intensities for RI and RII in the cytoplasm-RER compartment. Labeling intensities for RII in the nuclei and secretory granules were about 50% greater than in the cytoplasm-RER, and 3 to 4-fold greater than values for RI in these two compartments. Electrophoresis and autoradiography of the postnuclear parotid-tissue fraction, the contents of purified secretory granules and saliva collected from the main excretory duct, after photoaffinity labeling with [32P]-8-azido-cyclic AMP, revealed the presence of R subunits. Predominantly RII was present in the granule contents and saliva, while both RII and RI were present in the cell extracts. Additionally, R subunits were purified from saliva by affinity chromatography on agarose-hexane-cyclic AMP. These findings confirm the localization of cA-PK in parotid cell nuclei and establish the acinar secretory granules as the source of the cyclic AMP-binding proteins in saliva.     

Increased activity of cyclic AMP phosphodiesterase from frozen-thawed rat liver. A role of lysosomal protease in enzyme activation.

The activity of cyclic AMP phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in 105 000 X g supernatant fraction from frozen-thawed rat liver was 2.5 times higher than the corresponding preparation from fresh liver. This increased activity of frozen liver enzyme was accompanied by a decreased sensitivity of the enzyme to known activators such as alpha-tocopheryl phosphate and trypsin. Neither membrane-bound cyclic AMP phosphodiesterase, nor supernatant cyclic GMP phosphodiesterase increased in frozen liver preparation. It is unlikely that the activator protein of phosphodiesterase participated in the observed change of enzyme activity. Among rat tissues so far tested, the increased level of cyclic AMP phosphodiesterase was noted only in tissues rich in lysosome content. In the recombination experiment where phosphodiesterase from fresh liver was incubated with lysosomal fraction, stimulation of the enzyme activity was observed with a concomitant loss of sensitivity to above-mentioned activators. Since the stimulation by lysosomal fraction was effectively inhibited by cathepsin B1 inhibitors, leupeptin and antipain, it was deduced cathepsin-B1 (EC 3.4.12.3) type protease(s) was the main causative of activating the cyclic AMP phosphodiesterase. The freezing-thawing process of rat liver made the lysosomal membrane more permeable, and hence lysosomal proteases were released into soluble fraction during phosphodiesterase preparation. These results provide a warning not to use frozen liver for phosphodiesterase preparation, otherwise altered properties of the enzymes will be seen.     

Distribution of enzyme activities in subcellular fractions of bovine retina.

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

Isolation of a Golgi apparatus-rich fraction from rat liver. IV. Thiamine pyrophosphatase

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1–3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30–50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.     

Subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm.

The subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 80% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucleotide phosphodiesterase in sperm flagella were also briefly described.     

Subcellular localization of the enzyme that forms mannosyl retinyl phosphate from guanosine diphosphate [14C]mannose and retinyl phosphate.

The subcellular distribution of the enzyme catalysing the conversion of retinyl phosphate and GDP-[14C]mannose into [14C]mannosyl retinyl phosphate was determined by using subcellular fractions of rat liver. Purity of fractions, as determined by marker enzymes, was 80% or better. The amount of mannosyl retinyl phosphate formed (pmol/min per mg of protein) for each fraction was: rough endoplasmic reticulum 0.48 +/- 0.09 (mean +/- S.D.); smooth membranes (consisting of 60% smooth endoplasmic reticulum and 40% Golgi apparatus), 0.18 +/- 0.03; Golgi apparatus, 0.13 +/- 0.03; and plasma membrane 0.02.     

Distribution of cyclic AMP phosphodiesterase in adipose tissue from trained rats

Fat cells were isolated from sedentary and exercise trained female Sprague-Dawley rats and cyclic AMP phosphodiesterase (cyclic AMP-PDE) activities were determined from crude homogenates of the fat cells in the whole homogenate, P5, P48, and S48 fractions. Exercise training resulted in a significant increase in the mean specific activity of cyclic AMP-PDE (pmol X min-1 X mg-1) from the whole homogenate and S48 fraction at cyclic AMP concentrations of 4, 8, and 16 microM and in the P48 fraction at 8 and 16 microM cyclic AMP. Cyclic AMP-PDE kinetic plots according to Lineweaver-Burk for the calculation of Michaelis constants (Km) and maximum enzyme velocities (Vmax) were nonlinear, indicating both a low and high enzyme form. The Michaelis constants were significantly lower in trained rats than those of its control for the high Km form of cyclic AMP-PDE in the whole and soluble fractions and for the low Km form of the P5 particulate fraction. The Vmax of the high Km form of the P48 particulate fraction from trained animals was also significantly higher than that found in its control. Phosphodiesterase inhibition by methylxanthines in the various fractions was similar in both trained and sedentary animals. These changes in specific activity, Michaelis constants, and Vmax of cyclic AMP-PDE from crude homogenates of isolated fat cells from exercise trained animals may account for the decreased intracellular levels of cyclic AMP following catecholamine stimulation of isolated fat cells from trained rats.     

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.     

Distribution subcellulaire des protéine-kinases stimulables par l'AMP cyclique et des protéines réceptrices de l'AMP cyclique dans la thyroïde de porc

The distribution of cyclic AMP-dependent protein kinase activity in porcine thyroid glands has been studied. Enzyme activity catalyzing phosphorylation of exogenous substrate (protamine) from ATP, and cyclic AMP binding were determined in parallel in subcellular fractions purified by differential centrifugation and flotation on sucrose density layers. Both activities were found in all the studied fractions; they were quantitatively the highest in the cytosol but particles showed the highest specific activities.Latent protein-kinase activity was unmasked by action of detergents on microsomes (× 5–10 fold) and solubilized (85 to 99 p. cent of the initial total activity). Cyclic AMP binding capacity was also recovered in detergent-treated microsomal extracts in spite of reduced cyclic AMP binding in the presence of detergent.Protein kinase activity and cyclic AMP-binding proteins were less represented in purified nuclei than in microsomes. Again both activities were unmasked by detergent.Preparations highly enriched in Golgi membranes were compared to rough microsomal preparations. Higher protein kinase activity was detected in rough microsomes as compared to Golgi membranes, whereas the reverse was true for cyclic AMP binding. Both activities were equalized after detergent treatment. Since unmasking of protein kinase activity was the highest in Golgi membranes, this fraction contains more enzyme activity and cyclic AMP binding capacity than rough microsomes.The localization of endogeneous protein substrates of cyclic AMP-dependent protein kinases was investigated using purified soluble protein kinase subcellular fractions. The better endogeneous substrates seemed to be localized in the rough microsomal and in the nuclear fractions.     

Subcellualr localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm

The subcellular localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 980% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucelotide phosphodiesterase in sperm flagella were also briefly described.     

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.     

Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities of rat mammary tissue.

Cyclic nucleotide phosphodiesterase activity in mammary tissue from rats in midlactation was resolved by DEAE-cellulose chromatography into three functionally distinct fractions: a Ca2+/calmodulin-stimulated cyclic GMP phosphodiesterase, a cyclic GMP-stimulated low-affinity cyclic nucleotide phosphodiesterase, and a high-affinity cyclic AMP-specific phosphodiesterase. The absolute activities and relative proportions of high- and low-affinity enzymes resemble those found, for example, in liver, as distinct from those in excitable tissues. Three functional characteristics are described which are peculiar to mammary-tissue phosphodiesterases. Firstly, the concentration of free Ca2+ required to achieve half-maximal activation of the Ca2+/calmodulin-stimulated phosphodiesterase is somewhat higher than for the analogous enzyme in other tissues; secondly, the activity of this enzyme towards cyclic AMP relative to that towards cyclic GMP is unusually low, and thirdly, the low-affinity cyclic nucleotide phosphodiesterase is inhibited by low concentrations of free Ca2+.