3',5'-cyclic nucleotide phosphodiesterase from human brain

3':5'-Cyclic nucleotide phosphodiesterase was isolated from human brain and characterized. After the first stage of purification on phenyl-Sepharose, the enzyme activity was stimulated by Ca2+ and micromolar concentrations of cGMP. High pressure liquid chromatography on a DEAE-TSK-3SW column permitted to identify three ranges of enzymatic activity designated as PDE I, PDE II and PDE III. Neither of the three enzymes possessed a high selectivity for cAMP and cGMP substrates. The catalytic activity of PDE I and PDE II increased in the presence of Ca2+-calmodulin (up to 6-fold); the degradation of cAMP was decreased by cGMP. The Ca2+-calmodulin stimulated PDE I and PDE II activity was decreased by W-7. PDE I and PDE II can thus be classified as Ca2+-calmodulin-dependent phosphodiesterases. With cAMP as substrate, the PDE III activity increased in the presence of micromolar concentrations of cGMP (up to 10-fold), Ca2+ and endogenous calmodulin (up to 2-3-fold). No additivity in the effects of saturating concentrations of these compounds on PDE III was observed. Ca2+ did not influence the rate of cGMP hydrolysis catalyzed by PDE III. In comparison with PDE I and PDE II, the inhibition of PDE III was observed at higher concentrations of W-7 and was not limited by the basal level of the enzyme. These results do not provide any evidence in favour of the existence of several forms of the enzyme in the PDE III fraction. The double regulation of PDE III creates some difficulties for its classification.     

Autophosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II in intact nerve terminals

The autophosphorylation of purified Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) on a threonine-containing phosphopeptide common to both the alpha and beta subunits was previously shown to convert this enzyme into a catalytically active Ca2+-independent species. We now have examined the phosphorylation and activation of Ca2+/CaM kinase II in synaptosomes, a Ca2+-dependent neurosecretory system consisting of isolated nerve terminals. Synaptosomes were prelabeled with 32Pi and the alpha subunit of Ca2+/CaM kinase II was immunoprecipitated. Under basal incubation conditions the alpha subunit was phosphorylated. Depolarization of synaptosomes produced a rapid (2-5 s) Ca2+-dependent increase of about 50% in the state of phosphorylation of the alpha subunit. This was followed by a slower increase in the 32P content of the alpha subunit over the next 5 min of depolarization. The enhanced phosphorylation was characterized by an initial rise (2 s) and subsequent decrease (30 s) in the phosphothreonine content of the alpha subunit. In contrast, the phosphoserine content of the alpha subunit slowly increased during the course of depolarization. Thermolytic two-dimensional phosphopeptide maps of the alpha subunit demonstrated that depolarization stimulated the labeling of a phosphopeptide associated with autoactivation. In parallel experiments, unlabeled synaptosomes were depolarized, and lysates of these synaptosomes were assayed for Ca2+/CaM kinase II activity. Depolarization produced a rapid (less than or equal to 2 s) increase in Ca2+-independent Ca2+/CaM kinase II activity. This activity returned to basal levels by 60 s. Thus, depolarization of intact synaptosomes is associated with the transient phosphorylation of Ca2+/CaM kinase II on threonine residues, presumably involving an autophosphorylation mechanism and concomitantly the transient generation of the Ca2+-independent form of Ca2+/CaM kinase II.     

Calcium-dependent cyclic nucleotide phosphodiesterase. Inhibition of basal activity by heat-stable factors from rat cerebrum.

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca2+-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca2+-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in Km for cyclic GMP and a decrease in V. In the presence of calcium ions and purified activator protein, the Ca2+-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca2+-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was minimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca2+-independent activation of Ca2+-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca2+-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca2+-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phoshodiesterase activity.     

Inhibition of Ca2+-activated cyclic nucleotide phosphodiesterase reaction by a heat-stable inhibitor protein from bovine brain.

An inhibitor protein of cyclic nucleotide phosphodiesterase is demonstrated in bovine brain extract and separated from modulator binding protein, a recently discovered inhibitory factor of phosphodiesterase. The new inhibitor protein is similar to the cyclic AMP phosphodiesterase inhibitor from bovine retina (Dumler, I. L., and Etingof, F. N. 1976) Biochim. Biophys, Acta 429, 474-484) in its heat stability: it retains full activity upon heating in a boiling water bath for 2 min. The new inhibitor protein counteracts the activation of the Ca2+-activatable cyclic nucleotide phosphodiesterase by the Ca2+-dependent modulator protein without affecting the basal activity of the enzyme. The inhibition of phosphodiesterase by the inhibitor can be reversed by high concentrations of modulator protein but is not influenced by a 20-fold increase in Ca2+ concentration. In contrast, a Ca2+-independent form of cyclic nucleotide phosphodiesterase is not inhibited by the inhibitor protein. These results suggest that the heat-stable inhibitor protein is specific against the action of the Ca2+-dependent modulator protein. Gel filtration analyses on Sephadex G-75 and G-100 columns have shown that the inhibitor protein and the modulator protein may associate in the presence of Ca2+. The molecular weights determined by the gel filtration for the free inhibitor protein and the complex of the inhibitor and modulator protein are about 70,000 and 85,000, respectively.     

Properties of enzyme activities involved in protein phosphorylation-dephosphorylation of thyroid plasma membranes

Bovine thyroid tissue exhibited cAMP-dependent and Ca2+-dependent protein kinase activities as well as a basal (cAMP- and Ca2+-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kinase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent Km values of cAMP and Ca2+ for the membrane-bound protein kinase were 5 . 10(-8) M and 8.3 . 10(-4) M in the presence of 1 Mm EGTA), respectively. The apparent Km values of Mg2+ were 7.10-4M (without (in the cAMP and Ca2+), 5 . 10(-4) M (with cAMP) and 1.3 . 10(-3) M (with Ca2+), and those of ATP were 3.5 . 10(-5)M (with or without cAMP) and 8.5 . 10(-5) M (with Ca2+). The Ca2+-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using 32P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca2+, but was stimulated by a rather broad range (5-25 mM) of Mg2+ and Mn2+. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca2+-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.     

Inhibition of phospholipid/Ca2+-dependent protein kinase and phosphorylation of leukemic cell proteins by CP-46,665-1, a novel antineoplastic lipoidal amine

CP-46,665-1, an antineoplastic lipoidal amine, was found to inhibit phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C), with an IC50 (concentration causing a 50% inhibition) of 10 microM. Its inhibition of the enzyme was reversed by phosphatidylserine, but not by Ca2+. The agent also inhibited the enzyme activity which was further augmented by 12-0-tetradecanoylphorbol-13-acetate (TPA), mezerein or diolein. Phosphorylation of endogenous proteins from HL-60 cells by the enzyme, with or without being further augmented by TPA, was inhibited by CP-46,665-1 as well as by alkyllysophospholipid (an antineoplastic agent). CP-46,665-1, while without effect on cyclic AMP-dependent protein kinase, also inhibited myosin light chain kinase (a calmodulin/Ca2+-dependent protein kinase). The present findings suggest that inhibition of the Ca2+-effector enzymes may be related in part to the antimetastatic activity of the lipoidal amine.     

Calcium-independent activation of two plant leaf calcium-regulated protein kinases by unsaturated fatty acids

Two soluble Ca2+-dependent protein kinases from silver beet leaves (enzymes I and II) can be resolved from each other by chromatography on DEAE-cellulose. Enzyme II is substantially activated by arachidonic, oleic and linoleic acids in the absence of Ca2+ but not at high Ca2+ concentrations. Stearic and arachidic acids do not substantially activate either enzyme at low free Ca2+. Enzyme I differs from enzyme II in exhibiting a much lower degree of Ca2+-independent activation by unsaturated fatty acids.     

Catalytic and regulatory properties of two forms of bovine heart cyclic nucleotide phosphodiesterase.

The soluble supernatant fraction of bovine heart homogenates may be fractionated on a DEAE cellulose column into two cyclic nucleotide phosphodiesterases (EC 3.1.4.-):PI and PII phosphodiesterases, in the order of emergence from the column. In the presence of free Ca2+, the PI enzyme may be activated several fold by the protein activator which was discovered by Cheung((1971) J. Biol. Chem. 246, 2859-2869). The PII enzyme is refractory to this activator, and is not inhibited by the Ca2+ chelating agent, ethylene glycol bis (beta-aminoethyl ether)-N, N'-tetraacetate (EGTA). The activated activity of PI phosphodiesterase may be further stimulated by imidazole or NH+4, and inhibited by high concentrations of Mg2+. These reagents have no significant effect on either the PII enzyme or the basal activity of PI phosphodiesterase. Although both forms of phosphodiesterase can hydrolyze either cyclic AMP or cyclic GMP, they exhibit different relative affinities towards these two cyclic nucleotides. The PI enzyme appears to have much higher affinities toward cyclic GMP than cyclic AMP. Km values for cyclic AMP and cyclic GMP are respectively 1.7 and 0.33 mM for the non-activated PI phosphodiesterase; and 0.2 and 0.007 mM for the activated enzyme. Each cyclic nucleotide acts as a competitive inhibitor for the other with Ki values similar to the respective Km values. In contrast with PI phosphodiesterase, PII phosphodiesterase exhibits similar affinity toward cyclic AMP and cyclic GMP. The apparent Km values of cyclic AMP and cyclic GMP for the PII enzyme are approx. 0.05 and 0.03 mM, respectively. The kinetic plot with respect to cyclic GMP shows positive cooperativity. Each cyclic nucleotide acts as a non-competitive inhibitor for the other nucleotide. These kinetic properties of PI and PII phosphodiesterase of bovine heart are very similar to those of rat liver cyclic GMP and high Km cyclic AMP phosphodiesterases, respectively (Russel, Terasaki and Appleman, (1973) J. Biol. Chem. 248, 1334).     

Purification and characterization of two wheat-embryo protein phosphatases.

Two protein phosphatases (enzymes I and II) were extensively purified from wheat embryo by a procedure involving chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, DEAE-Sephacel and Ultrogel AcA 44. Preparations of enzyme I (Mr 197,000) are heterogeneous. Preparations of enzyme II (Mr 35,000) contain only one major polypeptide (Mr 17,500), which exactly co-purifies with protein phosphatase II on gel filtration and is not present in preparations of enzyme I. However, this major polypeptide has been identified as calmodulin. Calmodulin and protein phosphatase II can be separated by further chromatography on phenyl-Sepharose CL-4B. Protein phosphatases I and II do not require Mg2+ or Ca2+ for activity. Both enzymes catalyse the dephosphorylation of phosphohistone H1 (phosphorylated by wheat-germ Ca2+-dependent protein kinase) and of phosphocasein (phosphorylated by wheat-germ Ca2+-independent casein kinase), but neither enzyme dephosphorylates a range of non-protein phosphomonoesters tested. Both enzymes are inhibited by Zn2+, Hg2+, vanadate, molybdate, F-, pyrophosphate and ATP.     

Cyclic AMP phosphodiesterase activities in Xenopus laevis oocytes

Cyclic AMP phosphodiesterase activity has been identified in full-grown Xenopus oocytes in vivo and in vitro. About 50% of the in vitro phosphodiesterase activity was present in the solution fraction and 35% in a partially purified membrane fraction. Both activities exhibited high substrate affinity (Km about 10(-6) M). Sucrose gradient fractionation revealed two forms of phosphodiesterase: a 5 S form (peak I) and a 6.5 S form (peak II). Treatment with trypsin led to the activation of the soluble enzyme with the transformation of peak II into peak I. Ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, calcium dependent regulator, and Fluphenazine did not influence the enzyme activities suggesting that the oocyte phosphodiesterases were not Ca2+-dependent. Intact oocytes were induced to mature by exposure to progesterone; their phosphodiesterase activities and distribution tested in vitro were comparable to those of untreated oocytes.     

Hydrophobic association of calpains with subcellular organelles. Compartmentalization of calpains and the endogenous inhibitor calpastatin in tissues

Calpains I and II isolated from diverse tissues possess both Ca2+-independent, and Ca2+-dependent accessible hydrophobic regions. Possible subcellular organelle association of calpains involving these hydrophobic regions was studied. By homogenizing rat tissues directly in Ca2+ (50 microM), about 30-60% of the cytosolic calpain I and II activity reversibly associated with isolated subcellular fractions (microsomal greater than plasma membrane greater than nuclear). After binding to the particulate fraction, calpain II converted to a calpain I-like form exhibiting stronger Ca2+-independent binding to phenyl-Sepharose and a lower Ca2+ requirement for optimal activity. However, it retained its DEAE-cellulose chromatographic pattern, and precipitated with monospecific anti-calpain II antibodies. Although purified calpastatin (endogenous inhibitor) is known to form a Ca2+-dependent complex with calpains, it was not able to reverse the binding of calpains to the particulate fraction upon short incubation. It was, however, effective in blocking calpain binding when the isolated cytosolic fraction or a mixture of purified calpain and calpastatin was preincubated in the presence of Ca2+, and then added to the particulate fraction. Extraction of tissues under controlled conditions revealed that in fact calpains are already loosely associated with subcellular organelles even in the absence of Ca2+. This is the reason why in the crude homogenates with the addition of Ca2+, calpains strongly bind to the particulate fraction without interference by cytosolic calpastatin. Although calpastatin by complexing initially to calpain can prevent the association of this protease with subcellular organelles, it cannot dissociate calpains already bound to these subcellular fractions. By prior Ca2+-independent association with the hydrophobic proteins present in the subcellular fractions, calpains overcome the 3- to 30-fold inhibitory excess of calpastatin in tissues.     

Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber.

1. Three phosphodiesterases that are capable of hydrolysing 3':5'-cyclic nucleotides were purified from potato tubers. 2. The phosphodiesterases were fractionated by (NH4)2SO4 precipitation and CM-cellulose chromatography. The phosphodiesterases were resolved from each other and further purified by gel filtration in high- and low-ionic-strength conditions. 3. All three enzymes lacked significant nucleotidase activity. 4. Enzymes I and II had mol. wts. 240,000 and 80,000 respectively, determined by gel filtration, whereas enzyme III showed anomalous behaviour on gel filtration, behaving as a high- or low-molecular-weight protein in high- or low-ionic-strength buffers respectively. 5. All enzymes hydrolysed 2':3'-cyclic nucleotides as well as 3':5'-cyclic nucleotides. The enzymes also had nucleotide pyrophosphatase activity, hydrolysing NAD+ and UDP-glucose to various extents. Enzymes I and II hydrolyse cyclic nucleotides at a greater rate than NAD+, whereas enzyme III hydrolyses NAD+ at a much greater rate than cyclic nucleotides. All three enzymes hydrolysed the artificial substrate bis-(p-nitro-phenyl) phosphate. 6. The enzymes do not require the addition of bivalent cations for activity. 7. Both enzymes I and II have optimum activity at pH6 with 3':5'-cyclic AMP and bis-(p-nitrophenyl) phosphate as substrates. The products of 3':5'-cyclic AMP hydrolysis were 3'-AMP and 5'-AMP, the ratio of the two products being different for each enzyme and varying with pH. 8. Theophylline inhibits enzymes I and II slightly, but other methyl xanthines have little effect. Enzymes I and II were competitively inhibited by many nucleotides containing phosphomonoester and phosphodiester bonds, as well as by Pi. 9. The possible significance of these phosphodiesterases in cyclic nucleotide metabolism in higher plants is discussed.     

Cyclic nucleotide phosphodiesterases in larval brain of wild type and dunce mutant strains of Drosophila melanogaster: isoenzyme pattern and activation by Ca2+/calmodulin

Like adult heads and whole flies, larval brains of wild type Drosophila melanogaster contain two major soluble cyclic nucleotide phosphodiesterases, forms I and II. Larval brains of the learning-defective mutant strain, dunceM11, contain only the form I enzyme. In both wild type and dunce strains the form I enzyme is activated by Ca2+/calmodulin. A time-dependent loss of this Ca2+ activation was observed.     

Inhibition by gossypol of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis

Gossypol, a polyphenolic binaphthalene-dialdehyde extracted from cotton plants which possesses male antifertility action in mammals, is a potent inhibitor of phospholipid-sensitive Ca2+-dependent protein kinase from pig testis. Gossypol inhibited Ca2+-dependent activity of the enzyme without affecting its basal activity. The IC50 value (concentration causing 50% inhibition) was 31 microM when lysine-rich histone was used as substrate. Kinetic analysis indicated that the compound inhibited the enzyme non-competitively with respect to ATP (Ki = 31 microM) or lysine-rich histone (Ki = 30 microM), and competitively with respect to phosphatidylserine (Ki = 2.1 microM). With Ca2+, irrespective of the presence or absence of 1,3-diolein, the compound lowered Vmax and increased the apparent Ka for Ca2+. The compound also inhibited phosphorylation by the enzyme of high-mobility-group 1 protein (one of the endogenous substrates in the testis for the enzyme located in nucleosome), with an IC50 value of 88 microM. These results suggested that a phospholipid-sensitive Ca2+-dependent protein phosphorylation system in the testis is involved in the regulation of spermatogenesis.     

Regulation of type I adenylyl cyclase by calmodulin kinase IV in vivo.

Type I adenylyl cyclase is a neurospecific enzyme that is stimulated by Ca2+ and calmodulin (CaM). This enzyme couples the Ca2+ and cyclic AMP (cAMP) regulatory systems in neurons, and it may play an important role for some forms of synaptic plasticity. Mutant mice lacking type I adenylyl cyclase show deficiencies in spatial memory and altered long-term potentiation (Z. Wu, S. A. Thomas, Z. Xia, E. C. Villacres, R. D. Palmiter, and D. R. Storm, Proc. Natl. Acad. Sci. USA 92:220-224, 1995). Although type I adenylyl cyclase is synergistically stimulated by Ca2+ and G-protein-coupled receptors in vivo, very little is known about mechanisms for inhibition of the enzyme. Here, we report that type I adenylyl cyclase is inhibited by CaM kinase IV in vivo. Expression of constitutively active or wild-type CaM kinase IV inhibited Ca2+ stimulation of adenylyl cyclase activity without affecting basal or forskolin-stimulated activity. Type I adenylyl cyclase has two CaM kinase IV consensus phosphorylation sequences near its CaM binding domain at Ser-545 and Ser-552. Conversion of either serine to alanine by mutagenesis abolished CaM kinase IV inhibition of adenylyl cyclase. This suggests that the activity of this enzyme may be directly inhibited by CaM kinase IV phosphorylation. Type VIII adenylyl cyclase, another enzyme stimulated by CaM, was not inhibited by CaM kinase II or IV. We propose that CaM kinase IV may function as a negative feedback regulator of type I adenylyl cyclase and that CaM kinases may regulate cAMP levels in some cells.     

In Vitro Phosphorylation of Bovine Adrenal Chromaffin Cell Tyrosine Hydroxylase by Endogenous Protein Kinases

Under phosphorylating conditions, addition of Ca2+ or cyclic AMP to the 100,000 g supernatant of purified bovine adrenal chromaffin cells increases both the incorporation of 32P into tyrosine hydroxylase and the activity of the enzyme. Combining maximally effective concentrations of each of these stimulating agents produces an additive increase in both the level of 32P incorporation into tyrosine hydroxylase and the degree of activation of the enzyme. The increased phosphorylation by Ca2+ is due to stimulation of endogenous Ca2+-dependent protein kinase activity and not inhibition of phosphoprotein phosphatases. When the chromaffin cell supernatant is subjected to diethylaminoethyl (DEAE) chromatography to remove calmodulin and phospholipids, tyrosine hydroxylase is no longer phosphorylated or activated by Ca2+; on the other hand, phosphorylation and activation of tyrosine hydroxylase by cyclic AMP are not affected. Subsequent replacement of either Ca2+ plus calmodulin or Ca2+ plus phosphatidylserine to the DEAE-fractionated cell supernatant restores the phosphorylation, but not activation of the enzyme. Reverse-phase HPLC peptide mapping of tryptic digests of tyrosine hydroxylase from the 100,000 g supernatant shows that the Ca2+-dependent phosphorylation occurs on three phosphopeptides, whereas the cyclic AMP-dependent phosphorylation occurs on one of these peptides. In the DEAE preparation, either cyclic AMP alone or Ca2+ in the presence of phosphatidylserine stimulates the phosphorylation of only a single phosphopeptide peak, the same peptide phosphorylated by cyclic AMP in the crude supernatant. In contrast, Ca2+ in the presence of calmodulin stimulates the phosphorylation of three peptides having reverse-phase HPLC retention times that are identical to peptides phosphorylated by Ca2+ addition to the crude unfractionated 100,000 g supernatant. Rechromatography of the peaks from each of the in vitro phosphorylations, either in combination with each other or in combination with each of the seven peaks generated from phosphorylation of tyrosine hydroxylase in situ, established that cyclic AMP, Ca2+/phosphatidylserine, and Ca2+/calmodulin all stimulate the phosphorylation of the same reverse-phase HPLC peptide: in situ peptide 6. Ca2+/calmodulin stimulates the phosphorylation of in situ peptides 3 and 5 as well. Thus, tyrosine hydroxylase can be phosphorylated in vitro by protein kinases endogenous to the chromaffin cell. Phosphorylation occurs on a maximum of three of the seven in situ phosphorylated sites, and all three of these sites can be phosphorylated by a Ca2+/calmodulin-dependent protein kinase.     

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+.     

The role of calcium ion as a mediator of the effects of angiotensin II, catecholamines, and vasopressin on the phosphorylation and activity of enzymes in isolated hepatocytes.

Angiotensin II, catecholamines, and vasopressin are thought to stimulate hepatic glycogenolysis and gluconeogenesis via a cyclic AMP-independent mechanism that requires calcium ion. The present study explores the possibility that angiotensin II and vasopressin control the activity of regulatory enzymes in carbohydrate metabolism through Ca2+-dependent changes in their state of phosphorylation. Intact hepatocytes labeled with [32P]PO43- were stimulated with angiotensin II, glucagon, or vasopressin and 30 to 33 phosphorylated proteins resolved from the cytoplasmic fraction of the cell by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. Treatment of the cells with angiotensin II or vasopressin increased the phosphorylation of 10 to 12 of these cytosolic proteins without causing measurable changes in cyclic AMP-dependent protein kinase activity. Glucagon stimulated the phosphorylation of the same set of 11 to 12 proteins through a marked increase in cyclic AMP-dependent protein kinase activity. The molecular weights of three of the protein bands whose phosphorylation was increased by these hormones correspond to the subunit molecular weights of phosphorylase (Mr = 93,000), glycogen synthase (Mr = 85,000), and pyruvate kinase (Mr = 61,000). Two of these phosphoprotein bands were positively identified as phosphorylase and pyruvate kinase by affinity chromatography and immunoprecipitation, respectively. Incubation of hepatocytes in a Ca2+-free medium completely abolished the effects of angiotensin II and vasopressin on protein phosphorylation but did not alter those of glucagon. Treatment of hepatocytes with angiotensin II, glucagon, or vasopressin stimulated phosphorylase activity by 250 to 260%, inhibited glycogen synthase activity by 50%, and inhibited pyruvate kinase activity by 30 to 35% (peptides) to 70% (glucagon). The effects of angiotensin II and vasopressin on the activity of all three enzymes were completely abolished if the cells were incubated in a Ca2+-free medium while those of glucagon were not altered. The results imply that angiotensin II, catecholamines, and vasopressin control hepatic carbohydrate metabolism through a Ca2+-requiring, cyclic AMP-independent pathway that leads to the phosphorylation of important regulatory enzymes.     

Interaction of platinum and palladium 5-sulfo-8-mercaptoquinoline complexes with sarcoplasmic reticulum Ca2+-dependent ATPase]

5-Sulfo-8-mercaptoquinoline complexes of platinum and palladium (complexes I and II) effectively inhibit Ca2+-dependent ATPase from sarcoplasmic reticulum. However, in contrast to K2PtCl4, K2PdCl4 and other previously investigated platinum and palladium complexes, they do not interact with the thiol groups of the enzyme. The inhibiting effects of complexes I and II are reversible and competitive with respect to ATP. In aqueous solutions complexes I and II decrease the fluorescence of tryptophane with a simultaneous shift in fluorescence towards the long-wave region. The same effect is exerted by the complexes on the fluorescence of tryptophane residues in Ca2+-dependent ATPase preparations. An addition of tryptophane to the enzyme preparations preincubated with complexes I and II partly restores the enzyme activity. It is assumed that the inhibiting effect of complexes I and II is due to their non-covalent interactions with the trytophane residues vicinal to the ATPase center.     

Inhibition of adenylate cyclase by transglutaminase-catalyzed reactions in pigeon erythrocyte ghosts

We report the occurrence in pigeon erythrocytes of a soluble Ca2+-dependent transglutaminase (TGase) activity. The effect of the erythrocyte ghost protein modifications, determined by TGase-catalyzed reactions, on adenylate cyclase, phospholipid methyltransferase I and II activities and on the lipidic matrix fluidity of the membrane was investigated by using a purified guinea pig liver TGase preparation. The results showed a significant inhibitory effect of such modifications both on the basal and on the variously stimulated (by NaF, Gpp(NH)p alone or in the presence of 1-isoproterenol) adenylate cyclase activity. By contrast, both the phospholipid methylation and the fluidity of the lipidic matrix of the membrane were unaffected by TGase-mediated reactions. These data suggest a new possible inhibitory mechanism of the cyclic AMP synthesis which might be triggered by the enhancement of the cytosolic Ca2+ concentration.