Dependent Phosphodiesterase Research Articles

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 Ca 2+-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 Ca 2+-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 K m for cyclic GMP and a decrease in V . In the presence of calcium ions and purified activator protein, the Ca 2+-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca 2+-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was mimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca 2+-independent activation of Ca 2+-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca 2+-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca 2+-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phosphodiesterase activity.

Effects of acute hyperbaric exposure on the concentration of alanine and other metabolites in mouse tissues.

genate and subcellular fractions designated MI 0.8, Sup and M3 (Lapetina & Michell, 1973) and M,P (Friedel et a/., 1969) were 3.1, 14.7, 0.9, 1.4 and 12.4pg of P/h:pmol of NADH/min respectively. If, however, the true enzyme activities were assayed (lactate dehydrogenase with 0.1 %Triton X-100; phosphatidylinositol phosphodiesterase with 7m~-phosphatidylinositol, 2m~-Ca’+ and 3 mg of deoxycholate/ml) the corresponding ratios are 23.0, 23.8, 12.0, 20.0 and 27.4 respectively. The clear difference between the cell supernatant (Sup) and synaptosomal supernatant (M3) precludes any unequivocal demonstration of a bound activity aswe can never be sure that a given supernatant is of exactly the same enzyme composition as that entrapped in a membrane fraction. In summary, our data show that the reported differences between the Ca2+-dependent phosphatidylinositol phosphodiesterase activities of membrane and supernatant brain fractions can be explained by the properties of the soluble enzyme alone; there is no reason to assume a membrane-bound enzyme from present evidence. Additional important conclusions are, first, that the soluble activity can only hydrolyse phosphatidylinositol in a membrane if the membrane is suitably activated, e.g. by deoxycholate, but in uivo perhaps by some other amphiphilic compound; secondly, that once hydrolysis is so activated the rate is unlikely to be limited by Ca’+.

Involvement of Calcium in Cyclic Nucleotide Metabolism in Human Vascular Smooth Muscle

When cyclic nucleotide phosphodiesterase was purified from isolated smooth muscle layer of human aorta by DEAE-cellulose column chromatography, separated cyclic GMP phosphodiesterase activity was markedly stimulated in the presence of 10-20 micrometer of Ca2+ by a protein modulator which has similar physicochemical properties to troponin C. Synthetic compound, N-(6-aminohexl)-5-chloro-1-naphthalensulfonamide, which produced relaxations of arteries contracted by prostaglandin F2alpha or KCl was found to inhibit selectively this Ca2+-dependent cyclic GMP phosphodiesterase. This compound produced inhibition of superprecipitation of myosin B system obtained from mouse skeletal muscle and also inhibited adenosine triphosphatase activity of myosin B. Our data suggest that calcium is involved through a protein modulator in cyclic nucleotide metabolism of vascular smooth muscle and that the calcium-dependent protein modulator probably participates in the regulation of contractile response of vascular smooth muscle by affecting actomyosin ATPase activity.

Inhibitors of cyclic nucleotide phosphodiesterases produced by streptomyces. IV. Inhibition of cyclic nucleotide phosphodiesterases by reticulol.

Inhibition mechanism of rat cerebral cortex cyclic nucleotide phosphodiesterases (PDE) by reticulol was investigated. The inhibition of PDE by reticulol was not reduced in the presence of excess PDE activating factor (PAF) or/and Ca2+ ion. Reticulol showed lower Ki values for Ca2+-PAF dependent PDE than for Ca2+ independent PDE.

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: Comparison of adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate as substrates

A Ca 2+-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca 2+ and microgram quantities of a purified Ca 2+-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K m values of 180 and 8 μ m, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca 2+. This activity exhibited apparent K m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca 2+-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior. Apparent K m values of the Ca 2+-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K m for Mg 2+ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg 2+. At low cyclic nucleotide substrate concentrations i.e., 2.5 μ m, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.

Calcium-dependent cyclic nucleotide phosphodiesterase from glial tumor cells

A Ca 2+-dependent cyclic nucleotide phosphodiesterase has been identified in homogenates of C-6 glial tumor cells. The Ca 2+-dependent phosphodiesterase was resolved by ECTEOLA-cellulose chromatography into two fractions. One fraction contained a protein regulator of the enzyme which was identical to a homogeneous Ca 2+-binding protein (CDR) from porcine brain by the criteria of electrophoretic migration, biological activity, heat stability, and behavior in diverse chromatographic systems. The second fraction contained deactivated enzyme (CDR-dependent phosphodiesterase) which regained full activity upon the readdition of both Ca 2+ and CDR. In subcellular fractionation experiments both the CDR and the Ca 2+-dependent phosphodiesterase were predominantly located in the 100,000 g supernatant fraction. The apparent K m values of the phosphodiesterase for cyclic AMP (cAMP) and cyclic GMP (cGMP) were 10 and 1.2 μ m, respectively, when CDR was not rate limiting. Minor increases in the apparent K m for cAMP were observed at rate-limiting concentrations of CDR. At the ratio of CDR to CDR-dependent enzyme present in the C-6 cell homogenate, half-maximal activation was conferred by 4 μ m Ca 2+ for the hydrolysis of 25 μ m cGMP and by 8 μ m Ca 2+ for the hydrolysis of 25 μ m cAMP. Increased ratios of CDR to CDR-dependent phosphodiesterase increased the sensitivity of the enzyme to Ca 2+. The enzyme was more sensitive to CDR with cGMP as substrate than with cAMP, and more sensitive at high than at low cyclic nucleotide substrate concentrations. The quantity of enzyme in the assay also influenced the amount of CDR required for half-maximal activation.

Mechanism of stimulation of Ca 2+ plus Mg 2+-dependent phosphodiesterase from rat cerebral cortex by the modulator protein and Ca 2+

The activity of phosphodiesterase (“Ca 2+ plus Mg 2+-dependent” phosphodiesterase) of a preparation from brain was found to depend on the presence of both Ca 2+ and a protein factor called modulator. It was shown by gel filtration that the active enzyme-modulator complex (MW, about 200,000) was formed from the modulator (MW, 28,000) and an inactive enzyme (MW, about 150,000) in the presence of Ca 2+. When EGTA was added, this active enzyme-modulator complex dissociated into inactive enzyme and modulator. These results, together with the finding of Teo and Wang that Ca 2+ binds to the modulator, could explain the stimulatory effect of Ca 2+ on this enzyme as follows: The “Ca 2+ plus Mg 2+-dependent” phosphodiesterase may exist as the inactive free form in equilibrium with the active enzymemodulator (Ca 2+) complex, and Ca 2+, through binding to the modulator, may shift the equilibrium towards formation of the active enzyme-modulator (Ca 2+) complex, thereby increasing the activity of the mixture. On decreasing the concentration of Ca 2+, the process is reversible.

Cyclic 3′,5′-AMP phosphodiesterase of human blood lymphocytes

We have specified the main kinetic parameters of human blood lymphocyte cyclic 3′,5′-AMP phosphodiesterase using a crude extract (34 000 × g supernatant). 1. 1.|The pH optimum was found to be about 8.0 and the activity is classically inhibited by methyl-xanthines. Mg 2+ fulfills the cation requirement. Integrity of SH groups is essential. 2. 2.|Ca 2+ at low concentration (1 · 10 6-1 · 10 −4 M) has a stimulatory effect in the presence of Mg 2+ and a (Ca 2+ + Mg 2+)-dependent phosphodiesterase is assumed to occur. 3. 3.|Lymphocyte cyclic 3′,5′-AMP phosphodiesterase is neither subject to inhibition nor to stimulation by cyclic 3′,5′-GMP. 4. 4.|Activity-substrate concentration relationship revealed a low ( K m = 20 μM) and a high ( K m = 0.72 μM) affinity for cyclic AMP, suggesting the existence of several molecular forms of the enzyme. 5. 5.|We failed to observe cyclic 3′,5′-IMP production from cyclic 3′,5′-AMP in a lymphocyte homogenate, thus proving the unique degradative conversion of cyclic AMP into 5′-AMP. 6. 6.|None of the blastogenic or interferon inducers investigated (phytohaemagglutinins from Phaseolus vulgaris and Pisum sativum, streptolysin O, lipopolysaccharides from Salmonella typhi endotoxin) has been found to be a cyclic AMP phosphodiesterase effector at physiologically active concentrations. Such a property is discussed in connection with present researches on the role of cyclic 3′,5′-nucleotides in specific and non specific immunity processes.

Cyclic 3′,5′-nucleotide phosphodiesterase, IV. Two enzymes with different properties from brain

Two active peaks of phosphodiesterase, I and II, were resolved by a gel filtration column chromatography of the high speed supernatant of brain extract. The peak II represented Ca ++ plus Mg ++ dependent phosphodiesterase, the occurence of which in the supernatant of brain extract had been reported (1, 3), while the peak I may be called as “Ca ++ independent” and Mg ++ dependent phosphodiesterase from its nature. The former decomposed cyclic AMP, cyclic GMP, and cyclic UMP with the stimulatory effect of Ca ++ion. The latter, decomposing cyclic GMP at the comparable rate to cyclic AMP, showed negligible activity to cyclic UMP.

Calcium dependent phosphodiesterase activity and its activating factor (PAF) from brain: Studies on cyclic 3′,5′-nucleotide phosphodiesterase (III)

Crude extract from rat brains revealed two different activities of cyclic 3′,5′-nucleotides phosphodiesterase, namely the basal activity and the calcium dependent activity, and the total activity was shown as the sum of these two. The calcium dependent enzyme activity required for a heat-stable, nondialyzable factor (PAF) present in the brain extract. Regulation of calcium ion on the enzyme activity, in the presence of PAF, was observed within the range of pCa 5.9 to 4.9 and seems to be the physiological mechanism.

Partial purification and properties of A K +- and Mg 2+-dependent phosphodiesterase from Salmonella typhimurium

A phosphodiesterase (orthophosphoric diester phosphohydrolase, EC 3.1.4.1) which has been partially purified from the lysate of P22(C 1)-infected spheroplasts of Salmonella typhimurium is dependent on either K + or Mg 2+ for its activity and displays maximal activity in the presence of both. The enzyme is practically free from interfering activities and specifically hydrolyses single-stranded polyribonucleotides leading to the production of 5′-nucleotides. The homopolymers are hydrolysed in the following sequence: poly A > poly U > poly C . Poly C and tRNA are poorly hydrolysed, and poly I is not hydrolysed at all. The enzyme is most active at neutral or slightly alkaline pH. It is thermolabile but could be protected against heat denaturation by native and denatured DNA's as well as by poly A. During the hydrolysis of homopolymers no oligonucleotides could be detected as intermediate products, which indicates the exonucleolytic nature of the enzyme. Though there are some differences in properties, the behaviour of this phosphodiesterase is similar to that of ribonuclease II of Escherichia coli.