Purification and properties of adenosine 3′,5′-monophosphate phosphodiesterase from baker's yeast

Cyclic AMP phosphodiesterase from Saccharomyces cerevisiae was purified about 20,000-fold to homogeneity. The purified enzyme had a molecular weight of about 60,000 as estimated by gel filtration.The enzyme activity was optimal at pH 8.5–9.0 and was not stimulated by imidazole. Among cyclic 3′,5′-nucleotides, cyclic AMP was the most active substrate for the purified enzyme (K_m = 0.25 mM), but it was inhibitory at concentrations above 4 mm. N⁶,O^2′-dibutyryl cyclic AMP was not hydrolyzed at all.Unlike other cyclic AMP phosphodiesterases from various sources, the purified yeast enzyme did not require divalent metal ions for maximal activity and was rather inhibited in various degrees by added metal ions. The enzyme was not very sensitive to thiol inhibitors.The purified yeast enzyme was strongly inhibited by theophylline and slightly by caffeine. In contrast to the enzyme from S. carlsbergensis, the enzyme from S. cerevisiae was not inhibited at all by ATP or PP_i.The enzyme activity was not released into the growth medium, and the intracellular distribution studies indicated that the enzyme was located mainly in the cytosol fraction.     

Further studies on the properties of the rabbit reticulocyte adenosine 3',5'-cyclic monophosphate-dependent protein kinase I

The properties of cyclic AMP-dependent protein kinase I isolated from rabbit reticulocytes were further investigated. The enzyme catalyzes the phosphorylation of histone in the presence of ATP and Mg²⁺ and this reaction is stimulated by cyclic AMP. The pH optimum of the reaction was between 8.5 and 9.0, when assayed in the presence of cyclic AMP. No distinct pH optimum was observed in the absence of the cyclic nucleotide. The K_m values for ATP appeared to be very similar whether it was determined in the presence (K_m = 1.7 × 10⁻⁴m) or absence (K_m = 2.5 × 10⁻⁴m) of cyclic AMP. The rate of heat inactivation of the catalytic activity and the cyclic AMP binding activity of kinase I were found to be dependent on the presence of Mg²⁺, ATP, and/or cyclic AMP. In the presence of cyclic AMP, the rate of inactivation of the catalytic activity of kinase I at 53 ° was accelerated. On the other hand, the cyclic AMP binding activity appeared to be protected from heat inactivation by the cyclic nucleotide. When both ATP and Mg²⁺ were present in the heating mixture, no loss of catalytic and binding activities of kinase I were observed even up to 8 min of heating at 53 °. The cyclic AMP binding activity of kinase I was almost completely inhibited by mercuric acetate at a concentration of 1 mm, while the loss in catalytic activity was only 50%. These results substantiate our previous observation that kinase I contains two nonidentical subunits, a catalytic subunit and a cyclic AMP binding subunit.     

On the biochemical classification of yeast trehalases: Candida albicans contains two enzymes with mixed features of neutral and acid trehalase activities

Two enzymes endowed with trehalase activity are present in Candida albicans. The cytosolic trehalase (Ntc1p), displayed high activity in exponential phase regardless of the carbon source (glucose, trehalose or glycerol). Ntc1p activity was similar in neutral (pH 7.1) or acid (pH 4.5) conditions, strongly inhibited by ATP, weakly stimulated by divalent cations (Ca²⁺or Mn²⁺) and unaffected in the presence of cyclic AMP. The Ntc1p activity decreased in stationary phase, except in glycerol-grown cultures, but the catalytic properties did not change. In turn, the cell wall-linked trehalase (Atc1p) showed elevated activity in resting cells or in cultures growing on trehalose or glycerol. Although Atc1p is subjected to glucose repression, exhaustion of glucose in itself did not increased the activity. Significant Atc1p values could also be measured at neutral or acid pH, but Atc1p was insensitive to ATP, cyclic AMP and divalent cations. These results are in direct contrast with the current classification of yeast trehalases based on their optimum pH. They are also relevant in the light of the proposed use of trehalase inhibitors for the treatment of candidiasis.     

Cyclic AMP activation of a triglyceride lipase in broken cell preparations of rat heart

The effect of CAM [cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine (MIX)] on triacylglycerol (TG) lipase activity in extracts from heparin-perfused rat heart was determined. TG lipase activity in homogenate, 10,000g supernatant, 105,000g supernatant, ammonium sulfate supernatant, and the eluate from heparin-Sepharose was increased between 62 and 151% when incubated with a combination of 0.3 mM cyclic AMP, 5 mM MgCl2, and 2 mM ATP. The addition of Mg-ATP + cyclic AMP caused a greater activation of TG lipase in the various fractions than did Mg-ATP + MIX or cyclic AMP + MIX. These results suggest that activation may be mediated by the classical cyclic AMP-protein kinase cascade. Control and CAM-stimulated activities were increased by heparin and inhibited by NaCl and protamine sulfate. In the absence of serum in the assay, the CAM system caused a relatively greater stimulation of lipolytic activity in each fraction compared to when serum was present in the assay. However, the absolute values were 6.1 to 16.3-fold greater with serum in the assay than without serum. In a similar manner, TG lipase activity was stimulated by CAM between 1.75 and 4.26-fold at pH 7.4, and only between 1.62 and 2.51-fold at pH 8.1. However, the absolute values at pH 8.1 were 6.77 to 31.83-fold greater than those seen at pH 7.4. These data demonstrate, for the first time, the cyclic AMP activation of a TG lipase above basal levels in cell-free fractions of rat heart. It is intriguing to speculate that the intracellular fraction of lipoprotein lipase may play a role in the hormonal regulation of cardiac TG lipolysis.     

Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5′-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5′-monophosphate, and PP_i by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5′-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PP_i were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PP_i by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.     

Enzymes of cyclic nucleotide metabolism in invertebrate and vertebrate sperm.

Sperm from several invertebrates contained guanylate cyclase activity several-hundred-fold greater than that in the most active mammalian tissues; the enzyme was totally particulate. Activity in the presence of Mn²⁺ was up to several hundred-fold greater than with Mg²⁺ and was increased 3–10-fold by Triton X-100. Sperm from several vertebrates did not contain detectable guanylate cyclase. Sperm of both invertebrates and vertebrates contained roughly equal amounts of Mn²⁺-dependent adenylate cyclase activity; in invertebrate sperm, this enzyme was generally several hundred-fold less active than guanylate cyclase. Adenylate cyclase was particulate, was unaffected by fluoride, and was generally greater than 10-fold more active with Mn²⁺ than with Mg²⁺. Invertebrate sperm contained phosphodiesterase activities against 1.0 μm cyclic GMP or cyclic AMP in amounts greater than mammalian tissues. Fish sperm, which did not contain guanylate cyclase, had high phosphodiesterase activity with cyclic AMP as substrate but hydrolyzed cyclic GMP at a barely detectable rate. In sea urchin sperm, phosphodiesterase activity against cyclic GMP was largely particulate and was strongly inhibited by 1.0% Triton X-100. In contrast, activity against cyclic AMP was largely soluble and was weakly inhibited by Triton. The cyclic GMP and cyclic AMP contents of sea urchin sperm were in the range of 0.1–1 nmol/g. Sea urchin sperm homogenates possessed protein kinase activity when histone was used as substrate; activities were more sensitive to stimulation by cyclic AMP than by cyclic GMP.⁵     

GTP,as well as ATP,can act as a substrate for the intrinsic protein kinase activity of synaptic plasma membranes

Summary GTP as well as ATP can act as phosphate donor for the intrinsic protein kinase activity of synaptic plasma membranes. There are many similarities between the activities observed with ATP or GTP. Both need a divalent cation, Mg²⁺ being preferred, both are slightly inhibited by Na⁺, and more strongly by K⁺, both are inhibited by theophylline and adenosine. The K_m for GTP (0.13 mM) is similar to that ATP (0.12 mM). There are, however, some differences in properties. When GTP instead of ATP is the phosphate donor the pH optimum is 6.5 instead of 7.4. In addition NH ₄ ⁺ inhibits the transfer of phosphate from GTP but not from ATP. More importantly, cyclic AMP only stimulates the transfer of phosphate from ATP not from GTP. SDS gel electrophoresis reveals that similar membrane proteins are phosphorylated by GTP and ATP in the presence or absence of cyclic AMP. This suggests that there may be two different types of protein kinase in the synaptic plasma membrane which act on similar membrane proteins. One is stimulated by cyclic AMP and is specific to ATP while the other is unaffected by cyclic nucleotides and can use either ATP or GTP as phosphate donor.Deceased     

Rat thyroid phosphofructokinase. Comparison of the regulatory and molecular properties with those of rat muscle enzyme.

The kinetic and molecular properties of rat thyroid phosphofructokinase (specific activity 134 units/mg) were compared with those of rat muscle phosphofructokinase (specific activity 135 units/mg). Thyroid and muscle phosphofructokinase showed similar sedimentation patterns in sucrose density gradients; their affinity for DEAE-cellulose was similar but not identical. A comparison of the kinetic properties revealed differences in the pH optima. Striking differences in the kinetic properties were shown below pH 7.4; the thyroid enzyme was less inhibited by ATP or citrate and more sensitive to activation by cyclic 3':5'-AMP than the muscle enzyme. A study of the effects of some cyclic as well as linear mononucleotides, such as cyclic AMP, cyclic IMP, cyclic GMP, cyclic CMP, cyclic UMP, 5'-AMP, and 3'-AMP on thyroid phosphofructokinase showed that at concentrations as low as 1 micrometer only cyclic AMP and cyclic IMP were able to activate thyroid enzyme in the presence of low fructose-6-P and high ATP concentrations.     

Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate.

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.     

Effects of 5-hydroxytryptamine, cyclic AMP, AMP, and fructose 2,6-bisphosphate on phosphofructokinase activity in Hymenolepis diminuta

1. 5-HT (10(-4) M) had no effect on the activity of phosphofructokinase in Hymenolepis diminuta. Concentrations of ATP above 33 microM inhibited PFK activity; AMP and cyclic AMP relieved this inhibition. 2. Local levels of cyclic AMP may be indirectly modulated by NaF, guanylyl imidophosphate, or 5-HT in the presence of GTP, which stimulates adenylyl cyclase activity x2 in H. diminuta homogenates. 3. Fructose 2,6-bisphosphate (F2BP), a physiological regulator of PFK activity in rat liver, also relieved ATP-induced inhibition of PFK. F2BP was present in supernatants from the worms at about 20 mumol/g wet wt. 4. 5-HT may cause an increase in the rate of glycolysis in H. diminuta by elevating either cyclic AMP and/or AMP levels; these nucleotides can in turn increase PFK activity.     

Inhibition of adenylate cyclase in human blood platelets by 9-substituted adenine derivatives.

A series of 9-substituted adenine derivatives inhibited adenylate cyclase activity (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) of a particulate preparation of human blood platelets. A 3--6 fold elevation of adenylate cyclase activity by prostaglandin E1 (PGE1) was inhibited in a concentration-related manner by 9-(tetrahydro-5-methyl-2-furyl) adenine (SQ 22,538), 9-(tetrahydro-2-furyl) adenine (SQ 22,536), 9-cyclopentyladenine (SQ 22,534), 9-furfuryladenine (sQ 4647) and 9-benzyladenine (SQ 218611). The I50 values ranged from 21 microM for SQ 22,538 to 140 microM for SQ 21,611. These same adenine derivatives reversed the inhibition by PGE1 of ADP-induced aggregation and the PGE1-stimulated elevation of adenosine 3':5'-monophosphate (cyclic AMP). The reversal of platelet aggregation inhibition by SQ 22,536 and SQ 4647 was concentration-related with I50 values of 30 microM in each case, whereas SQ 22,534 and SQ 21,611 reversed inhibition by 30% at 100 microM. SQ 22,536, SQ 22,534 and SQ 21,611 also blocked the increase in cyclic AMP levels in a concentration-related manner with I50 values of 1, 4 and 60 microM, respectively. SQ 4647 inhibited the elevation of cyclic AMP by more than 85% at 1000 microM. The adenine derivatives had no effect on platelet aggregation or on cyclic AMP levels in the absence of PGE1. These results provide additional evidence that the inhibition of platelet aggregation by PGE1 is mediated by cyclic AMP.     

Synthesis of adenosine 3′,5′-monophosphate by guanylate cyclase,a new pathway for its formation

The 105 000 × g supernatant fractions from homogenates of various rat tissues catalyzed the formation of both cyclic GMP and cyclic AMP from GTP and ATP, respectively. Generally cyclic AMP formation with crude or purified preparations of soluble guanylate cyclase was only observed when enzyme activity was increased with sodium azide, sodium nitroprusside, N-methyl-N′-nitro-N-nitrosoguanidine, sodium nitrite, nitric oxide gas, hydroxyl radical and sodium arachidonate. Sodium fluoride did not alter the formation of either cyclic nucleotide. After chromatography of supernatant preparations on Sephadex G-200 columns or polyacrylamide gel electrophoresis, the formation of cyclic AMP and clycic GMP was catalyzed by similar fractions. These studies indicate that the properties of guanylate cyclase are altered with activation. Since the synthesis of cyclic AMP and cyclic GMP reported in this study appears to be catalyzed by the same protein, one of the properties of activated guanylate cyclase is its ability to catalyze the formation of cyclic AMP from ATP. The properties of this newly described pathway for cyclic AMP formation are quite different from those previously described for adenylate cyclase preparations. The physiological significance of this pathway for cyclic AMP formation is not known. However, these studies suggest that the effects of some agents and processes to increase cyclic AMP accumulation in tissue could result from the activation of either adenylate cyclase or guanylate cyclase.     

Cyclic nucleotide phosphodiesterase activities from isolated fat cells: correlation of subcellular distribution with effects of nucleotides and insulin

Cyclic nucleotide phosphodiesterasc activities were determined in fractions of fat cell homogenates, prepared either by differential centrifugation or by centrifugation on discontinuous sucrose gradients.In the supernatant fraction (150,000g supernatant in 0.25 m sucrose, or 92,000g supernatant in 0.32m sucrose): (a) there was 70% of the cyclic AMP phosphodiesterase activity of the whole homogenate, and over 90% of the cyclic GMP phosphodiesterase activity; (b) double reciprocal kinetic plots were nonlinear for both substrates; (c) cyclic (GMP, 0.02-2 μm, activated hydrolysis of 10 μm cyclic AMP; (d) 25 or 50 μm cyclic GMP noncompetitively inhibited hydrolysis of 5–20 μm cyclic AMP (K_i = 38 μm); (e) cyclic AMP, 0.1 μm, slightly activated hydrolysis of 10 μm cyclic GMP; (f) 10 or 20 μm cyclic AMP competitively inhibited hydrolysis of 5–20 μm cyclic GMP (K_i = 18 μm).In the particle fraction (1000g, 1000-16,000g, and 16,000–150,000g pellets in 0.25m sucrose, or 0.8-1.2m sucrose interface at 92,000g): (a) there was 30% of the cyclic AMP phosphodiesterase activity of whole homogenate, but less than 5% of the cyclic GMP phosphodiesterase; (b) the double reciprocal kinetic plot of hydrolysis of cyclic AMP was nonlinear; (c) cyclic GMP, 0.02-2μm, did not affect hydrolysis of 10 μm cyclic AMP; (d) 5 or 10 μm cyclic GMP competitively inhibited hydrolysis of 5–20 μm cyclic AMP (K_i = 1.9 μm).Incubation of fat cells with insulin, 40 ng/ml, increased the maximum velocity of particulate high-affinity cyclic AMP phosphodiesterase, but did not affect the supernatant activity. Addition of insulin after homogenization of the cells had no effect on any phosphodiestesterase activity.     

Studies on nucleotidases in plants. Dimerization of the crystalline mung bean nucleotide pyrophosphatase by 5'-adenosine monophosphate and the properties of the dimerized enzyme.

The addition of AMP to the crystalline and homogeneous mung bean nucleotide pyrophosphatase [EC 3.6.1.9]altered its electrophoretic mobility. AMP was tightly bound to the enzyme and was not removed on passage through a column of Sephadex G-25 or on electrophoresis. The molecular weight of the native and AMP-modified enzymes were 65,000 and 136,000, respectively. The properties of the native enzyme such as the pH (9.4) and temperature (49 °C) optima, inhibition by EDTA, reversal of EDTA-inhibition by Zn²⁺ and Co²⁺, were not altered on dimerization by AMP. The AMP-modified enzyme had a linear time-course of reaction, unlike the native enzyme which exhibited a biphasic time-course of reaction. The AMP-modified enzyme was irreversibly denatured by urea. AMP concentrations larger than 100 μM inhibited linearly the activity of the AMP-modified enzyme. ADP and ATP inhibited the activity in a sigmoidal manner. K_m and V of the native and AMP-modified enzymes were, 0.25 mm and 0.58 mm; and 3.3 and 2.5, respectively.     

Purification and characterization of an acid phosphatase from Trichoderma harzianum

An acid phosphatase from Trichoderma harzianum was purified in a single step using a phenyl-Sepharose chromatography column. A typical procedure showed 22-fold purification with 56% yield. The purified enzyme showed as a single band on SDS-PAGE with an apparent molecular weight of 57.8 kDa. The pH optimum was 4.8 and maximum activity was obtained at 55°C. The enzyme retained 60% of its activity after incubation at 55°C for 60 min. The K _m and V _max values for p-nitrophenyl phosphate (p-NPP) as a substrate were 165 nM and 237 nM min^?1, respectively. The enzyme was partially inhibited by inorganic phosphate and strongly inhibited by tungstate. Broad substrate specificity was observed with significant activities for p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate and phenyl phosphate.     

Role of Silicon in Diatom Metabolism : Cyclic Nucleotide Levels, Nucleotide Cyclase, and Phosphodiesterase Activities during Synchronized Growth of Cylindrotheca fusiformis

Adenylate cyclase, guanylate cyclase, and the cyclic nucleotide phosphodiesterases of Cylindrotheca fusiformis were characterized in crude and partially purified preparations. Both cyclases were membrane-bound and required Mn²⁺ for activity, though Mg²⁺ gave 50% activity with adenylate cyclase. Properties of adenylate cyclase were similar to those of higher eukaryotic cyclases in some respects, and in other respects were like lower eukaryotic cyclases. Guanylate cyclase was typical of other lower eukaryotic enzymes.

Two phosphodiesterase activities were found, one selective for cyclic AMP, the other for cyclic GMP. The 5′-nucleoside monophosphate was the major product of both activities and each of the enzymes had distinctive divalent cation requirements, pH optima, and kinetic parameters. Both phosphodiesterases were similar to those of other lower eukaryotes with one notable difference: the cyclic AMP enzyme was inhibited by calcium.

Changes in the cyclic nucleotide levels were quantitated in light-dark and silicon-starvation synchronized cultures using a more sensitive radioimmunoassay than used in a previously published study (Borowitzka and Volcani 1977 Arch Microbiol 112: 147-152). Contrary to the previous report, the cyclic GMP level did not change significantly in either synchrony. The cyclic AMP level increased dramatically very early in the period of DNA replication with the peak cyclic AMP accumulation substantially preceding that of DNA synthesis in both synchronies. There was no significant change in the activity of either cyclase or either phosphodiesterase during either synchrony. Thus, the mechanism for the rise in cAMP level remains unclear.

     

Identification and solubilization of a cAMP-independent protein kinase from mouse L-cell smooth membranes

1. Smooth membranes have been prepared from mouse L-cells and found to contain an endogenous protein kinase activity. 2. The enzyme(s) responsible for this activity use ATP, but no other nucleoside triphosphates, to phosphorylate endogenous membrane proteins as well as exogenously-added protein substrates such as phosvitin and casein. 3. Mg2+ is required for enzyme activity, maximal activity is observed at pH 7.5-8.0 and the kinase is not dependent on, or stimulated by, cyclic 3'-5' AMP. 4. The kinase activity is not decreased by the Walsh heat-stable inhibitor of cyclic 3'-5' AMP-dependent protein kinases. 5. Fifty percent or more of the membrane-associated kinase activity can be solubilized by extracting membranes with buffer containing 0.6 M NaCl. 6. The solubilized enzyme resembles the membrane-associated activity in its Mg2+ requirement, pH optimum and independence of cyclic 3'-5' AMP. 7. Phosvitin and casein are better exogenous substrates than histones or protamine for phosphorylation by the enzyme in either the membrane-associated or solubilized state.     

Redox modulation of the response of NADH oxidase activity of rat liver plasma membranes to cyclic AMP plus ATP

NADH oxidase activity of rat liver plasma membranes was inhibited by lowconcentrations (1-100 nM) of ATP. The inhibition was amplified by additionof nanomolar concentrations (0.1-10) of cyclic AMP. The inhibition wascomplex and related to a marked increase in the Km for NADH at high NADHconcentrations together with a concomitant decrease in the Vmax. In theabsence of added or residual ATP, cyclic AMP was without effect. Theresponse of cyclic AMP + ATP was inhibited by low concentrations of theselective inhibitor of cyclic AMP-dependent protein kinase, H-89 but not bystaurosporin. The Vmax but not the Km was modified by treating the plasmamembranes with a mild oxidizing agent, N-chlorosuccinamide, or with thereducing agent, dithiothreitol. In the presence of dithiothreitol, the Vmaxwas reduced by cyclic AMP + ATP. In contrast, in the presence ofN-chlorosuccinamide, the Vmax was increased by cyclic AMP + ATP relative tocyclic AMP + ATP alone. Thus, the effect of cyclic AMP + ATP on the Vmaxcould be either an increase or a decrease depending on whether the membraneswere oxidized or reduced. The results demonstrate regulation of NADH oxidaseactivity of rat liver plasma membranes through cyclic AMP-mediatedphosphorylation by membrane-located protein kinase activities where thefinal response is dependent on the oxidation-reduction status of the plasmamembranes.     

Evidence that the mitochondrial activator of phosphorylated branched-chain 2-oxoacid dehydrogenase complex is the dissociated E1 component of the complex

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate). The half-maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon-stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 microgram/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon-stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.