Variations in the levels of cyclic adenosine 3':5'-monophosphate and in the activities of adenylate cyclase and cyclic adenosine 3':5'-monophosphate phosphodiesterase during aerobic morphogenesis of Mucor rouxii

Particulate cell fractions of mycelium of Mucor rouxii contain adenylate cyclase activity which can be partially solubilized by 2% Lubrol PX. The enzyme requires Mn²⁺ and its activity is not modified by NaF or guanosine nucleotides. Mycelial extracts also contain cyclic adenosine 3′:5′-monophosphate phosphodiesterase activity, 60% of which is soluble. This activity shows characteristic low K_m (1 μm) for cyclic AMP and does not hydrolyze cyclic guanosine 3′:5′-monophosphate. It requires Mn²⁺ ions for maximal activity and is not inhibited by methylxanthines or activated by imidazole. Both enzymatic activities vary during the aerobic life cycle of the fungus. The spores have the highest levels of adenylate cyclase and cAMP phosphodiesterase, which decrease during the aerobic development. At the round cell stage, phosphodiesterase activity reaches 40% of the activity of the spores and varies only slightly thereafter. At this stage the specific activity of adenylate cyclase is 25% of the activity of ungerminated spores, and from this stage on, the activity increases up to the end of the logarithmic phase. Intracellular levels of cyclic AMP have been measured during aerobic germination. The variations of the intracellular level are tentatively explained by unequal variations in the activities of adenylate cyclase and cyclic AMP phosphodiesterase. A continuous increase of the extracellular cyclic AMP level during aerobic development has also been found, which cannot be accounted for solely by variations in the cyclase and diesterase activities.     

Factors affecting interconversion between kinetic forms of ribulose diphosphate carboxylase-oxygenase from spinach

Ribulose diphosphate carboxylase was found to exist in two distinct kinetic forms in spinach leaf extracts. One form displayed an apparent K_m for CO₂ in excess of 200 μm and is likely to be the form purified and studied by many previous workers. However, if leaf extracts were prepared in the presence of Mg²⁺ and atmospheric levels of CO₂, the recently described high-affinity form was obtained. It had a K_m for CO₂ of about 20 μm, was quite stable even at 25 °C, and its properties were consistent with it being the form which operates in photosynthesis in vivo. Mg²⁺ was also able to convert the high-K_m (CO₂) form to the low-K_m (CO₂) form when it was added to an extract which had been prepared in its absence. Mg²⁺ was more effective in causing this conversion if bicarbonate was added as well. This activating effect of bicarbonate is a probable cause of previously reported apparent homotropic effects of bicarbonate on ribulose diphosphate carboxylase activity. It is possible that the apparently high-K_m (CO₂) form is not intrinsically active and appears to have activity only by virtue of the low-K_m (CO₂) form produced by contact with Mg²⁺ and bicarbonate (or CO₂) during the course of the assay. Extracts prepared with ribose 5-phosphate in the absence of Mg₂₊ also showed low-K_m (CO₂) carboxylase activity initially, but the presence of this sugar phosphate was deleterious during storage at 25 °C, where it promoted conversion to the apparently high-K_m (CO₂) form.Effects on the affinity of ribulose diphosphate carboxylase for CO₂ were paralleled by effects on the activity of the associated ribulose diphosphate oxygenase. Treatments which produced the low-K_m (CO₂) form of the carboxylase also resulted in high oxygenase activity, and it is possible that the apparently high-K_m (CO₂) form of the carboxylase has little, if any, oxygenase activity associated with it.The carboxylase and oxygenase activities of the low-K_m (CO₂) form showed broad and quite similar responses to pH variation, and the oxygenase had a K_m for O₂ of 0.22 mm.The stability of the low-K_m (CO₂) form in the presence of Mg²⁺ and bicarbonate was quite sufficient for it to be partially purified by Sepharose chromatography. The significance of the low-K_m (CO₂) form is discussed with respect to activation of photosynthesis by Mg²⁺.     

Adenylate cyclase in cilia from Paramecium: Localization and partial characterization

A particulate adenylate cyclase was identified in the excitable ciliary membrane from Paramecium tetraurelia. MnATP was preferentially used as substrate, the K_m was 67 μM, V_max was 1 nmol cAMP.min^?1.mg^?1, a marked temperature optimum of 37°C was observed. Adenylate cyclase was not inhibited by 100 μM EGTA or 100 μM La³⁺, whereas under these conditions guanylate cyclase activity was abolished. Fractionation of ciliary membrane vesicles by a Percoll density gradient yielded two vesicle populations with adenylate cyclase activity. In contrast, calmodulin/Ca-dependent guanylate cyclase was associated with vesicles of high buoyant density only.     

Conversion of a NADPH-dependent aldehyde reducing enzyme into aldose reductase

• 1.1. Aldose reductase, aldehyde reductase and high-K_m, aldose reductase were purified from the inner medulla of dog kidney.
• 2.2. Compared with aldose reductase, high-K_m aldose reductase had a lower isoelectric point, a lower activity for aldo-sugars and a lower sensitivity for aldose reductase inhibitors, and it was not activated by sulfate ions. Both reductases had the same molecular weight (38,500) and immunochemical properties.
• 3.3. High-K_m aldose reductase was easily converted into an aldose reductase-like enzyme, namely a generated reductase upon incubation in neutral buffer solution.
• 4.4. The generated reductase was identical with aldose reductase with respect to the isoelectric point, substrate specificity, activation by sulfate ions and IC₅₀ values for aldose reductase inhibitors. The generated reductase revealed immunochemical identity with aldose reductase as well as high-K_m aldose reductase.

     

Activation of purified guanylate cyclase by nitric oxide requires heme comparison of heme-deficient,heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containg forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 μM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent K_m for GTP in the presence of excess Mn²⁺ or Mg²⁺ was 10 μM and 85–120 μM, respectively, for unactivated guanylate cyclase. The apparent K_m for GTP in the presence of Mn²⁺ was not altered but the K_m in the presence of Mg²⁺ was lowered to 58 μM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg²⁺ or Mn²⁺. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO nitroso compounds.     

Alterations of cAMP metabolism and hormone responsiveness of cloned differentiated rat liver cells (RL-PR-C) upon spontaneous transformation

In normal Rat Liver Primary Culture (RL-PR-C) liver cells, cAMP was low prior to confluency, then rose continuously as cells became contact inhibited. In contrast, spontaneously transformed RL-PR-C cells did not become contact inhibited, and cAMP decreased steadily with increasing cell density. Normal cells released large amounts of cAMP into the extracellular fluid at all densities, while transformed cells did not do so at any density. Neither exogenous db-cAMP nor phosphodiesterase inhibitors reversed the uncontrolled growth of transformed cells, nor did conditioned media from contact-inhibited normal cells.While both normal and transformed RL-PR-C hepatocytes produced large amounts of cAMP in response to epinephrine and cholera toxin, transformed cells were much more sensitive to these agents; however, only normal cells responded to glucagon. Although the plasma membrane adenylate cyclase of transformed hepatocytes responded better than did that of normal cells to epinephrine, cholera toxin and fluoride, the basal cyclase activity of transformed cells was only about half that of normal cells. The adenylate cyclase of transformed cells did not respond to glucagon, although the number of glucagon receptors of such cells far exceeded that of normal cells. The V_max of cyclic nucleotide phosphodiesterase of normal hepatocytes was five times that of transformed cells, although the K_m was unchanged.The data indicate that spontaneous transformation of diploid differentiated RL-PR-C hepatocytes leads to cultural hormone receptor and cAMP changes similar to those seen in undifferentiated fibroblasts and other cells transformed by viruses and chemical carcinogens. Although there are significant changes in various parameters of cAMP metabolism upon transformation, decreased cAMP per se does not seem to be responsible for transformation. Furthermore, it is possible that following transformation, these hepatocytes lose some factor necessary for coupling of the glucagon receptor to adenylate cyclase.     

Fatty acid esters of testosterone in rat brain: identification, distribution, and some properties of enzymes which synthesize and hydrolyze the esters

[4-¹⁴C]Testosterone was converted to an unknown compound with a much higher R_f on thin layer chromatogram than the substrate when it was incubated with a rat brain microsomal preparation. Evidence from its mass, infrared, and ultraviolet spectra indicated that the enzymic product is a mixture of fatty acid esters of testosterone. Saponification of the product yielded testosterone and a mixture of C_12:0, C_14:0, C_16:0, C_18:0, and C_18:1 fatty acids. The enzymic product was identical to testosterone laurate and testosterone stearate which were synthesized chemically. The enzyme system had a pH optimum at 4.9 with acetate buffer. The apparent K_m was 8.3 × 10^?5m for testosterone and 5.0 × 10^?5m for palmityl CoA. An enzyme which hydrolyzes testosterone[1-¹⁴C]oleate was also detected in rat brain. Most of this activity was in the nuclear and mitochondrial fractions. This enzyme had an optimum pH at 6.5 with phosphate buffer and its apparent K_m was 2.1 × 10^?4m. A low level of synthetic activity was found in fetal brain tissue which increased and reached a maximum at 3 weeks of age. The synthetic activity rapidly decreased with further increase in age. Hydrolytic activity was nearly undetectable in fetal rat brain, increased gradually until the animal reaches 3 weeks old, and remained at this level. Both synthetic and hydrolytic enzyme activities were higher in the brain than in other tissues examined.     

Phosphodiesterase activity and cyclic AMP content during early germination ofMucor rouxii spores

Incubation ofMucor rouxii spores in complex medium under aerobic conditions resulted in a very rapid transient increase in the level of soluble, low-K_m cAMP phosphodiesterase. Maximum activity was reached after 2–4 min. Simultaneously, the cAMP content increased quickly, reproducing the profile exhibited by phosphodiesterase. Activation of the enzymein vitro by a cAMP-dependent phosphorylation process showed that its stimulation was minimal at those times when its activity was highest. The correlation between cellular cAMP content, phosphodiesterase activity, and sensitivity of the enzyme to activation by phosphorylation suggests that thein vivo regulation of phosphodiesterase activity by cAMP-dependent phosphorylation may be the mechanism for shutoff of the cAMP signal elicited by glucose.     

Plasma membrane-mediated effects of extracellular pH on the growth of neuroblastoma cells

Mouse neuroblastoma cells (Neuro-2A) were cultured at pH values between 6.5 and 8.0. Acidification of the culture medium resulted in a reversible inhibition of growth. To explore the possibility that the effect of extracellular pH on growth is mediated by the plasma membrane, plasma membrane microviscosity, cellular cAMP levels and adenylate cyclase activities were measured after exposing the cells to various pH levels. Upon lowering the extracellular pH from 7.6 to 6.5, plasma membrane microviscosity decreases within a few hours to 50%. Concomitantly cellular cAMP levels increase more than three-fold, while adenylate cyclase activity is enhanced three-fold as well, accompanied by a small increase in K_m of the enzyme. All these effects are reversible. These results are compatible with the idea that the growth-regulatory role of the extracellular pH is mediated at the level of the plasma membrane, probably by altered membrane-adenylate cyclase interactions.     

Effect of hormonal and neuronal agents on adenylate cyclase from smooth muscle of the gastric antrum

Smooth muscle adenylate cyclase of a membrane preparation of canine gastric antrum has been characterized, and the effect of hormonal and neuronal agents examined. The enzyme is active in the presence of Mg²⁺ or Mn²⁺, but is inhibited by Ca²⁺. The K_m is 0.5 mM ATP, similar to the K_m of skeletal muscle adenylate cyclase. The enzyme is activated by isoproterenol but not norepinephrine, consistent with a β₂-catecholamine receptor-adenylate cyclase interaction. Secretin activates the enzyme in concentrations as low as 1 · 10^?11 M, while glucagon was effective only at 1 · 10^?6 M. Prostaglandin E₁ and E₂ have a biphasic effect with activation of adenylate cyclase at 1 · 10^?5 M and a small but significant inhibition of enzyme activity at 1 · 10^?11 M.     

Decrease of platelet aggregation and spreading via inhibition of the cAMP phosphodiesterase by trapidil

Trapidil (N,N-diethyl-5-methyl[l,2,4]triazolo[l,5-α]pyrimidine-7-amine) inhibits platelet spreading and aggregation induced by arachidonic acid (AA), a stable analogue of prostaglandin (PG) endoperoxides (U46619), ADP, and low concentrations of thrombin, but not by A23187 and high concentrations of thrombin. Trapidil does not affect platelet adenylate cyclase but inhibits the cAMP PDE by approx. 50%. PDE inhibition proceeds via a competitive mechanism (K_i = 0.52 mM) and is not mediated by calmodulin inhibition. Trapidil does not change the platelet basal cAMP level but potentiates an increase of cAMP induced by the stable prostacyclin analogue (6β-PGI_i). These results suggest that trapidil antiplatelet effects may be due to the inhibition of platelet PDE.     

Effects of ethanol on gastric mucosal adenosine 3', 5' monophosphate (cAMP)

The effects of ethanol on the gastric mucosal adenosine 3′, 5′-monophosphate (cAMP) system were evaluated. The activity of adenylate cyclase (AC), phosphodiesterase (PDE), and tissue content of cAMP were determined in the presence of ethanol. NaF stimulated AC in rat gastric mucosa was inhibited in vitro and in vivo by 20% ethanol. Basal AC activity was so low (0.05 ± 0.10 pmoles cAMP formed/min/mg protein) that consistent results without NaF could not be obtained. The PDE activity (172 ± 11 pmoles cAMP consumed/min/mg protein) was approximately 350 fold greater than the basal AC activity. All levels of ethanol tested (2.0–20.0%) significantly inhibited (p<0.05) PDE in vitro. Gastric mucosal levels of cAMP are not measurably altered by ethanol in vivo (5–20%).     

Increased proline transport resulting from growth of normal and Kirsten sarcoma virus-transformed BALB 3T3 cells in the presence of N(6), O(2')-dibutyryl cyclic adenosine 3',5'-monophosphate.

Proline transport in Kirsten sarcoma virus-transformed BALB 3T3 (Ki-3T3) cells was increased approximately twofold by 0.5 mm dibutyryl cAMP (dbcAMP), and the increase was observed whether transport was assayed in the presence or absence of cycloheximide. Two days of exposure to the analog was required for maximum stimulation. Increased proline transport contributed almost entirely to the increased incorporation of [¹⁴C]proline into noncollagen protein but for only 13% of the increased incorporation into collagen of dbcAMP-treated Ki-3T3 cells. Proline transport was further characterized using an assay system containing 0.1 mm cycloheximide, which did not affect transport over a 30-min period. The K_m for proline was decreased from 6.5 to 3.4 mm by dbcAMP treatment of Ki-3T3. Proline transport in Ki-3T3 proceeds almost entirely via the A system, and the effect of dbcAMP appears to be on this system specifically since glycine and glutamine transport, which are heterogeneous, were not affected but transport of N-methylaminoisobutyrate, a specific A system substrate, was increased by dbcAMP treatment. Although 0.5 mm butyrate increased proline transport in Ki-3T3 cells to a similar degree as dbcAMP, the effect of the latter appeared related to its action as a cAMP analog since N⁶-monobutyryl cAMP, having a stable butyryl group, and 8-bromo-cAMP also increased proline transport while dbcGMP did not. The rate of proline transport in normal BALB 3T3 cells was only 30–40% lower than that of Ki-3T3 cells at various growth stages, and dbcAMP and 8-bromo-cAMP treatment also increased proline transport in the normal cells. The results of these studies suggest that dbcAMP and other cAMP analogs induce the synthesis of an altered component of the A system for amino acid transport and that the effect of these compounds is unrelated to the effect of transformation on proline transport.     

Characterization of high affinity GTPase activity correlated to β-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes

β-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The β-adrenergic agonist isoproterenol (10 μM) increased the V_max of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its K_m value (≈ 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5′-(3-O-thio)triphosphate (GTPγS), isoproterenol increased cAMP formation to the same extent as that observed with AlF₄^?. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (≈ 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the ‘turn-off’ step for the adenylyl cyclase activation seen following β-adrenergic stimulation of rat parotid glands.     

Occurrence and involvement of adenylate cyclase activity in the first step of tobacco mosaic virus infection of Nicotiana tabacum cv Xanthi nc leaves

Adenylate cyclase activity and its involvement in a physiopathological process (virus infection) were observed in a higher plant, Nicotiana tabacum cv Xanthi nc. The enzyme was characterized in leaves by measuring the conversion of [α-³²P]ATP into cyclic [α-³²P]AMP using a cell membrane preparation. The basal enzyme activity was 1–2 pmol/min per mg protein, was linear with time and protein concentration, and had a temperature optimum between 20 and 25% C. The K_m for ATP was 2 mM in the presence or absence of stimulators. GTP (10⁻⁷ M) increased both basal and sodium fluoride-stimulated activities. During the hypersensitive reaction which follows tobacco mosaic virus (TMV) infection, we detected in the first 10 min a 40–80% increase in the basal activity. These results indicate that cAMP could play an important role by mediating the viral and plant host-cell interaction. The rapid pulse-release of cAMP leads us to propose that this nucleotide may, as in animal tissues, represent a secondary messenger in higher plants.     

Diffusional and electrostatic effects on apparent kinetic parameters of reactions catalyzed by enzyme immobilized on the external surface of a support

Diffusional and electrostatic effects on the apparent maximum reaction rate V_m^app and the apparent Michaelis constant K_m^app were investigated theoretically for a system in which an enzyme immobilized on the external surface of a solid support catalyzes a reaction according to Michaelis-Menten kinetics. In such a system, the dependence of V_m^app and K_m^app on the substrate concentration can be expressed analytically. When the support and substrate carry charges of the same sign, resulting in a repulsive force between them, both V_m^app and K_m^app decrease with increasing substrate concentration, but they never decrease below the respective intrinsic values. On the other hand, when the support and substrate carry charges of opposite sign and therefore an attractive force occurs, V_m^app decreases towards its intrinsic value, while K_m^app decreases to values below its intrinsic value in the region of high substrate concentration.     

Purification and partial characterization of beef liver gluconolactonase.

Gluconolactonase is isolated and purified from beef liver. The molecular weight is estimated at 233,000 and that of its six similar subunits is 39,400. The pH maximum is 7.1 in 50 mm Tris-acetate buffer at 27 °C. K_m and V_m values of 9.1 mm and 1.62 mmol/min/ mg, respectively, were obtained at 27 °C in 50 mm Tris-HCl buffer. This enzyme requires a divalent metal for activity, with manganese being preferred over magnesium. A subcellular fractionation study indicates that gluconolactonase is located primarily in the cytosol, and its hepatic concentration is 2.3 μmol/kg of hepatic tissue.     

Effect of the structure of lipids favoring disordered domain formation on the stability of cholesterol-containing ordered domains (lipid rafts): identification of multiple raft-stabilization mechanisms

Despite the importance of lipid rafts, commonly defined as liquid-ordered domains rich in cholesterol and in lipids with high gel-to-fluid melting temperatures (T_m), the rules for raft formation in membranes are not completely understood. Here, a fluorescence-quenching strategy was used to define how lipids with low T_m, which tend to form disordered fluid domains at physiological temperatures, can stabilize ordered domain formation by cholesterol and high-T_m lipids (either sphingomyelin or dipalmitoylphosphatidylcholine). In bilayers containing mixtures of low-T_m phosphatidylcholines, cholesterol, and high-T_m lipid, the thermal stability of ordered domains decreased with the acyl-chain structure of low-T_m lipids in the following order: diarachadonyl > diphytanoyl > 1-palmitoyl 2-docosahexenoyl = 1,2 dioleoyl = dimyristoleoyl = 1-palmitoyl, 2-oleoyl (PO). This shows that low-T_m lipids with two acyl chains having very poor tight-packing propensities can stabilize ordered domain formation by high-T_m lipids and cholesterol. The effect of headgroup structure was also studied. We found that even in the absence of high-T_m lipids, mixtures of cholesterol with PO phosphatidylethanolamine (POPE) and PO phosphatidylserine (POPS) or with brain PE and brain PS showed a (borderline) tendency to form ordered domains. Because these lipids are abundant in the inner (cytofacial) leaflet of mammalian membranes, this raises the possibility that PE and PS could participate in inner-leaflet raft formation or stabilization. In bilayers containing ternary mixtures of PO lipids, cholesterol, and high-T_m lipids, the thermal stability of ordered domains decreased with the polar headgroup structure of PO lipids in the order PE > PS > phosphatidylcholine (PC). Analogous experiments using diphytanoyl acyl chain lipids in place of PO acyl chain lipids showed that the stabilization of ordered lipid domains by acyl chain and headgroup structure was not additive. This implies that it is likely that there are two largely mutually exclusive mechanisms by which low-T_m lipids can stabilize ordered domain formation by high-T_m lipids and cholesterol: 1), by having structures resulting in immiscibility of low-T_m and high-T_m lipids, and 2), by having structures allowing them to pack tightly within ordered domains to a significant degree.     

Studies on the developmental pattern of the enzymes converting glucose 6-phosphate to myo-inositol in the rat

The myo-inositol level of plasma was determined during pre- and postnatal development of the rat. Fetal concentrations exceeded those of maternal rats by nearly 10-fold. Immediately after birth, the myo-inositol level decreased but was maintained at values 3–4 times that of the lactating dams. The cyclitol content of rat milk was high and rose during lactation to a maximum of 1.6 mM.The biosynthesis of myo-inositol from glucose 6-phosphate is catalyzed by glucose 6-phosphate:l-myo-inositol-1-phosphate cyclase and l-myo-inositol-1-phosphate phosphatase. The activity of both enzymes was monitored in fetal and neonatal liver, maternal liver, placenta, and mammary gland. Results indicated that the fetal liver accounted for over 48% of the total carcass cyclase and 26% of the total carcass phosphatase activity. Developmental changes correlated well with the pattern of myo-inositol in fetal rat plasma. Similarly, the enzymes of the myo-inositol biosynthetic pathway increased in rat mammary gland in close agreement with the myo-inositol content of milk and diminished to prelactation activities within 24 hr after the onset of involution.The myo-inositol level of colostrum and milk of five human subjects was highest (2.8 mM) before birth and decreased to 40% of that level 5 days postpartum, where it remained for at least 3 weeks. Even after 7 months of lactation, the milk of one subject contained 3–4-fold more myo-inositol than all commercial infant formulas analyzed.     

Comparison of phospholipid effects on insulin-sensitive low Km cyclic AMP phosphodiesterase in adipocyte plasma membranes and microsomes

Both adipocyte plasma membranes and microsomes possess insulin-sensitive low K_m cyclic AMP phosphodiesterase activity. The activity of the enzyme from both sources was susceptible to activation by several anionic phospholipids. Activators of the plasma membrane enzyme were lysophosphatidylglycerol > lysophosphatidylcholine > lysophosphatidylserine > phosphatidylserine > phosphatidylglycerol. These same phospholipids activated the microsomal enzyme but the extent of activation by each phospholipid was reversed. Neutral phospholipids and other anionic phospholipids were without effect. The phospholipids had no effect on high K_m cAMP phosphodiesterase in either membrane. The results suggest that the phospholipid headgroup was an important determinant for enzyme activation by phospholipid. The increased susceptibility of the plasma membrane enzyme to lysophospholipid may be attributed to a difference in the plasma membrane enzyme compared to the microsomal membrane enzyme or to differences in plasma membrane and microsomal membrane phospholipid composition and their ability to regulate low K_m cAMP phosphodiesterase activity.