Guanylate cyclase activity of human lymphocytes from peripheral blood, thymus, and tonsils. A comparative study

The subcellular repartition and the distinctive properties of guanylate cyclase (EC 4.6.1.2) vary according to the lymphocyte population studied and according to the presence of detergent. Guanylate cyclase of non-adherent peripheral lymphocytes and of thymus lymphocytes is recovered by more than 90% in the soluble fraction of the homogenate. Kinetics according to the substrate (5'-GTP-Mn2+) is Michaelian, the Ca2+ ion acts as an activator, especially in the case of blood lymphocytes, and the detergent has no effect on the enzyme activity. On the other hand, the guanylate cyclase of tonsil lymphocytes reside in the particulate fraction. It has non-Michaelian kinetics for the substrate, a strong stimulating effect of detergent, and an inhibitory effect of Ca2+. A comparison of the enzymatic activities of unseparated and of non-adherent tonsil lymphocytes obtained from the same donor points to a correlation between their T and B properties: predominant soluble activity in the T population and particulate guanyl cyclase activity in the B subset.     

Conditions of activation of guanylate cyclase from human platelets

Preincubation (50 min, 0 degree C) with nitroprusside increases 12-fold the activity of human platelet guanylate cyclase. The stimulating effect of nitroprusside is enhanced two-fold by dithiothreitol (2 mM) and by 60% by hemoglobin (20 micrograms/ml). Storage of guanylate cyclase preparations (105000 g supernatant) for 2-3 days at 4 degrees C causes a progressive increase of the enzyme activity and diminishes the stimulating effect of nitroprusside. After storage of guanylate cyclase preparations for 3 days, hemoglobin (20 micrograms/ml) augments the stimulating effect of nitroprusside by 130%. It is concluded that the degree of activation of guanylate cyclase by nitroprusside reflects the functional state of the enzyme.     

Purification of soluble guanylate cyclase enzyme from human platelets

Soluble guanylate cyclase enzyme was purified from human platelets. The soluble fraction of the lysed platelets was sequentially chromatographed over DEAE-sepharose, GTP-agarose and HPLC size-exclusion columns. About 0.1 mg of purified enzyme could be obtained from 2000 ml of platelet rich plasma. The purified enzyme had the specific activity of 205 nmoles cGMP/mg/min with Mn2+ as cofactor. The enzyme eluted at the 160,000 daltons position from the size-exclusion column. Electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions revealed two subunits of 83,000 and 71,000 daltons respectively.     

Guanylate cyclase in olfactory cilia from rat and pig

A guanylate cyclase was identified in cilia from rat and pig olfactory epithelia. Enzyme activities were 200-250 and 90-100 pmol/min.mg-1, respectively. Activity required the presence of non-ionic detergents, e.g., 0.1% Lubrol PX. MnGTP, not MgGTP was used as a substrate. Furthermore, 0.9 mM free Mn2+ was necessary for optimal activity indicating a regulatory site for a divalent cation. The guanylate cyclase displayed sigmoidal Michaelis-Menten kinetics suggesting cooperativity between MnGTP and enzyme. S0.5 was 160 microM MnGTP. The Hill coefficient of 1.7 indicates that more than one class of substrate-binding sites interact in a positive cooperative manner. ATP inhibited the enzyme and linearized plots of substrate kinetics with MnGTP. SH-Blocking agents reversibly inhibited enzyme activity. Sodium azide and nitroprusside were without effect as were several odorants. A guanylate cyclase activity in cilia from tracheal tissue had properties similar to the olfactory enzyme.     

Hydrogen peroxide release from human blood platelets

The release of hydrogen peroxide from human blood platelets after stimulation with particulate membrane-perturbing agents has been determined by fluorescence using scopoletin as the detecting agent. Platelet suspensions containing less than 1 polymorphonuclear leukocyte/10⁸ platelets showed a significant release of hydrogen peroxide (6.11 nmol/10⁹ platelets per 20 min, S.D., 0.26, n=9) after addition of zymosan or latex particles, compared to unstimulated platelets. The release of hydrogen peroxide was only observed when the scopoletin was added to the platelet suspensions during the stimulation. Any attempt to determine hydrogen peroxide release in the supernatant at the end of the incubation with zymosan or latex failed. A NADH-dependent production of hydrogen peroxide was observed by measuring the difference of oxygen uptake in the presence and absence of catalase (500 units), which was not inhibited by potassium cyanide (1 mM). By this method the NADH-dependent cyanide-insensitive peroxide production and release was 6.0 nmol/10⁹ platelets per 20 min from resting platelets (S.D., 2, n=6) vs. 15 nmol/10⁹ platelets per 20 min from stimulated platelets (S.D., 2, n=6).     

Guanylate cyclase, a cell surface receptor

Guanylate cyclase appears to represent a central member of a diverse family of proteins involved in cell signaling mechanisms including the protein kinases, a low Mr ANP receptor, and possibly adenylate cyclase (based on limited sequence identity with the yeast enzyme). A membrane form of guanylate cyclase represents a new model for cell surface receptors, although such a model was once envisioned for adenylate cyclase (79). In original models for adenylate cyclase, hormone was thought to bind with either the enzyme or with an unknown protein to enhance cyclic AMP production (79). Guanylate cyclase appears to fall into the first adenylate cyclase model where binding of a ligand to an extracellular site on the enzyme transmits a signal to an intracellular catalytic site. The production of cyclic GMP, a second messenger, and of pyrophosphate are then increased. The protein tyrosine kinase family of receptors (80) and possibly another forthcoming family of cell surface receptors containing protein tyrosine phosphatase activity (81-83) contain a single transmembrane domain like guanylate cyclase. Furthermore, the protein tyrosine kinases are activated by ligand binding to the extracellular domain. However, the activation of guanylate cyclase, unlike these cell surface receptors, results in the formation of a low molecular weight second messenger.     

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors.     

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.     

Guanylate cyclase from the rat renal medulla. Physical properties and comparison with adenylate cyclase.

Guanylate cyclase from the rat renal medulla is found in both the soluble and particulate fractions of the cell. Sucrose density gradient centrifugation and gel filtration in H2O and D2O indicate that the enzyme from the soluble cell fraction has the following properties: S20w, 6.3 S; Stokes radius, 54 A; partial specific volume, 0.75 ml/g; mass, 154,000 daltons; f/fo, 1.4; axial ratio (prolate ellipsoid), 7. The addition of 0.1% Lubrol PX to this fraction activates the enzyme and changes thartial specific volume, 0.74 ml/g; mass, 148,000 daltons; f/fo, 1.6; axial ratio (prolate ellipsoid), 11. These findings show that detergent activates the enzyme by changing its conformation and not simply by dispersing nonsedimentable membrane fragments. The dimensions of this guanylate cyclase in detergent are very similar to those of detergent-solubilized adenylate cyclase from the same tissue (Neer, E.J. (1974) J. Biol. Chem. 249, 6527-6531). Guanylate cyclase can be solubilized from the particulate cell fraction with 1% Lubrol PX but has properties quite different from those of the guanylate cyclase in the soluble cell fraction. It is a large aggregate with a value of S20,w of about 10 S, Stokes radius of 65 A, and a mass of approximately 300,000 daltons. However, the peaks of guanylate cyclase activity in column effluents and sucrose density gradients are very broad indicating a mixture of different size proteins. The conditions used to solubilize guanylate cyclase from the particulate fraction also solubilize adenylate cyclase, and the two activities can be separated on the same sucrose gradient. Studies of this sort require a rapid, accurate guanylate cyclase assay. We have developed an assay for guanylate cyclase activity which meets these criteria by adapting the competitive protein binding assay for guanosine cyclic 3':5' monophosphate originally described by Murad et al. (Murad, F., Manganiello, V., and Vaughn, M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 736-739).     

Guanylate cyclase activity in Escherichia coli mutants defective in adenylate cyclase.

Guanylate cyclase, which catalyzes the synthesis of guanosine 3',5'-monophosphate, has been assayed in several strains of Escherichia coli. They include wild-type cells and mutants defective in adenylate cyclase, which is responsible for the synthesis of adenosine 3',5'-phosphate. Our results demonstrate that adenylate cyclase and guanylate cyclase are two different enzymes in E. coli and suggest that the gene that encodes adenylate cyclase also plays a regulatory role in the synthesis of guanylate cyclase.     

Guanylate cyclase activity in permeabilized Dictyostelium discoideum cells

Dictyostelium discoideum cells respond to chemoattractants by transient activation of guanylate cyclase. Cyclic GMP is a second messenger that transduces the chemotactic signal. We used an electropermeabilized cell system to investigate the regulation of guanylate cyclase. Enzyme activity in permeabilized cells was dependent on the presence of a nonhydrolysable GTP analogue (e.g., GTPγS), which could not be replaced by GTP, GDP, or GMP. After the initiation of the guanylate cyclase reaction in permeabilized cells only a short burst of activity is observed, because the enzyme is inactivated with a t_1.2 of about 15 s. We show that inactivation is not due to lack of substrate, resealing of the pores in the cell membrane, product inhibition by cGMP, or intrinsic instability of the enzyme. Physiological concentrations of Ca²⁺ ions inhibited the enzyme (half-maximal effect at 0.3 μM), whereas InsP₃ had no effect. Once inactivated, the enzyme could only be reactivated after homogenization of the permeabilized cells and removal of the soluble cell fraction. This suggests that a soluble factor is involved in an autonomous process that inactivates guanylate cyclase and is triggered only after the enzyme is activated. The initial rate of guanylate cyclase activity in permeabilized cells is similar to that in intact, chemotactically activated cells. Moreover, the rate of inactivation of the enzyme in permeabilized cells and that due to adaptation in vivo are about equal. This suggests that the activation and inactivation of guanylate cyclase observed in this permeabilized cell system is related to that of chemotactic activation and adaptation in intact cells. © 1996 Wiley-Liss, Inc.     

Adenyl cyclase in human platelets: activity and responsiveness

A clinical study was conducted whereby the activity of adenyl cyclase in the human platelet was demonstrated. The study showed that this activity can be stimulated and inhibited in vitro. Platelets were isolated from normal donors. The laboratory procedures involved in the study are described in detail. It seems that many of the biologic processes which occur in the human platelet are dependent on the breakdown of ATP (adenosine-tri-phosphate) to, among other substances, AMP (adenosine-3',5' monophosphate). Activity of the adenyl cyclase was stimulated by fluoride, prostaglandin E1, and glucagon; it was inhibited by thrombin, epinephrine, norepinephrine, and serotonin. PG (prostaglandin) E1 at concentrations of 20 ng/ml and above increased adenyl cyclase in 7 experiments by 3-5 times. Even at concentrations as low as 2 ng/ml., PGE2 caused perceptable stimulation. The PGE, while stimulating adenyl cyclase activity, also inhibited aggregation of platelets by a variety of substances. Results of the study suggest that adenosine-3',5' monophosphate may be important in the regulation of platelet adhesiveness.     

Isolation of palmitoyl-CoA hydrolases from human blood platelets.

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].     

Guanylate cyclase of isolated bovine retinal rod axonemes.

The guanylate cyclase activity of axoneme--basal apparatus complexes isolated from bovine retinal rods has been investigated. The Mg2+ and Mn2+ complexes of GTP4- serve as substrates. Binding of an additional mole of Mg2+ or Mn2+ per mole of enzyme is required. Among cations which are ineffective are Ca2+, Ni2+, Fe2+, Fe3+, Zn2+, and Co2+. The kinetics are consistent with a mechanism in which binding of Mg2+ or Mn2+ to the enzyme must precede binding of MgGTP or MnGTP. The apparent dissociation constants of the Mg--enzyme complex and the Mn--enzyme complex are 9.5 x 10(-4) and 1.1 x 10(-4) M, respectively. The apparent dissociation constants for binding of MgGTP and MnGTP to the complex of the enzyme with the same metal are 7.9 x 10(-4) and 1.4 x 10(-4) M, respectively. The cyclase activity is maximal and independent of pH between pH 7 and 9. KCl and NaCl are stimulatory, especially at suboptimal concentrations of Mg2+ or Mn2+. Ca2+ and high concentrations of Mg2+ and Mn2+ are inhibitory. Ca2+ inhibition appears to require the binding of 2 mol of Ca2+ per mol of enzyme. The dissociation constant of the Ca2--enzyme complex is estimated to be 1.4 x 10(-6) M2. The axoneme--basal apparatus preparations contain adenylate cyclase activity whose magnitude is 1--10% that of the guanylate cyclase activity.     

Purification of guanylate cyclase from human platelets and effect of arachidonic acid peroxide.

Guanylate cyclase of human platelets was separated from cyclic nucleotide and GTP hydrolytic activities with a 104-fold purification over the homogenate. The purified guanylate cyclase preparation requires neither the GTP regenerating system nor cyclic GMP but is stimulated by about 2-fold by 2.5 mM cyclic GMP. The molecular weight of the enzyme was estimated as 180,000 and the Km value for GTP was 95 μM. Arachidonic acid peroxide stimulated the purified enzyme by increasing maximum velocity without changing Km value.