Receptor-mediated regulation of guanylate cyclase activity in spermatozoa

Two peptides, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), which activate sperm respiration and motility and elevate cyclic GMP concentrations in a species-specific manner, were tested for effects on guanylate cyclase activity. The guanylate cyclase of sea urchin spermatozoa is a glycoprotein and it is localized entirely on the plasma membrane. When intact sea urchin sperm cells were incubated with the appropriate peptide for time periods as short as 5 s and subsequently homogenized in detergent, guanylate cyclase activity was found to be as low as 10% of the activity of cells not treated with peptide. The peptides showed complete species specificity and analogues of one peptide (speract) caused decreases in enzyme activity coincident with their receptor binding properties. The peptides did not inhibit enzyme activity when added after detergent solubilization of the enzyme. When detergent-solubilized spermatozoa were incubated at 22 degrees C, guanylate cyclase activity declined in previously nontreated cells to the peptide-treated level. The rate of decline was dependent on temperature and protein concentration. When spermatozoa were first incubated with 32P, the decrease in guanylate cyclase activity was accompanied by a shift in the apparent molecular weight of a major plasma membrane protein (160,000-150,000) and a loss of 32P label from the 160,000 band. Other agents (Monensin A, NH4Cl) which were capable of stimulating sperm respiration and motility also caused decreases of guanylate cyclase activity when added to intact but not detergent-solubilized spermatozoa. The maximal decrease in guanylate cyclase activity occurred 5-10 min after addition of these agents. The enzyme response to Monensin A required extracellular Na+ suggestive that the ionophore caused the effect on guanylate cyclase activity by virtue of its ability to catalyze Na+/H+ exchange. These studies demonstrate that guanylate cyclase activity of sperm cells can be altered by the specific interaction of egg-associated peptides with their plasma membrane receptors.     

Purification and Characterization of Sperm Creatine Kinase and Guanylate Cyclase of the Sea Urchin Hemicentrotus pulcherrimus

Creatine kinase and guanylate cyclase were purified from Hemicentrotus pulcherrimus spermatozoa. The molecular weight of the purified sperm tail creatine kinase was estimated to be 137,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sperm tail guanylate cyclase was purified by chromatography on a WGA-Sepharose column connected to a Concanavalin A-Sepharose column, and a Superose 12 HR column. The molecular weight of the tail guanylate cyclase was estimated to be 128,000 by SDS-PAGE. The specific activity of the purified enzyme was 8.25 μmol of cGMP formed/min/mg protein. Sperm-activating peptide I (SAP-I) causes an electrophoretic mobility shift of H. pulcherrimus sperm guanylate cyclase from 131 kDa to 128 kDa. The 131 kDa form of guanylate cyclase was co-purified with a 76 kDa protein, whose molecular mass is similar to that of a SAP-I receptor. The purified 131 kDa form of guanylate cyclase had higher activity than the 128 kDa form. The 131 kDa and 128 kDa forms of guanylate cyclase contained 23.83 ± 0.65 and 4.16 ± 0.45 moles of phosphate per mol protein (mean ± S.D.; n = 3), respectively. The activities of guanylate cyclase and creatine kinase increased during the testis development. During spermatogenesis, sperm tail creatine kinase was detected immunohistochemically only in mature spermatozoa.     

Sea urchin sperm guanylate cyclase antibody. Cross-reactivity various rat tissue guanylate cyclases.

After the repeated injection of sea urchin sperm guanylate cyclase into rabbits, antibodies to the enzyme were formed. These antibodies inhibited the particulate or the Triton-dispersed forms of the sperm enzyme by greater than 97%. The sperm adenylate cyclase, cyclic GMP phosphodiesterase, adenosine triphosphatase, guanosine triphosphatase, and 5'-nucleotidase enzymes were not affected by the antiserum. The antiserum inhibited the Triton-dispersed guanylate cyclase from rat heart, liver, lung, spleen, and kidney but did not inhibit the soluble form of the enzyme from any of these tissues. The inhibition of the Triton-dispersed enzyme in these tissues was partial, however, ranging from 30% (liver) to 70% (heart). These results provide evidence that adenylate cyclase is antigenically different from guanylate cyclase, and that the soluble form of guanylate cyclase is antigenically different from a particulate form of the enzyme in various rat tissues.     

Purification and properties of the phosphorylated form of guanylate cyclase

Guanylate cyclase is dephosphorylated in response to the interaction of egg peptides with a spermatozoan surface receptor (Suzuki, N., Shimomura, H., Radany, E. W., Ramarao, C. S., Ward, G. E., Bentley, J. K., and Garbers, D. L. (1984) J. Biol. Chem. 259, 14874-14879). Here, the phosphorylated form of guanylate cyclase was purified to apparent homogeneity from detergent-solubilized spermatozoan membranes by the use of GTP-agarose, DEAE-Sephacel, and concanavalin A-Sepharose chromatography. To prevent dephosphorylation of the enzyme during purification, glycerol (35%) was required in all buffers. Following purification, a single protein-staining band of Mr 160,000 was obtained on sodium dodecyl sulfate-polyacrylamide gels. The final specific activity of the purified enzyme was 83 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C, an activity 5-fold higher than that observed with the purified, dephosphorylated form of guanylate cyclase. A preparation containing protein phosphatase from spermatozoa, or highly purified alkaline phosphatase (from Escherichia coli), catalyzed the dephosphorylation of the enzyme; this resulted in a subsequent decrease in guanylate cyclase activity and a shift in the Mr from 160,000 to 150,000. The phosphate content of the high Mr form of the enzyme was 14.6 mol/mol protein whereas the phosphate content of the low Mr form was 1.6 mol/mol protein. All phosphate was localized on serine residues. The Mr 160,000 form of guanylate cyclase demonstrated positive cooperative kinetics with respect to MnGTP while the Mr 150,000 form displayed linear, Michaelis-Menten type kinetics. The phosphorylation state of the membrane form of guanylate cyclase, therefore, appears to dictate not only the absolute activity of the enzyme but also the degree of cooperative interaction between catalytic or GTP-binding sites.     

The incorporation of a purified, membrane-bound form of guanylate cyclase into phospholipid vesicles and erythrocytes

The purified membrane-bound form of guanylate cyclase was incorporated into artificial unilamellar phospholipid vesicles. The rate and extent of enzyme incorporation into the vesicles was dependent upon the phospholipid concentration and the time period of incubation. The enzyme was incorporated at a significantly faster rate after removal of carbohydrate with endoglycosidase H. The incorporation of the enzyme led to a 10-fold decrease in the apparent maximal velocity and a 2-fold increase in the apparent Michaelis constant for MnGTP. Extraction of liposomes containing guanylate cyclase with 0.2% Lubrol PX resulted in the recovery of 85% of the original amount of added activity, suggesting that the decrease in maximal velocity was not due to enzyme denaturation. Phosphatidylcholine liposomes differentially effected the activity of the membrane-form of guanylate cyclase, dependent on the nature of the fatty acid present on the phospholipid. Specific activities ranged between 458 nmol/min per mg and 2.6 mumol/min per mg, dependent upon the fatty acids present. Liposomes containing the membrane-bound form of guanylate cyclase were subsequently fused with erythrocytes using poly(ethylene glycol) 4000 in attempts to introduce the enzyme into intact cells. The enzyme was successfully introduced into the erythrocytes; greater than 90% of the enzyme activity was subsequently shown to be associated with erythrocyte membranes. Cyclic GMP concentrations of erythrocytes increased from essentially nondetectable to 4 pmol/10(9) cells after introduction of the enzyme. These results demonstrate that guanylate cyclase can be incorporated into liposomes in an active state and that such liposomes can be used to introduce the enzyme into cells where it can subsequently function to generate cyclic GMP.     

Selective activation of particulate guanylate cyclase by a specific class of porphyrins

Guanylate cyclase was activated 3- to 10-fold by hemin in a dose-dependent manner in membranes prepared from homogenates of rat lung, C6 rat glioma cells, or B103 rat neuroblastoma cells. Maximum activation was observed with 50 to 100 microM hemin with higher concentrations being inhibitory. Activation was observed when Mg2+-GTP but not when Mn2+-GTP was used as the substrate. Increased enzyme activity reflected selective activation of the particulate form of guanylate cyclase; hemin inhibited the soluble form of guanylate cyclase 70 to 90% over a wide range of concentrations. Activation was not secondary to proteolysis since a variety of protease inhibitors failed to alter stimulation by hemin. Protophorphyrin IX had little effect on particulate guanylate cyclase activity and sodium borohydride almost completely abolished hemin-dependent activation. These data suggest a requirement for the ferric form of the porphyrin-metal chelate for activation. However, agents which interact with the iron nucleus of porphyrins, such as cyanide, had little effect on the ability of hemin to activate guanylate cyclase. The stimulatory effects of hemin were observed in the presence of detergents such as Lubrol-PX, and highly purified particulate enzyme could be activated to the same extent as enzyme in native membranes. These data suggest that the interaction of porphyrins with particulate guanylate cyclase is complex in nature and different from that with the soluble enzyme.     

Receptor-mediated activation of spermatozoan guanylate cyclase

The sea urchin egg peptides speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Arg-Leu-NH2) bind to spermatozoa of the homologous species (Lytechinus pictus or Arbacia punctulata, respectively) and cause transient elevations of cyclic GMP concentrations (Hansbrough, J. R., and Garbers, D. L. (1981) J. Biol. Chem. 256, 1447-1452). The addition of these peptides to spermatozoan membrane preparations caused a rapid and dramatic (up to 25-fold) activation of guanylate cyclase. The peptide-induced activation of guanylate cyclase was transient, and the subsequent decline in enzyme activity coincided with conversion of a high Mr (phosphorylated) form of guanylate cyclase to a low Mr (dephosphorylated) form. When membranes were incubated at pH 8.0, the high Mr form was converted to the low Mr form without substantial changes in basal enzyme activity. However, the peptide-stimulated activity of the low Mr form of guanylate cyclase was much less than the peptide-stimulated activity of the high Mr form. Activation of the low Mr form by peptide was not transient and persisted for at least 10 min. In addition, the pH 8.0 treatment that caused the Mr conversion of guanylate cyclase also caused an increase in the peptide-binding capacity of the membranes. We propose a model in which activation of the membrane form of guanylate cyclase is receptor-mediated; the extent of enzyme activation is modulated by its phosphorylation state.     

Retention of the speract receptor by isolated plasma membranes of sea urchin spermatozoa

Membrane vesicle preparations enriched in plasma membrane marker proteins, such as adenylate cyclase, were prepared from spermatozoa of the sea urchin, Lytechinus pictus. These membranes, prepared by nitrogen cavitation and subsequent sucrose gradient centrifugation, retained the capacity to bind [125I]-Bolton-Hunter speract (nonspecific binding was less than 5% of specific binding). Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), Tyr-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly, Tyr-Asp-Leu-Thr-Thr-Gly-Gly-Gly-Val-Gly and Gly-Phe-Ala-Leu-Gly-Gly-Gly-Val-Gly caused a 50% decrease in [125I]-Bolton-Hunter speract binding at 10, 600, 1260 and 3160 nM concentrations, respectively. One analogue (Phe-Asp-Leu-Asn-Gly-Gly-Gly), which had no biological activity, failed to compete at concentrations as high as 10 microM. To demonstrate that the binding was due to the isolation of membranes with an intact receptor, the speract analogue (Gly-Gly-Gly-Gly-Tyr-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) was synthesized, radiolabeled with 125I at the position of tyrosine, and covalently cross-linked to the receptor with disuccinimidyl suberate. A single radiolabeled band at an apparent molecular weight of 77,000 was detected on Na X dodecyl X SO4 gels. These studies are the first to identify a receptor for egg-associated peptides in isolated spermatozoan membranes.     

A Two-Step Procedure for Obtaining Highly Purified Particulate Guanylate Cyclase From Rat Lung

Abstract

Cuanylate cyclase (CTP pyrophosphate lyase (cyclizing), EC 4.6.1.21, the enzyme catalyzing the formation of cyclic CMP, from GTP, exists in both soluble and membrane-bound forms.1-6 These enzymes have been implicated as key regulatory components in a variety of biological events such as secretion7-10, and smooth muscle relaxation.11-14 Understanding the role these enzymes play in cellular regulation is predicated upon obtaining purified preparations of guanylate cyclase. Several procedures for purifying the soluble enzyme to apparent homogeneity from a variety of tissues have been reported.15-19 The particulate enzyme has been purified to apparent homogeneity from sea urchin sperm20,21 but, to date, homogeneous preparations of particulate guanylate cyclase from mammalian tissues have not been obtained. Here we report our efforts in purifying particulate guanylate cyclase about 8,200- to 52,000-fold from homogenates of rat lung. Portions of this work have appeared in abstract form.     

Biosynthesis and glycosylation of the insulin receptor. Evidence for a single polypeptide precursor of the two major subunits

The biosynthesis and carbohydrate processing of the insulin receptor were studied in cultured human lymphocytes by means of metabolic and cell surface labeling, immunoprecipitation with anti-receptor autoantibodies, and analysis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. In addition to the two major subunits of Mr = 135,000 and Mr = 95,000, two higher molecular weight bands were detected of Mr = 210,000 and Mr = 190,000. The Mr = 210,000 band and the two major subunits were labeled by [3H]mannose, [3H]glucosamine, [3H]galactose, and [3H]fucose, and were bound by immobilized lentil, wheat germ, and ricin I lectins. On the other hand, the Mr = 190,000 band was labeled only by [3H]mannose and [3H]glucosamine and was bound only by lentil lectin. All four components could be labeled with [35S] methionine; however, in contrast with the other three polypeptides, the Mr = 190,000 band was not labeled by cell surface iodination with lactoperoxidase, suggesting that it is not exposed at the outer surface of the plasma membrane. Pulse-chase studies with [3H]mannose showed that the Mr = 190,000 was the earliest labeled component of the receptor; radioactivity in this band reached a maximum 1 h after the pulse, clearly preceded the appearance of the other components, and had a very brief half-life (t1/2 = 2.5 h). The Mr = 210,000, Mr = 135,000, and Mr = 95,000 bands were next in appearance and reached a maximum 6 h in the chase period. Monensin, an ionophore which interferes with maturation of some proteins, blocked both the disappearance of the Mr = 190,000 protein and the appearance of the Mr = 135,000 and Mr = 95,000 subunits. The mannose incorporated in the Mr = 190,000 component was fully sensitive to treatment with endoglycosidase H while that in the Mr = 210,000 band and the two major subunits was only partially sensitive. Tryptic fingerprints of the 125I-labeled Mr = 210,000 band suggested that this component contains peptides of both the Mr = 135,000 and Mr = 95,000 subunits. In conclusion, the Mr = 190,000 component appears to represent the high mannose precursor form of the insulin receptor that undergoes carbohydrate processing and proteolytic cleavage to generate the two major subunits. In addition, the Mr = 210,000 band is probably the fully glycosylated form of the precursor that escapes cleavage and is expressed in the plasma membrane.     

Comparison of particulate guanylate cyclase in cells with and without atrial natriuretic peptide receptor binding activity

Summary A line of kidney cells (PK,) which does not possess measurable ANP binding but has an active particulate guanylate cyclase has been identified. The physical characteristics of this enzyme were compared with those of particulate guanylate cyclase and ANP receptors isolated from rat lung. Although receptor and enzyme appear to reside on the same protein in the lung while the cyclase from PK₁ cells does not possess ANP binding activity, these proteins exhibit identical physical characteristics. Guanylate cyclase from PK₁ cells and rat lung and ANP receptor from lung co-eluted during gel filtration chromatography, with a Stokes radius of 6.1 nm. Also, these activities co-migrated through sucrose density gradients with S_20,w values of 10.4 to 10.9. Using these parameters, a molecular weight of about 270 kD was estimated for all three activities. Furthermore, these enzyme activities exhibited similar mobilities in isoelectric focusing gels, with a pI of 6.1. Thus, although particulate guanylate cyclase from lung presumably possesses receptor binding activity, it is physically identical to a form of this enzyme associated with no measurable binding activity. Possible explanations for these observations are discussed.     

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     

Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.     

Amiloride increases the sensitivity of particulate guanylate cyclase to atrial natriuretic factor

The natriuretic agent amiloride induces a shift of the dose-response curve of particulate guanylate cyclase to atrial natriuretic factor (ANF) to the left. The ANF concentration for half-maximal activation of guanylate cyclase is shifted from 20 to 3 nM in the presence of 100 microM amiloride. This effect is observed with GTP*Mn2+, but not with GTP*Mg2+ as substrate. Amiloride derivatives, which inhibit a specific Na+-channel, also shift the dose-response curve to the left. These data suggest that some of the effects of amiloride may be mediated by an increased sensitivity of particulate guanylate cyclase to ANF.     

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-containing forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 microM 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 Km for GTP in the presence of excess Mn2+ or Mg2+ was 10 microM and 85-120 microM, respectively, for unactivated guanylate cyclase. The apparent Km for GTP in the presence of Mn2+ was not altered but the Km in the presence of Mg2+ was lowered to 58 microM 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 and nitroso compounds.     

Highly purified particulate guanylate cyclase from rat lung: characterization and comparison with soluble guanylate cyclase

Guanylate cyclase was purified 1000-fold from washed rat lung particulate fractions to a final specific activity of 500 nmoles cyclic GMP produced/min/mg protein by a combination of detergent extraction and chromatography on concanavalin A-Sepharose, GTP-agarose, and blue agarose. Particulate guanylate cyclase has a molecular weight of 200 000 daltons, a Stokes radius of 48 A and a sedimentation coefficient of 9.4 while the soluble form has a molecular weight of 150 000 daltons, a Stokes radius of 44 A, and a sedimentation coefficient of 7.0. Whereas the particulate enzyme is a glycoprotein with a specific affinity for concanavalin A and wheat germ agglutinin, the soluble form of guanylate cyclase did not bind to these lectins. Purified particulate guanylate cyclase did not cross-react with a number of monoclonal antibodies generated to the soluble enzyme. While both forms of the enzyme could be regulated by the formation of mixed disulfides, the particulate enzyme was relatively insensitive to inhibition by cystine. With GTP as substrate both forms of the enzyme demonstrated typical kinetics, and with GTP analogues negative cooperativity was observed with both enzyme forms. These data support the suggestion that the two forms of guanylate cyclase possess similar catalytic sites, although their remaining structure is divergent, resulting in differences in subcellular distribution, physical characteristics, and antigenicity.     

Purification and characterization of two types of atrial natriuretic factor receptors from bovine adrenal cortex: guanylate cyclase-linked and cyclase-free receptors

Atrial natriuretic factor (ANF) receptors with and without guanylate cyclase activity were simultaneously purified to apparent homogeneity from bovine adrenal zona glomerulosa cell membrane fractions. The particulate guanylate cyclase which co-purified with the ANF receptor showed one of the highest specific activity reported. The receptors with or without the guanylate cyclase activity showed high affinities to ANF (99-126). The receptor without the cyclase showed a high affinity to truncated ANF analogs, ANF (103-123) and ANF (105-121), whereas the cyclase-linked receptor had a much lower affinity to these analogs. Both of the receptors migrated as a single band with a molecular weight of 135,000 daltons on SDS-gel electrophoresis under non-reducing conditions. The 135,000 daltons band of the receptor without the cyclase was shifted to a 62,000 daltons band under reducing conditions, but the band for the cyclase-linked receptor was not shifted. These results demonstrated the presence of two subtypes of ANF receptor in bovine adrenal cortex and indicate two different modes of intracellular action of ANF.     

Adenine nucleotide regulation of particulate guanylate cyclase from rat lung.

Adenine nucleotides activate basal particulate guanylate cyclase in rat lung membranes. Activation is specific for adenine and not guanine, cytidine or uridine nucleotides. The concentration of adenine nucleotides yielding half-maximum activation of particulate guanylate cyclase is 0.1 mM and this nucleotide activates the enzyme by increasing maximum velocity 11-fold without altering affinity for substrate. Activation is specific for particulate guanylate cyclase, since soluble enzyme is inhibited by adenine nucleotides. Similarly, activation is specific for magnesium as the enzyme substrate cation cofactor, since adenine nucleotides inhibit particulate guanylate cyclase when manganese is used. Adenine nucleotide regulation of particulate guanylate cyclase may occur by a different molecular mechanism compared to other activators, since the effects of these nucleotides are synergistic with those of detergent, hemin and atrial natriuretic peptides. Cystamine inhibits adenine nucleotide activation of particulate guanylate cyclase at concentrations having minimal effects on basal enzyme activity suggesting a role for critical sulfhydryls in mechanisms underlying nucleotide regulation of particulate guanylate cyclase. Purification and quantitative recovery of particulate guanylate cyclase by substrate affinity chromatography results in the loss of adenine nucleotide regulation. These data suggest that adenine nucleotides may be important in the regulation of basal and activated particulate guanylate cyclase and may be mediated by an adenine nucleotide-binding protein which is separate from that enzyme.     

Arylsulfatase a from normal human lung and lung tumors showed different patterns of microheterogeneity

Arylsulfatase A was purified from human lung to apparent homogeneity as determined by electrophoresis in the presence of sodium dodecyl sulfate. The enzyme from normal lung as well as that from lung adenocarcinoma showed considerable microheterogeneity when examined by isoelectric focussing, with an isoelectric point (pI) ranging from 5.1 to 4.6. The tumor enzyme was more heterogeneous and contained more acidic components than the normal lung enzyme. The cause of the charge heterogeneity was examined by treatment with exogenous hydrolases. Upon treatment with sialidase, phosphatase or endo-beta-N-acetylglucosaminidase H (endoglycosidase H), the acidic enzyme forms shifted to an alkaline region on isoelectric focussing gels. Combined treatment of the arylsulfatase A with endoglycosidase H and sialidase resulted in complete loss of the most acidic components to give the less acidic components with pI 5.1, 5.0, and 4.9. These results strongly suggest that the charge heterogeneity of arylsulfatase A is due not only to sialylation but also to phosphorylation at the carbohydrate moiety of the enzyme, and the extent of substitution by acidic groups is markedly increased in the tumor enzyme.     

ANF stimulation of detergent-dispersed particulate guanylate cyclase from bovine adrenal cortex

Particulate guanylate cyclase from bovine adrenal cortex can be stimulated by ANF. A 2-fold stimulation of the enzyme was obtained with 100 nM ANF and a half-maximal stimulation, with a 5 nM dose. The stimulation by ANF persisted for at least 30 min. Various detergents, such as Triton X-100, Lubrol PX, cholate, CHAPS, digitonin and zwittergent, stimulated several-fold the activity of particulate guanylate cyclase. However, only Triton X-100 dispersed particulate guanylate cyclase without affecting its response to ANF. The dose-response curve of ANF stimulation of the particulate and the Triton X-100 dispersed enzyme was similar. The dispersion of a fully responsive guanylate cyclase to ANF will help us to uncover the type of interactions between guanylate cyclase and ANF. It will also be used as a first step for the purification of an ANF-sensitive particulate guanylate cyclase.