Production and characterization of monoclonal antibodies against particulate guanylate cyclase in porcine kidney

Three monoclonal antibodies (Ig G1 type) to particulate guanylate cyclase from porcine kidney cortex have been produced by fusing spleen cells from immunized BALB/c mouse with P3X63 myeloma cells. The antibodies were detected by their ability to bind immobilized antigen and by immunoprecipitation of enzyme activity. After subcloning by limiting dilution, hybridomas were injected intraperitoneally into mice to produce ascitic fluid. The antibodies recognized a 180,000 dalton protein in Lubrol-PX extract of porcine kidney cortex membrane, and when immobilized on Sepharose 4B, they co-precipitated both [125I]human atrial natriuretic peptide (ANP)-receptor complex and guanylate cyclase activity. The antibodies caused a greater increase in generation of cGMP than that of ANP.     

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.     

Human soluble guanylate cyclase: functional expression, purification and structural characterization

Soluble guanylate cyclase is an enzyme that catalyzes formation of cGMP from GTP and is a member of the nucleotide cyclase family of enzymes. sGC is a receptor for endogenous and exogenous nitric oxide and is activated several-fold upon its binding, constituting a core enzyme in the nitric oxide signal transduction pathway. cGMP generated by sGC is an important second messenger that regulates activity of several enzymes triggering such important physiologic reactions as vasodilation, smooth muscle relaxation and platelet aggregation. We report here the functional expression of the human isoform of soluble guanylate cyclase in HighFive insect cells using a baculovirus expression system. Highly active recombinant protein was obtained without heme reconstitution or supplementation of the cell growth medium and the level of protein expression was found to be heavily affected by the composition of the growth medium. We have successfully purified highly active sGC (sp act up to 940 nmol/min/mg) from adherent cultures using a three-column, 1-day procedure. The UV-Vis spectrum of the isolated protein shows a Soret band at 431 nm, consistent with a histidine-ligated, 5-coordinate heme as previously reported. Far UV CD spectroscopy, intrinsic tryptophan fluorescence, fluorescence of the hydrophobic dye bis-ANS, size-exclusion chromatography, and small angle X-ray scattering (SAXS) were used to characterize the structural properties of the purified sGC. We used two hierarchical neural network methods to predict the secondary structure of sGC and found it to be consistent with the observed CD spectrum of sGC.     

Purification and characterization of guanylate cyclase from Caulobacter crescentus

Guanylate cyclase has been purified 60-fold from cell extracts of the bacterium $\text{Caulobacter crescentus}$. It has a molecular weight of approximately 140,000 and is dependent upon Mn²⁺ for activity. Enzymic activity is unaffected by cyclic AMP, cyclic GMP or N⁶,O^2′-dibutyryl cyclic AMP but is stimulated by N²,O^2′-dibutyryl cyclic GMP. The partially purified preparation of guanylate cyclase does not contain detectable adenylate cyclase activity.     

Functional characterization of two nucleotide-binding sites in soluble guanylate cyclase

Soluble guanylate cyclase is a heterodimeric hemoprotein composed of alpha- and beta-subunits with a homologous motif to the nucleotide-binding sites of adenylate cyclases. Homology modeling of guanylate cyclase, based on the crystal structure of adenylate cyclase, reveals a single GTP-binding site and a putative second site pseudosymmetric to the GTP-binding site. However, the role of this pseudosymmetric site has remained unclear. Using equilibrium dialysis, we identified two nucleotide-binding sites with high and low affinity for alpha,beta-methylene guanosine 5'-triphosphate (GMP-CPP). In contrast, 2'-dADP occupied both sites with equivalent affinities. Adenosine-5'-beta,gamma-imido triphosphate (AMP-PNP), which competitively inhibited the cyclase reaction, bound solely to the high affinity site, indicating the role of this site as the catalytic site. The function of the low affinity site was examined using allosteric activators YC-1 and BAY 41-2272. YC-1 significantly reduced the affinity of 2'-dADP, probably by competing for the same site as 2'-dADP. BAY 41-2272 totally inhibited the specific binding of one molecule of 2'-dADP as well as GMP-CPP. This suggests that the activators compete with these nucleotides for the low affinity site. Infrared and EPR analyses of the enzymic CO- and NO-hemes also supported the suggested role of the low affinity site as a target for the activators. Our results imply that the low affinity site is the pseudosymmetric site, which binds YC-1 or BAY 41-2272.     

Purification and characterization of particulate guanylate cyclase from sea urchin spermatozoa

The particulate form of guanylate cyclase from sea urchin spermatozoa was purified to apparent homogeneity by chromatography on GTP-Sepharose and DEAE-Sepharose and by preparative gel electrophoresis. The sedimentation coefficient (S20,w) was 6.8 and the Stokes radius was 5.1 nm, from which a native molecular weight of 157,000 was calculated. A single protein or periodic acid-Schiff staining band of 135,000 Da was observed after Na dodecyl SO4 gel electrophoresis. Antibody was produced to guanylate cyclase and was shown by electrophoretic transfer experiments (Western blot) to interact with only the Mr = 135,000 band in cases where all of the detergent-extracted protein from spermatozoa was added to the Na dodecyl SO4 gels. Although guanylate cyclase was normally bound to concanavalin A-Sepharose, after endoglycosidase H treatment it failed to bind. Treatment of the enzyme with endoglycosidase H did not alter guanylate cyclase activity, but the apparent size of the enzyme decreased to 72,000 Da on Na dodecyl SO4 gels. An analysis of carbohydrate composition indicated that the oligosaccharides contained N-acetylglucosamine, mannose, galactose, and 2-aminoerythritol in molar ratios (1:3:0.75:2); after endoglycosidase H treatment the enzyme contained essentially no carbohydrate. Major amino acids in the enzyme were aspartic (Asn) and glutamic (Gln) which accounted for approximately 25 mol % of the enzyme amino acid composition. The purified enzyme displayed linear kinetics on double reciprocal plots and had a KMnGTP = 133 microM, KM2+ = 138 microM, KiMnGTP = 122 microM, KiMn2+ = 127 microM, and a V max in excess of 15 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C. Sodium nitroprusside did not stimulate the enzyme in either the presence or absence of added hemeproteins. These results indicate that the particulate form of guanylate cyclase from sea urchin spermatozoa is a glycoprotein which is distinctly different than the soluble form of the enzyme found in mammalian tissues.     

Antiproliferative monoclonal antibodies: detection and initial characterization

Two monoclonal antibodies (MAB) are described which inhibit in vitro cellular proliferation in the absence of complement or effector cells. These MAB were produced by hybridomas made from mice immunized against human B lymphoma cells. The MAB were detected by using a colorimetric assay that quantifies proliferation based on the conversion of a yellow tetrazolium salt to a purple formazan product, a reaction that occurs only in metabolically active cells with intact mitochondrial enzymes. A human B lymphoblastoid cell was used as the screening target. RBC4 is an IgM MAB that modulates and immunoprecipitates the transferrin receptor. RBG5 is an IgG1 that binds to a nonmodulating cell surface determinant different from the transferrin receptor. Both MAB are active at low concentrations (RBC4, 0.5 microgram/ml and RBG5, 0.01 microgram/ml). Immunofluorescence staining of cell lines by RBC4 and RBG5 shows little correlation with inhibition by the antibodies. They differentially inhibit the proliferation of a panel of T, B, and myeloid cell lines. Both antibodies inhibit the proliferation of alloantigen or mitogen-activated human peripheral blood lymphocytes (PBL). Unstimulated PBL are not affected by either MAB. The RB MAB each cause different morphologic changes of target cells. Whereas RBC4-inhibited cells exhibit nonspecific changes, RBG5 causes a progressive increase in the size and nuclear number of a subset of inhibited cells.     

Human liver catalase: cloning,expression and characterization of monoclonal antibodies

We isolated a cDNA encoding liver catalase from a human liver cDNA library. The cDNA had a high degree of sequence similarity to the corresponding enzyme from other sources. It was expressed in E. coli using the pET15b vector. The protein produced was enzymatically active after purification, and its kinetic parameters closely resembled those of other mammalian catalases. Monoclonal antibodies were generated against the purified catalase; six antibodies recognizing different epitopes were obtained, one of which inhibited the enzyme. The cross reactions of the antibodies with brain catalases from human and other mammalian tissues were investigated, and all the immunoreactive bands obtained on Western blots had molecular masses of about 58 kDa. Similarly fractionated extracts of several mammalian cell lines all gave a single band of molecular mass 58 kDa. These results indicate that mammalian livers and human cell lines contain only one major type of immunologically reactive catalase, even though some of catalases have been previously reported to differ in certain properties.     

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.     

Preparation of heme-free soluble guanylate cyclase

Soluble guanylate cyclase (sGC), a heterodimer consisting of alpha- and beta-subunit, is the key enzyme of the NO/cGMP signaling pathway. The heme moiety ligated to the beta-subunit via His(105) is crucial for the activation of the enzyme by NO. In addition to this NO binding capability, the heme status of the enzyme influences the activity of non-NO sGC activators and sGC inhibitors. Different sGC activity profiles were observed in the presence, absence, or the oxidized form of heme. Modulating the heme status is therefore crucial for the investigation of the mechanism of sGC activation. Here, we present a simple and reliable procedure for the removal of the heme moiety of sGC that is capable of eliminating any traces of unbound heme and detergent from the sample mixture in one single step. Samples containing 15 microg sGC and the non-ionic detergent Tween 20 (2%) were incubated at 37 degrees C for 10 min and loaded onto centrifugal ion exchange columns. After centrifugation, heme was bound entirely to the ion exchanger and could not be eluted, even after incubation with 1M NaCl. Tween 20 was found completely within the flowthrough. Heme-free sGC was eluted from the ion exchanger after application of 300 mM NaCl. The absence of the heme moiety was confirmed by UV/Vis spectra and determination of the enzymatic activity. In summary, the described procedure is suitable for the preparation of very small amounts of highly purified heme-free sGC for the investigation of the mechanism of action of different types of sGC activators.     

EPR characterization of axial bond in metal center of native and cobalt-substituted guanylate cyclase

The nature of the metal-proximal base bond of soluble guanylate cyclase from bovine lung was examined by EPR spectroscopy. When the ferrous enzyme was mixed with NO, a new species was transiently produced and rapidly converted to a five-coordinate ferrous NO complex. The new species exhibited the EPR signal of six-coordinate ferrous NO complex with a feature of histidine-ligated heme. The histidine ligation was further examined by using the cobalt protoporphyrin IX-substituted enzyme. The Co2+-substituted enzyme exhibited EPR signals of a broad g perpendicular;1 component and a g;1 component with a poorly resolved triplet of 14N superhyperfine splittings, which was indicative of the histidine ligation. These EPR features were analogous to those of alpha-subunits of Co2+-hemoglobin in tense state, showing a tension on the iron-histidine bond of the enzyme. The binding of NO to the Co2+-enzyme markedly stimulated the cGMP production by forming the five-coordinate NO complex. We found that N3- elicited the activation of the ferric enzyme by yielding five-coordinate high spin N3- heme. These results indicated that the activation of the enzymes was initiated by NO binding to the metals and proceeded via breaking of the metal-histidine bonds, and suggested that the iron-histidine bond in the ferric enzyme heme was broken by N3- binding.     

Production of monoclonal antibodies and enzyme immunoassay for typical adenylate cyclase activator,Forskolin

The ratio of hapten to bovine serum albumin in an antigen conjugate was exactly determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against forskolin was produced by fusing splenocytes immunized with a forskolin-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.57%. A very small cross-reaction appeared with 1-deoxy, 9-deoxy and 1,9-dideoxy forskolin derivatives. The full measuring range of the assay extends from 6 ng to 200 ng of forskolin. The competitive ELISA assay used for this analysis was found to be more sensitive than TLC (10 g), GLC (30 ng) and HPLC (1 g) methods.     

Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells

Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni²⁺-NTA, and POROS Q columns obtained approximately 100 mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M_r = 144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS–PAGE corresponding to the α (M_r 77,000) and β (M_r 70,000) sGC subunits. It showed an A₄₃₀/A₂₈₀ = 1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k_cat/K_m = 1.7 × 10⁻³ s⁻¹ μM⁻¹ that increased to 5.8 × 10⁻¹ s⁻¹ μM⁻¹ upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure–function relationships.     

Tritrichomonas foetus: preparation of monoclonal antibodies with effector function

Monoclonal antibodies were prepared against Tritrichomonas foetus and characterized with regard to binding and immune effector activities. Nine of 27 monoclonal antibodies which reacted with T. foetus appeared to bind to the surface of live parasites. Immunoelectron microscopy confirmed the surface binding of two of these. At least six of these surface reactive monoclonal antibodies facilitated complement mediated lysis of T. foetus and one acted as an opsonin for phagocytosis by peripheral blood bovine monocytes. Five surface reactive monoclonal antibodies identified a molecule of approximately 150,000 relative molecular weight on Western blots of whole parasite preparations. These results collectively suggest the 150,000 relative molecular weight molecule may be an important target antigen for the immune response to T. foetus.     

A monoclonal antibody to vasopressin: preparation, characterization, and application in immunocytochemistry

The hypothalamo-neurohypophysial system, containing the hormones oxytocin (OT) and vasopressin (VP) and their associated carrier proteins, the neurophysins (NPS), has been the subject of extensive investigation for more than 40 years. This system has been reinvestigated during the last decade by application of immunocytochemical methods employing the rabbit antisera to the hormones and NPS. In this study we describe the preparation and characterization of a monoclonal antibody to VP and its application in immunohistochemistry. The antibody did not cross-react with OT or arginine vasotocin (AVT). Its antigenic determinants as characterized by absorption with various VP analogs included two aromatic amino acids: Phe in position 3, and to a lesser extent Tyr in 2. Tissue fixation with formaldehyde resulted in inadequate immunostaining as compared to glutaraldehyde, most likely due to interference with the aromatic amino acid determinants by the former fixative.     

Cation-dependent prolactin activation of guanylate cyclase

Prolactin enhanced guanylate cyclase [E.C.4.6.1.2] two- to threefold in ovary, testis, mammary gland, liver and kidney. Dose response relationships revealed that maximal activation of this enzyme was at a concentration of one nanomolar and that increasing prolactin's concentration to the millimolar range caused no further increase in activity. There was an absolute cation requirement for prolactin's enhancement of guanylate cyclase. Calcium or manganese allowed prolactin to increase guanylate cyclase activity. Greater enhancement of this enzyme's activity by prolactin was observed when manganese was the co-factor. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of prolactin.     

NO-independent stimulators of soluble guanylate cyclase

SARs around a novel type of guanylate cyclase stimulator which act by a mechanism different from classical NO-donors are described. Several pyrazolopyridinylpyrimidines are shown to relax aortic rings and revealed a long-lasting blood pressure lowering effect in rats after oral application.