Immunoassay of the adenosine deaminase complexing proteins of human tissues and body fluids.

A sensitive immunoassay for the adenosine deaminase binding protein (complexing protein) of human kidney has been developed. Impetus for the development of the assay was provided by the observations that (a) antibody to complexing protein does not react with the catalytically active adenosine deaminase monomer, and (b) binding of antibody to complexing protein does not affect the binding or catalytic activity of the enzyme monomer. Preformed immune precipitate prepared from rabbit anti-kidney complexing protein serum and goat anti-rabbit gamma-globulin serum is used to selectively insolubilize complexing protein. Quantitation is accomplished by measuring the intrinsic adenosine deaminating activity or adenosine deaminase binding capacity of the protein held in the immune precipitate. As little as 1 ng of kidney complexing protein can be accurately quantitated with the assay. The assay was used to demonstrate that complexing proteins from liver, lung, spleen, fibroblasts, plasma, and urine react with antibody to kidney complexing protein. The shared capacity to bind adenosine deaminase coupled with their antigenic similarity suggests that the complexing proteins of a number of human tissues and body fluids may be products of the same gene.     

Adenosine deaminase-complexing protein from bovine kidney. Isolation of two distinct subunits

We have isolated a complex of two proteins from bovine kidney that bind to adenosine deaminase immobilized on Sepharose 4B. One protein, with Mr = 110,000, comigrates on both PAGE and SDS-PAGE gels with complexing protein isolated from rabbit kidney by the method of Schrader et al. (Schrader, W.P., Harder, C. M., and Schrader, D. K. (1983) Comp. Biochem. Physiol. B Comp. Biochem. 75, 119-126). The second protein has a Mr = 70,000. Both proteins bind to the adenosine deaminase-Sepharose but not to a control resin of bovine serum albumin bound to Sepharose. Based on a comparison of partial and complete denaturation on SDS-PAGE the two proteins appear to be bound to each other. At adenosine concentrations of 0.5-1 mM the isolated complexing protein increases small subunit adenosine deaminase catalytic activity by 20-30%. There may be some inhibition of catalytic activity at low adenosine concentrations. We have designated the 110,000 Mr protein CP-I, the 70,000 Mr protein CP-II and the complex of these two CP.     

Characterization of an insoluble adenosine deaminase complexing protein from human kidney

Evidence for the presence of an insoluble form of adenosine deaminase complexing protein in human kidney has been obtained. An initial study demonstrated that binding of monomeric adenosine deaminase to particulate material from kidney was saturable and could be blocked by preincubating the enzyme with soluble complexing protein. Treatment of particulate material with deoxycholate, followed by immunoassay of the detergent extract, confirmed the presence of an insoluble form of complexing protein in the kidney. Several other human organs examined by this technique contained smaller amounts of insoluble complexing protein. Complexing protein isolated from the soluble and particulate fractions of kidney homogenates were found to be structurally similar. The proteins had the same subunit M_r and showed complete crossreactivity with antiserum to soluble complexing protein. Indirect immunoperoxidase staining of renal cortical tissue revealed that complexing protein was concentrated in the brush border of the proximal tubules. These results indicate that (a) the soluble and insoluble forms of complexing protein from human kidney may be products of the same gene(s) and (b) a portion of the complexing protein in human kidney is bound to the brush border membranes of cells lining the proximal tubules.     

Evidence for receptor-mediated uptake of adenosine deaminase in rabbit kidney

We investigated the subcellular location of adenosine deaminase-complexing protein in the proximal renal tubules of rabbit kidney and its interaction with intravenously infused monomeric calf adenosine deaminase. Cortical tissue from non-infused animals, stained in suspension by the peroxidase-antiperoxidase method for complexing protein and embedded in resin, was examined by transmission electron microscopy. Positive staining indicated the presence of complexing protein on the surface of microvilli in the proximal tubules. Sections (1 micron) of resin-embedded cortex from infused rabbits, stained first for complexing protein and then for adenosine deaminase, were examined by light microscopy. After staining for complexing protein by indirect immunofluorescence, the sections were photographed and then immersed in buffer containing 6 M guanidine hydrochloride plus 2-mercaptoethanol for 3 hr at 60 degrees C to remove bound antibodies. The sections were then stained by the peroxidase-antiperoxidase method for infused enzyme. Vesicle-like apical structures, the basal membrane area and, as previously reported, the brush border of proximal tubule cells were positive for complexing protein. Vesicle-like structures and brush borders positive for complexing protein were also stained for adenosine deaminase. The basal membrane area did not stain. These results support the hypothesis that complexing protein can act as a receptor for adenosine deaminase.     

Adenosine deaminase complexing proteins of the rabbit

1. Complexing proteins isolated from the soluble and particulate fractions of rabbit kidney homogenates are structurally similar to complexing protein from human kidney. 2. The distribution of soluble and particulate complexing protein in other rabbit tissues is also similar to humans. 3. As in human kidney, complexing protein is localized in the glomeruli and proximal tubules of rabbit kidney. 4. The rabbit appears to be an appropriate animal model for the study of the adenosine deaminase complexing proteins in humans.     

Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase.

Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the amino acid level except at their N termini and contain three dsRNA binding motifs, a putative nuclear targeting signal, and a possible deaminase motif. Antibodies raised against the protein encoded by the partial bovine clone specifically recognize the calf thymus dsRNA adenosine deaminase. Furthermore, the antibodies can immunodeplete a calf thymus extract of dsRNA adenosine deaminase activity, and the activity can be restored by addition of pure bovine deaminase. Staining of HeLa cells confirms the nuclear localization of the dsRNA-specific adenosine deaminase. In situ hybridization in rat brain slices indicates a widespread distribution of the enzyme in the brain.     

Purification of an adenosine deaminase complexing protein from human plasma.

A protein which specifically complexes with adenosine deaminase (complexing protein) has been purified to homogeneity from human plasma. This protein was compared with complexing protein isolated from human kidney. The two proteins produce electrophoretically different forms of high molecular weight adenosine deaminase when combined with the Mr = 36,000 enzyme monomer from erythrocytes. This difference may, at least in part, be due to the greater sialic acid content of complexing protein from plasma. By other criteria, including amino acid composition, total carbohydrate content, and subunit structure, the two proteins are quite similar. In addition, plasma complexing protein shows complete cross-reactivity with anti-kidney complexing protein serum. These results suggest that plasma and kidney complexing proteins are products of the same gene.     

Localization of adenosine deaminase and adenosine deaminase complexing protein in rabbit brain

Adenosine deaminase and adenosine deaminase complexing protein have been localized in rabbit brain. Brains fixed in paraformaldehyde or in Clarke's solution were blocked coronally. Blocks from brains fixed in paraformaldehyde were either frozen in liquid nitrogen or embedded in paraffin. Tissue fixed in Clarke's solution was embedded in paraffin. Sections from each block were stained by the peroxidase-antiperoxidase method for adenosine deaminase or complexing protein using affinity-purified goat antibodies. Adenosine deaminase and complexing protein did not co-localize. Adenosine deaminase was detected in oligodendroglia and in endothelial cells lining blood vessels, whereas complexing protein was concentrated in neurons. The subcellular location and appearance of the peroxidase reaction product associated with individual cells was also quite distinctive. The cell bodies of adenosine deaminase-positive oligodendroglia were filled with intense deposits of peroxidase reaction product. In contrast to oligodendroglia, the reaction product associated with most neurons stained for complexing protein was concentrated in granular-appearing cytoplasmic deposits. In some instances, these deposits were clustered about the nuclear membrane. Staining of neurons in the granular layer of cerebellum was an exception. Granule cells were lightly outlined by peroxidase reaction product. Cerebellar islands, also referred to as glomeruli, were stained an intense uniform brown. These results raise the possibility that oligodendroglia and blood vessel endothelia, through the action of adenosine deaminase, might play a role in controlling the concentration of extracellular adenosine in brain. They do not, however, support the suggestion that complexing protein aids in adenosine metabolism by positioning adenosine deaminase on the plasma membrane.     

Specific receptors for atriopeptin III in rabbit lung

Binding studies revealed the presence of a single class of high affinity binding sites for atriopeptin III on rabbit lung membranes. An apparent dissociation constant (Kd) of 0.32 nM and a binding capacity of 166 fmol/mg protein was determined. Binding was time-dependent and saturable. The relative binding affinities of atrial peptide analogs correlated well with their potencies in eliciting relaxation of norepinephrine-contracted rabbit aorta strips. Unrelated peptide hormones did not compete for the atriopeptin binding site on rabbit lung membranes. The atrial peptide binding data are similar to those obtained for other tissues and indicate the presence of a physiologically relevant atrial peptide receptor in lung.     

Histamine H1-receptor in the retina: species differences

Histamine H1-receptors in membranes of the various mammalian retinas were studied by [3H]mepyramine binding assay. Specific [3H]mepyramine bindings to bovine, pig, dog and human retinas were observed with the dissociation constants (KD), 3.8 +/- 1.2 nM, 1.8 +/- 0.6 nM, 2.6 +/- 0.6 nM and 3.0 +/- 0.9 nM, respectively, which were similar to those found in brains. But there was no detectable specific binding in the guinea-pig and rabbit retinas. The number of binding sites (Bmax) ranged from negligible value to 290.7 +/- 51.7 fmole/mg protein(human retina). Some H1-antagonists acted as potent agents in competing with [3H]mepyramine binding to bovine and pig retinas. These results indicated that histamine H1-receptors exist in some mammalian retina and have similar characteristics to those in brain membranes, but they distributes in the wide difference of the binding capacities among the species, while in brain variations were smaller.     

Assay optimization and kinetic profile of the human and the rabbit isoforms of 11beta-HSD1

Assay conditions for the 11beta-hydroxysteroid dehydrogenase have been optimized by adding phospholipids in the media buffer to increase and stabilize the enzymatic activity. The presence of phospholipids greatly facilitates the study of the binding of cortisone and NADPH at the enzyme catalytic site. Kinetic analyses conducted with the human and rabbit enzyme isoforms suggest that both enzymes behave according to an ordered sequential bi-bi mechanism where the NADPH is the first to bind at the active site followed by cortisone. The equilibrium dissociation constant, K(i)a as well as the apparent Michaelis-Menten constants K(m)a, K(m)b, k(cat)a, and k(cat)b for NADPH and cortisone, have been determined to be 147.5 microM, 14.4 microM, 43.8 nM, 0.21 min(-1), and 0.27 min(-1), respectively, for the human enzyme and 41.1 microM, 3.1 microM, 161.7 nM, 0.49 min(-1), and 0.52min(-1), respectively, for the rabbit enzyme.     

Slight differences between adenosine deaminases from different species an immunochemical study.

IgGs against adenosine deaminase from rat brain, rat liver, mouse duodenum and human erythrocyte were purified from rabbit antisera with yields of 82-87%. The inhibition of adenosine deaminase by the antienzyme is studied, and it is demonstrated that rat and mouse antibodies are tight-binding inhibitors. These antibodies inhibit either the rat or the mouse enzymes and do not inhibit the human erythrocytes enzyme. The human antibody does not inhibit either the human or the rat or mouse enzyme. These results indicate that some differences in antigenic behaviour near the active site must be encountered among species. Comparing the sequenced of the two products corresponding to two adenosine deaminase genes recently sequenced (human and murine) a hypothesis concerning the localization of the adenosine deaminase active site is proposed.     

Biochemical evidence for the presence of an amiloride binding protein in adult alveolar type II pneumocytes.

An amiloride binding protein in adult rat and rabbit alveolar type II (ATII) cells was characterized using three different antibodies against epithelial Na+ channel proteins. We found that 1) polyclonal antibodies raised against epithelial Na+ channel proteins from bovine kidney cross-react with a 135-kDa protein in ATII membrane vesicles on Western blots; 2) using the photoreactive amiloride analog, 2'-methoxy-5'-nitrobenzamil (NMBA), in combination with anti-amiloride antibodies, we found that NMBA specifically labeled the same M(r) protein; and 3) monoclonal anti-idiotypic antibodies directed against anti-amiloride antibodies also recognized this same M(r) protein on Western blots. We also demonstrated a low benzamil affinity binding site (apparent Kd = 370 nM) in rabbit ATII cell membranes and both high and low benzamil affinity binding sites (apparent Kd = 6 nM and 230 nM) in bovine kidney membranes using [3H]Br-benzamil as a ligand. Pharmacological inhibitory profiles for displacing bound [3H]Br-benzamil were also different between ATII cells and bovine kidneys. These observations indicate that adult ATII pneumocytes express a population of epithelial Na+ channels having a low affinity to benzamil and amiloride and a pharmacological inhibitory profile different from that in bovine kidney.     

Synthetic peptide SLTCLVKGFY competes with beta-endorphin for naloxone-insensitive binding sites on rat brain membranes

The synthetic decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain and its synthetic fragment VKGFY were found to compete with 125I-labeled beta-endorphin for high-affinity naloxone-insensitive binding sites on membranes isolated from the rat brain cortex (K(i)=1.18+/-0.09 and 1.58+/-0.11 nM, respectively). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to [Met(5)]enkephalin and [Leu(5)]enkephalin as well. The K(d) values characterizing the specific binding of 125I-labeled immunorphin and its fragment Val-Lys-Gly-Phe-Tyr to these binding sites were determined to be 2.93+/-0.27 nM and 3.17+/-0.29 nM, respectively.     

The antigen identified by a mouse monoclonal antibody raised against human renal cancer cells is the adenosine deaminase binding protein

The antigen recognized by a mouse monoclonal antibody (mAb S27) raised against a human renal cancer cell line has been identified as the adenosine deaminase binding protein. mAb S27 immunoprecipitates binding protein purified from a soluble fraction of human kidney. It also recognizes the mature 120,000-dalton membrane form of binding protein from [35S]methionine-labeled human fibroblasts, HepG2 cells, and the renal cancer cell line against which the antibody was raised. A rabbit polyclonal antibody raised against purified kidney binding protein completely precipitates mAb S27-reactive material from labeled membrane extracts. mAb S27 does not precipitate the initially synthesized 110,000 molecular weight precursor of binding protein in fibroblasts and recognizes only a small portion of binding protein precursor in labeled HepG2 cells suggesting that the antigenic determinant recognized by mAb S27 may be a post-translational modification present on the mature form of binding protein or that mAb S27 recognizes molecules in a certain conformation. Glycopeptides derived from purified soluble kidney binding protein or exogenously added adenosine deaminase do not inhibit the immunoprecipitation of binding protein by mAb S27, indicating that the mature oligosaccharide chains of binding protein are not the determinant recognized by mAb S27 and that bound adenosine deaminase does not mask the antigenic sites on binding protein. The fact that monoclonal antibody S27, previously shown (Ueda, R., Ogata, S., Morissey, D. M., Finstad, C. L., Szkudlavek, J., Whitmore, W. F., Oettgen, H. F., Lloyd, K. O., and Old, L. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5122-5126) to detect a cell surface antigen on cultured renal cancer cells, is directed against the adenosine deaminase binding protein confirms and extends the earlier observation (Andy, R.J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925) that binding protein is located on the cell surface.     

Modulation of A2A adenosine receptor(s) by K+(ATP) channels in bovine brain striatal membranes

The modulation of adenosine receptor with K+(ATP) channel blocker, glibenclamide, was investigated using the radiolabeled A2A-receptor selective agonist [3H]CGS 21680. Radioligand binding studies in bovine brain striatal membranes (BBM) indicated that unlabeled CGS 21680 displaced the bound [3H]CGS 21680 in a concentration-dependent manner with a maximum displacement being approximately 65% at 10(-4) M. In the presence of 10(-5) M glibenclamide, unlabeled CGS 21680 increased the displacement of bound [3H]CGS 21860 by approximately 28% at 10(-4) M. [3H]CGS 21680 bound to BBM in a saturable manner to a single binding site (Kd = 10.6+/-1.71 nM; Bmax = 221.4+/-6.43 fmol/mg of protein). In contrast, [3H]CGS 21680 showed saturable binding to two sites in the presence of 10(-5) M glibenclamide; (Kd = 1.3+/-0.22 nM; Bmax = 74.3+/-2.14 fmol/mg protein; and Kd = 8.9+/-0.64 nM; Bmax = 243.2+/-5.71 fmol/mg protein), indicating modulation of adenosine A2A receptors by glibenclamide. These studies suggest that the K+(ATP) channel blocker, glibenclamide, modulated the adenosine A2A receptor in such a manner that [3H]CGS 21680 alone recognizes a single affinity adenosine receptor, but that the interactions between K+(ATP) channels and adenosine receptors.     

Characterization of Membrane-Bound and Solubilized High-Affinity Binding Sites for 5'-N-Ethylcarboxamido[3H]adenosine from Bovine Cerebral Cortex

Abstract: A high-affinity binding site for 5'- N -ethylcarboxamido[³H]adenosine ([³H]NECA) from bovine cerebral cortex has been characterized in its membrane-bound and solubilized state after gel filtration on Sepharose CL-6B. For detection of this site in membranes, it was necessary to remove metabolites with high affinities for this site enzymatically, e.g., adenosine by addition of adenosine deaminase and inosine by addition of nucleoside phosphorylase. The pore-forming peptide antibiotic alamethicin further enhanced binding of [³H]NECA to this site in membranes. In contrast to adenosine receptors and the adenotin-like low-affinity binding protein, this novel site was extremely sensitive against treatment with the sulfhydryl alkylating agent N -ethylmaleimide. In competition experiments, this site could be differentiated from adenosine receptors by its high affinity for adenine nucleotides and its lack of affinity for adenosine receptor antagonists. Inosine and its derivative S -(4-nitrobenzyl)-6-thioinosine were relatively potent ligands with K _i values in the high nano- and low micromolar range, respectively. We conclude that the high-affinity NECA binding site described previously in bovine striatum is not exclusively located in the striatum, but can also be detected in membrane preparations and soluble extracts of bovine brain cortex.     

Separation of human from mouse and monkey adenosine deaminase by ion-exchange chromatography following retroviral-mediated gene transfer

A method for the chromatographic separation of human adenosine deaminase (ADA) from murine and monkey ADA is described. This procedure was developed in order to detect the expression of low or moderate levels of human ADA following retroviral-mediated gene transfer of cloned human ADA gene sequences into both mouse and monkey cells. Protein separation was achieved on a Mono Q (HR 5/5) anion-exchange column using the Pharmacia fast protein liquid chromatography system and was found to be a highly reproducible method yielding enzymatically active protein. An increasing linear gradient extending from 0.05 to 0.5 M potassium chloride (pH 7.5) was used to elute the enzyme. Under these conditions, most human ADA does not bind to the column and elutes in the low-salt buffer (0.05 M KCl), while murine ADA elutes at 0.12 M KCl and monkey ADA at 0.15 M KCl. The column fractions were assayed for ADA activity, and the characteristic isozyme banding patterns for human, mouse, and monkey ADA were confirmed by starch gel electrophoresis. This procedure allows the rapid and reproducible separation of human ADA from that of other species and yields partially purified enzymatically active protein.     

Kinetic and functional properties of [3H]ZM241385, a high affinity antagonist for adenosine A2A receptors

We have characterized the binding of [2-(3)H]-4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-[1,3,5]-triazin-5-ylamino]ethyl)phenol ([(3)H]ZM241385) to adenosine A(2A) receptors in membranes of rat striatum and transfected CHO cells. Saturation experiments showed that [(3)H]ZM241385 binds to a single class of binding sites with high affinity (K(d) = 0.23 nM and 0.14 nM in CHO cell and striatal membranes, respectively). The membranes of CHO cells required pretreatment with adenosine deaminase (ADA) to achieve high-affinity binding, while ADA had no influence on the ligand binding properties in striatal membranes. The binding of [(3)H]ZM241385 was fast and reversible, achieving equilibrium within 20 minutes at all radioligand concentrations. The kinetic analysis of the [(3)H]ZM241385 interaction with A(2A) receptors indicated that the reaction had at least two subsequent steps. The first step corresponds to a fast equilibrium, which also determines the antagonist potency to competitively inhibit CGS21680-induced accumulation of cAMP (first equilibrium constant K(A) = 6.6 nM). The second step corresponds to a slow process of conformational isomerization (equilibrium constant K(i) = 0.03). The combination of the two steps gives the dissociation constant K(d) = 0.20 nM based on the kinetic data, which is in good agreement with the directly measured value. The data obtained shed light on the mechanism of the [(3)H]ZM241385 interaction with adenosine A(2A) receptors from different sources in vitro. The isomerization step of the A(2A) antagonist radioligand binding has to be taken into account for the interpretation of the binding parameters obtained from the various competition assays and explain the discrepancy between antagonist affinity in saturation experiments versus its potency in functional assays.