Existence of a tightly regulated water channel in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae strain Sigma1278b possesses two putative aquaporins, Aqy1-1p and Aqy2-1p. Previous work demonstrated that Aqy1-1p functions as a water channel in Xenopus oocyte. However, no function could be attributed to Aqy2-1p in this system. Specific antibodies were used to follow the expression of Aqy1-1p and Aqy2-1p in the yeast. Aqy1-1p was never detected whatever the growth phase and culture conditions tested. In contrast, Aqy2-1p was detected only during the exponential growth phase in rich medium containing glucose. Aqy2-1p expression was repressed by hyper-osmotic culture conditions. Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2-1p is located on the endoplasmic reticulum (ER) as well as on the plasma membrane. In microsomal vesicles enriched in ER, a water channel activity due to Aqy2-1p was detected by stopped-flow analysis. Our results show that the expression of aquaporins is tightly controlled. The physiological relevance of aquaporin-mediated water transport in yeast is discussed.

Polymorphism of Saccharomyces cerevisiae aquaporins.

Aquaporin water channels facilitate the transmembrane diffusion of water and higher organisms possess a large number of isoforms. The genome of the yeast Saccharomyces cerevisiae contains two highly similar aquaporin genes, AQY1 and AQY2. AQY1 has been shown to encode a functional water channel but only in certain laboratory strains. Here we show that the AQY2 gene is interrupted by an 11 bp deletion in 23 of the 27 laboratory strains tested, with the exception of strains from the sigma 1278b background, which also exhibit a functional Aqy1p. However, although the AQY2 gene from sigma 1278b is highly homologous to functional aquaporins, we did not observe Aqy2p-mediated water transport in Xenopus oocytes. A survey of 52 yeast strains revealed that all industrial and wild yeasts carry the allele encoding a functional Aqy1p, while none of these strains appear to have a functional Aqy2p. We conclude that natural and industrial conditions provide selective pressure to maintain AQY1 but apparently not AQY2.

Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain.

The yeast YPR192w gene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nine S. cerevisiae strains. The osmotic water permeability coefficient (Pf) of X. laevis oocytes expressing the gene cloned from the Sigma1278b strain (AQY1-1) was 5.7 times higher than the Pf of oocytes expressing the gene cloned from other strains (AQY1-2). Aqy1-1p, initially cloned without its C-terminus (Aqy1-1DeltaCp), mediated an approximately 3 times higher water permeability than the full-length protein. This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy. Pf measurements in yeast spheroplasts confirmed the presence of functional water channels in Sigma1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin.

A yeast recombinant aquaporin mutant that is not expressed or mistargeted in Xenopus oocyte can be functionally analyzed in reconstituted proteoliposomes.

We have recently identified AQPcic (for aquaporin cicadella), an insect aquaporin found in the digestive tract of homopteran insects and involved in the elimination of water ingested in excess with the dietary sap (Le Cahérec, F., Deschamps, S., Delamarche, C., Pellerin, I., Bonnec, G., Guillam, M. T., Gouranton, J., Thomas, D., and Hubert, J. F. (1996) Eur. J. Biochem. 241, 707-715). Like many other aquaporins, AQPcic is inhibited by mercury reagents. In this study, we have demonstrated that residue Cys82 is essential for mercury inhibition. Another mutant version of AQPcic (AQP-C134S), expression of which in Xenopus laevis failed to produce an active molecule, was successfully expressed in Saccharomyces cerevisiae. Using stopped-flow analysis of reconstituted proteoliposomes, we demonstrated that the biological activity and Hg sensitivity of yeast-expressed wild type and mutant type AQPcic was readily assessed. Therefore, we propose that the yeast system is a valid alternative to Xenopus oocytes for studying particular mutants of aquaporin.

Evidence for the presence of aquaporin-3 in human red blood cells.

A facilitated diffusion for glycerol is present in human erythrocytes. Glycerol transporters identified to date belong to the Major Intrinsic Protein (MIP) family of integral membrane proteins, and one of them, aquaporin-3 (AQP3), has been characterized in mammals. Using an antibody raised against a peptide corresponding to the rat AQP3 carboxyl terminus, we examined the presence of AQP3 in normal and Colton-null (aquaporin-1 (AQP1)-deficient) human erythrocytes. Three immunoreactive bands were detected on immunoblots of both normal and Colton-null red cells, very similar to the bands revealed in rat kidney, a material in which AQP3 has been extensively studied. By immunofluorescence, anti-AQP3 antibodies stained the plasma membranes of both normal and Colton-null erythrocytes. Glycerol transport was measured on intact erythrocytes by stopped-flow light scattering and on one-step pink ghosts by a rapid filtration technique. Glycerol permeability values, similar in both cell types, suggest that AQP1 does not represent the major path for glycerol movement across red blood cell membranes. Furthermore, pharmacological studies showed that Colton-null red cells remain sensitive to water and glycerol flux inhibitors, supporting the idea that another proteinaceous path, probably AQP3, mediates most of the glycerol movements across red cell membranes and represents part of the residual water transport activity found in AQP1-deficient red cells.

Purified vesicles of tobacco cell vacuolar and plasma membranes exhibit dramatically different water permeability and water channel activity.

The vacuolar membrane or tonoplast (TP) and the plasma membrane (PM) of tobacco suspension cells were purified by free-flow electrophoresis (FFE) and aqueous two-phase partitioning, with enrichment factors from a crude microsomal fraction of >/=4- to 5-fold and reduced contamination by other cellular membranes. For each purified fraction, the mean apparent diameter of membrane vesicles was determined by freeze-fracture electron microscopy, and the osmotic shrinking kinetics of the vesicles were characterized by stopped-flow light scattering. Osmotic water permeability coefficients (Pf) of 6.1 +/- 0.2 and 7.6 +/- 0.9 microm . s(-1) were deduced for PM-enriched vesicles purified by FFE and phase partitioning, respectively. The associated activation energies (Ea; 13.7 +/- 1.0 and 13.4 +/- 1.4 kcal . mol(-1), respectively) suggest that water transport in the purified PM occurs mostly by diffusion across the lipid matrix. In contrast, water transport in TP vesicles purified by FFE was characterized by (i) a 100-fold higher Pf of 690 +/- 35 microm . s(-1), (ii) a reduced Ea of 2.5 +/- 1.3 kcal . mol(-1), and (iii) a reversible inhibition by mercuric chloride, up to 83% at 1 mM. These results provide functional evidence for channel-mediated water transport in the TP, and more generally in a higher plant membrane. A high TP Pf suggests a role for the vacuole in buffering osmotic fluctuations occurring in the cytoplasm. Thus, the differential water permeabilities and water channel activities observed in the tobacco TP and PM point to an original osmoregulatory function for water channels in relation to the typical compartmentation of plant cells.

Function and regulation of seed aquaporins.

The discovery of water channel proteins named aquaporins has shed new light on the molecular mechanisms of transmembrane water transport in higher plants. As with their animal counterparts, plant aquaporins belong to the large MIP family of transmembrane channels. An increasing number of aquaporins is now being identified on both the vacuolar and plasma membranes of plant cells, but their integrated function remains unclear. Aquaporin α-TIP is specifically expressed in the membrane of protein storage vacuoles in seeds of many plant species. α-TIP was previously shown to undergo phosphorylation in bean seeds. The functional significance of this process was further investigated after heterologous expression of the protein in Xenopus oocytes. Using site-directed mutagenesis of α-TIP and in vitro and in vivo phosphorylation by animal cAMP-dependent protein kinase, it is shown that, in oocytes, direct phosphorylation of α-TIP occurs at three distinct sites and stimulates its water channel activity. In addition to aquaporin phosphorylation, other mechanisms that target aquaporin function are used by living cells to regulate their membrane water permeability. These are the fine control of aquaporin gene expression and, in animal cells only, the regulated trafficking of water channel-containing vesicles. The present work and studies by others on the phosphorylation of nodulin-26, an ion channel protein homologous to α-TIP, provide novel insights into the mechanisms of plant membrane protein regulation. These studies might help identifying and characterizing novel membrane-bound protein kinases and phosphatases. Finally, an integrated function for seed vacuolar aquaporins is discussed. During germination, the rehydration of seed cells, the drastic changes in vacuole morphology, the breakdown and the mobilization of storage products from the vacuole may create osmotic perturbations in the cytoplasm. The fine tuning of TIP aquaporin activity may help control the kinetics and amplitude of osmotic water flows across the tonoplast to achieve proper cytoplasm osmoregulation and control of vacuolar volume.

Purification and functional reconstitution of the human CHIP28 water channel expressed in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was used for heterologous expression of the human CHIP28 water Aquaporin-1 channel (Aquaporin-1). A nine-amino-acid epitope of the influenza hemagglutinin protein (HA epitope), recognized by the monoclonal antibody 12CA5, was chosen to tag CHIP28 at its N-terminus. Epitope-tagged CHIP28 was purified from yeast extracts by immunochromatography on protein A/ 12CA5-coupled beads, after KI extraction and detergent solubilization, then concentrated by anion exchange chromatography. Purified protein was reconstituted in proteoliposomes and was shown to function as a water channel by stopped-flow spectrophotometry. This study demonstrates that the yeast has the capacity to produce functional aquaporins at levels sufficient for biochemical and biophysical analyses.

Glycerol permeability of mutant aquaporin 1 and other AQP-MIP proteins: inhibition studies.

In a recent work, we showed that the aquaporins 1 (AQP1) are permeable to certain small solutes such as glycerol. Here, we have further investigated the permeation pathway of glycerol through human AQP1 (hAQP1) by the use of mutants (C189S, H180A, H209A) and inhibitors such as P-chloromercuribenzene sulphonate (pCMBS), CuSO4 or phloretin, in comparison with other AQP-MIP (where MIP denotes major intrinsic protein) proteins: hAQP2, plant water channel gammaTIP and bacterial glycerol permease facilitator, GlpF. Glycerol movements were measured in Xenopus laevis oocytes. Apparent glycerol permeability coefficients (P'gly) were calculated from the rates of oocyte swelling upon exposure to an isoosmotic medium containing an inwardly directed gradient of glycerol and from [3H]glycerol uptake measurements. Similar P'gly values were obtained for hAQP1 and hAQP2 6 to 8 times greater than control indicating that hAQP2 also transports glycerol. P'gly of hAQP2-injected oocytes was pCMBS and CuSO4 sensitive. In contrast, the P'gly value of gammaTIP was close to that of control, indicating that gammaTIP does not transport glycerol. The hAQP1-C189S, -H180A and -H209A mutants gave P'gly values similar to those obtained for wild hAQP1, indicating that these mutations did not affect glycerol movements. However, the H209A mutant has an osmotic water permeability coefficient (Pf) value decreased by 50%. The inhibitory effect pCMBS on P'gly was maintained for the 2 His mutants and, more interestingly, was also conserved for the C189S mutant. CuSO4 significantly inhibited P'gly of oocytes expressing hAQP1, hAQP1-C189S, -H180A, and -H209A mutants and had no effect on P'gly of GlpF-injected oocytes. Phloretin was shown to inhibit by around 80% the glycerol fluxes of wild and mutant hAQP1, hAQP2 and to fully inhibit glycerol uptake in GlpF-injected oocytes.

Functional expression of the human CHIP28 water channel in a yeast secretory mutant.

The temperature-sensitive Saccharomyces cerevisiae mutant strain NY17, deficient in the secretory pathway (sec6-4 mutation), is used for the heterologous expression of the human CHIP28 water channel. After a heat-shock, the protein is present in partially purified post-golgi secretory vesicles. Immunodetection and water transport studies, directly made on the vesicles, showed that CHIP28 is highly expressed and active in the yeast membranes.

Evidence for a glycerol pathway through aquaporin 1 (CHIP28) channels.

Permeabilities to glycerol and small non-electrolytes of three Aquaporin 1 CHIP (AQP1) water channels were measured in AQP1 cRNA-injected Xenopus laevis oocytes and in human AQP1 channels reconstituted in proteoliposomes. By an "osmotic" swelling assay, significant increases of ethylene glycol, glycerol and 1,3-propanediol apparent permeability coefficients (P'solutes) were found in oocytes expressing human, rat and frog AQP1. p-Chloromercuribenzene sulphonate (pCMBS) and CuSO4 inhibited, by 95% and 58% respectively, apparent glycerol permeability (P'gly) in oocytes expressing human AQP1. pCMBS inhibition was reversed by beta-mercaptoethanol and CuSO4 inhibition was partly reversed by the Cu(2+)-binding peptide Gly-Gly-His. Tritiated glycerol uptakes confirmed the augmented P'gly value of AQP1 cRNA-injected oocytes. In contrast, no increases of urea, meso-erythritol, D-or L-threitol, xylitol and mannitol uptakes were detected. Stopped-flow light scattering experiments performed with human AQP1 proteoliposomes also revealed a much greater increase of P'gly than did those with protein-free liposomes; the initial rate of proteoliposomes also swelling was inhibited by 96.2% with HgCl2 and by 72.5% with CuSO4. In AQP1 cRNA-injected oocytes and in proteoliposomes, the value of the glycerol reflection coefficient was 0.74-0.80, indicating that water and glycerol share the same pathway. All these results provide strong evidence that water and certain small solutes permeate the AQP1 channels expressed at the surface of X. laevis oocytes or reconstituted in proteoliposomes. The urea exclusion suggests that the selectivity of the AQP1 channels not only depends on the size of the solutes but probably also on their flexibility and their ability to form H-bonds.

Effect of aging on zinc and histidine transport across rat intestinal brush-border membranes.

The effects of aging on intestinal absorption of zinc and L-histidine were investigated in adult (10-month-old) and senescent (30-month-old) Wistar rats' brush-border membrane vesicles isolated from jejunum and ileum. Kinetic parameters of the zinc transport by the jejunal brush-border membrane were Jmax = 126 +/- 24 nmol.min-1.mg-1 protein and Km = 490 +/- 126 microM (10-month-old rats, n = 7). The transport of zinc was the same in the jejunum and the ileum of adult animals. In senescent rats, the zinc uptake was significantly lower in the distal part of the intestine than in the proximal one. A comparison of zinc uptake in 10- and 30-month-old rats showed that the transport capacity of the jejunum did not change with age but the ileal transport capacity decreased by 50%. This reduced uptake was associated with an increased cholesterol content of the brush-border membrane. The major site of L-histidine absorption was the jejunum, in both the 10- and 30-month-old animals. L-Histidine was co-transported with Na+. The kinetic parameters of the L-histidine carrier in the presence of Na+ were Jmax = 6.5 +/- 1.0 nmol.min-1.mg-1 protein and Km = 190 +/- 29 microM in the jejunum of 10-month-old rats (n = 12). Increasing the extra-vesicular concentration of zinc (0 --> 1 mM) reduced the uptake of L-histidine, and conversely increasing the concentration of L-histidine (0 --> 1 mM) reduced that of zinc: there was no evidence of transport of a complexed form [zinc-L-histidine] in brush-border membranes of the small intestine. During aging, the transport capacity of L-histidine by the jejunum decreased, whereas the ileal transport capacity was conserved. The modifications of absorptive capacity for zinc and L-histidine at the membrane level (loss of ileal function for zinc, and loss of jejunal function for amino acid) indicate that the normal aging of intestinal epithelial cells cannot be regarded as a decline in the overall transport of nutriments but as a combination of highly specific modifications of the various transport systems.

The low molecular weight protein which co-purifies with alpha-latrotoxin is structurally related to crustacean hyperglycemic hormones.

LMWP is the low molecular weight protein which copurifies with alpha-latrotoxin, the main neurotoxin from the black widow venom. It contains 70 residues and three disulfides. We found that its primary structure, including its 6 half-cystines, can be aligned with the amino acid sequences of crustacean hyperglycemic hormones (CHHs) which contain 72-73 residues and three disulfides. To further investigate this structural relationship, we produced a recombinant analog of LMWP in which the unique Met was changed in Leu (LMWPM35L). LMWPM35L was produced as a folded fusion protein in the periplasm of Escherichia coli and was generated in vitro by treating the fusion protein with cyanogen bromide. We showed that LMWPM35L and CHHs have an identical disulfide pairing pattern and possess some alpha-helical structure, as deduced from a comparison of their circular dichroism spectra. In addition, LMWPM35L and CHHs are consensually predicted to possess a helical structure within the region 13-17. Together, the data indicate that CHHs are structurally related to LMWPM35L and presumably also to LMWP. Finally, preliminary studies showed that LMWPM35L is not toxic to mice and does not form channels in lipid bilayers, two well-known properties of alpha-latrotoxin preparations.

Mechanisms of zinc transport into pig small intestine brush-border membrane vesicles.

1. The purpose of the present work was to examine certain membrane transport mechanisms likely to carry zinc across the brush-border membrane of pig small intestine, isolated in a vesicular form. 2. In initial velocity conditions, saturation kinetics revealed a great effect of pH on zinc transport: optimal conditions were observed with an intravesicular pH of around 6.6 with or without a H+ gradient; however, this did not allow us to conclude the existence of a neutral exchange between Zn2+ and H+ ions. 3. By measuring 36Cl uptakes, the presence of the Cl(-)-HCO3- or Cl(-)-OH-antiporter with typical 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) sensitivity was detected in vesicles; zinc did not alter this anionic exchange activity. A 65Zn time course, performed in conditions identical with those for 36Cl uptake, was DIDS insensitive and was greatly inhibited by an outward OH- gradient. This could argue against a transport of zinc as a complex with Cl- and HCO3- through the anion antiporter. 4. When external Cl- and HCO3- were replaced by SCN-, able to form a Zn(SCN)4(2-) complex, we observed a stimulating effect of outward HCO3- gradients on 65Zn uptake but neither DIDS nor diphenylamine-2-carboxylate (DPC) inhibited the transport in these conditions. This suggested that the intestinal anion antiporter was not a major route for zinc reabsorption. 5. The tripeptide Gly-Gly-His at low concentrations stimulated 65Zn uptake, then inhibited it in a dose-dependent manner either in the presence of an inward H+ gradient or in the presence of a membrane potential 'negative inside' or in both situations. These conditions are necessary for the active transport of the peptide and this strongly suggests that zinc can be transported as a [Gly-Gly-His-Zn] complex, utilizing the peptide carrier system.

Studies on the catabolism of glycated haemoglobin: Glycated globin is not a substrate for the glucosyl-galactosyl-hydroxylysine glucosidase but for a peptidase activity present in rat kidney and spleen.

Rat kidney and spleen glucosyl-galactosyl-hydroxylysine glucosidase (EC.3.2.1.107) whose specificity for the hydroxylysine-linked disaccharide units present in collagens depends upon the substrate's free amino group was tested for its glycosidase activity on the ketoamine form of glycated [(14)C]Glc-Globin. The most stable preparations of the enzyme from normal and diabetic rat tissues, partially purified by ultracentrifugation and ammonium sulphate fractionation, were used. These glucosidase preparations did not release any significant amount of radioactive neutral hexose. But a radioactive glycopeptide of about 1,400 Da was released from [(14)C]Glc-Globin at a pH optimum of 4.0. It appears to be released by a peptidase activity present in the kidney and spleen of normal and diabetic rats.

Zinc binding in intestinal brush-border membrane isolated from pig.

Zinc binding to brush-border membrane vesicles isolated from pig jejunum was investigated by a rapid filtration method, for long incubation periods (up to 180 min). Zn2+ influx revealed a large accumulation of the metal, reaching an apparent intravesicular volume of 160 microliters/mg protein at equilibrium, a volume 45-times that of an osmotically reactive sugar, sorbitol (3.6 microliters/mg protein). Changes in medium osmolarity had no effect on zinc accumulation. These results suggested a large degree of zinc binding to vesicular components (membrane or core). 65Zn efflux measurements led to the conclusion that two vesicular pools of zinc existed: a small external pool, accessible to different chelators (EGTA) or competitive cations, and a large intravesicular pool. Accumulated 65Zn was quickly removed from its internal sites only after the membrane had been permeabilized by the cation ionophore A23187 in association with an exchange molecule or a chelator. Scatchard plot analyses revealed, on one hand a first class of high-affinity extravesicular zinc binding sites (Ka = 8.6.10(3) M-1, n = 0.455 nmol Zn2+/mg protein) and a second class of extravesicular sites having a very low affinity (Ka = 22 M-1, n = 25.35 nmol Zn2+/mg protein) and, on the other hand one type of intravesicular sites (Ka = 3.3.10(4) M-1, n = 550 nmol Zn2+/mg protein). The intravesicular sites have a high affinity for zinc and are specific, since only nonlabelled zinc (or cadmium) but not calcium present in the bathing medium is exchanged with the internally accumulated labelled cation.

31P-NMR study of pig intestinal brush-border membrane structure: effect of zinc and cadmium ions.

31P-NMR experiments on intact pig small intestine brush-border membrane vesicles (BBMV) and detergent-solubilized membranes gave direct insights into the organization of the phospholipids (PL) and their interaction with zinc and cadmium ions. Various endogenous PL were identified from well resolved BBM micelle spectra. These experiments revealed a strong interaction of Zn2+ and Cd2+ with the negatively charged phosphatidylinositol and phosphatidylserine. In BBM micelles, a progressive time-dependent PL degradation occurred in the absence of ions and indicated the presence of active phospholipases. The presence of zinc inhibited the degradation process whereas cadmium had the opposite influence. 31P spectra of BBMV were carefully characterized. Neither zinc nor cadmium affected the PL bilayer structural organization. A degradation of PL, monitored by the increase of the inorganic phosphate (Pi) signal, also occurred in vesicles but to a lesser extent than in micelles. A 2/3 internal, 1/3 external PL asymmetry was observed in the absence and presence of ions.

Studies of zinc transport into brush-border membrane vesicles isolated from pig small intestine.

Zinc transport into brush-border membrane vesicles was investigated by measuring uptake rates at a very short incubation time (2 seconds), during the initial linear uptake. A divalent cation chelator (EGTA) was added to the stop and washout solutions in order to remove the zinc bound to the external surface of the vesicles. Under these conditions, we showed that zinc enters the vesicles by (1) a saturable carrier-mediated process, and (2) an unsaturable pathway. The kinetic parameters we calculated were an affinity of 0.215 +/- 0.039 mM, a Jmax of 17.2 +/- 1.7 nmol.min-1.(mg protein)-1 and an unsaturable constant of 0.025 +/- 0.006 (n = 6). The imposition of an outwardly directed K+ gradient (negative inside) did not affect the Jmax value of the zinc uptake but increased the Km value significantly. This suggests that, at least a portion of zinc which crosses the membrane does not do so in a cationic form. Zinc uptake was decreased or increased according to the nature of accompanying anions (Cl-, SO4(2)-, SCN-) in the absence of any membrane potential. With highly permeant anions such as thiocyanates, zinc uptake was considerably augmented, suggesting a movement of zinc in a complexed form involving the presence of negative species. We also showed that cadmium competitively inhibited the zinc uptake; we measured a Ki value of 0.21 mM, indicating a similar affinity of cadmium for the carrier as zinc itself. By contrast, the presence of calcium had little effect on zinc entry into vesicles. The calcium ionophore A23187 had only a slight stimulating effect on zinc uptake. These results indicate that zinc and calcium transports are probably independent of each other.

Neutral aminopeptidase: a potential marker enzyme of the amphibian urinary bladder epithelial cell apical membrane.

Antidiuretic hormone induces, in the apical plasma membrane of amphibian urinary bladder epithelial cells, the exocytotic insertion of intramembranous particle aggregates that probably contain water channels. Purification of the apical membrane is a way to characterize the aggregates. The isolation of such purified membranous fractions involves the use of specific exogenous or endogenous markers. One of them could be the neutral aminopeptidase (AP), whose activity was detected in urinary bladder. Enrichment in AP activity was observed in plasma membrane preparations compared to cell homogenates (X2.7). However, a large part of the enzyme activity was also recovered in the soluble fraction of the preparation, suggesting large proteolysis of the protein. The enzyme presents a low optimal pH (6.4) and a high specificity for proline-p-nitroanilide as compared to the AP present in kidneys and intestines. To localize the protein in the amphibian bladder epithelium, an immunological approach was necessary due to the low activity of the enzyme in this tissue. The low enzymatic activity also prevented the purification of sufficient amounts of the urinary bladder AP as antigen, and we prepared antibodies against purified AP from frog or toad kidneys where the activity is 60 times higher than in the bladder. The serum specificity was verified by spot immunodetection, Western blot, inhibition capacity of antibodies, and immunoadsorption on a solid support with the renal enzyme. The sera were found to be able to react with native as well as denatured forms of the kidney enzyme. Antibodies cross-reacted with several peptides of low molecular weight (40-60 kDa) from urinary bladder plasma membrane proteins (Western blot).(ABSTRACT TRUNCATED AT 250 WORDS)