BH3 domain-independent apolipoprotein L1 toxicity rescued by BCL2 prosurvival proteins.

The potent trypanolytic properties of human apolipoprotein L1 (APOL1) can be neutralized by the trypanosome variant surface antigen gene product known as serum resistance-associated protein. However, two common APOL1 haplotypes present uniquely in individuals of West African ancestry each encode APOL1 variants resistant to serum resistance-associated protein, and each confers substantial resistance to human African sleeping sickness. In contrast to the dominantly inherited anti-trypanosomal activity of APOL1, recessive inheritance of these two trypanoprotective APOL1 alleles predisposes to kidney disease. Proposed mechanisms of APOL1 toxicity have included BH3 domain-dependent autophagy and/or ion channel activity. We probed these potential mechanisms by expressing APOL1 in Xenopus laevis oocytes. APOL1 expression in oocytes increased ion permeability and caused profound morphological deterioration (toxicity). Coexpression of BCL2 family members rescued APOL1-associated oocyte toxicity in the order MCL1 ∼ BCLW > BCLXL ∼ BCL2A1 ≫ BCL2. Deletion of nine nominal core BH3 domain residues abolished APOL1-associated toxicity, but missense substitution of the same residues abolished neither oocyte toxicity nor its rescue by coexpressed MCL1. The APOL1 BH3 domain was similarly dispensable for the ability of APOL1 to rescue intact mice from lethal trypanosome challenge. Replacement of most extracellular Na(+) by K(+) also reduced APOL1-associated oocyte toxicity, allowing demonstration of APOL1-associated increases in Ca(2+) and Cl(-) fluxes and oocyte ion currents, which were similarly reduced by MCL1 coexpression. Thus APOL1 toxicity in Xenopus oocytes is BH3-independent, but can nonetheless be rescued by some BCL2 family proteins.

Acute regulation of mouse AE2 anion exchanger requires isoform-specific amino acid residues from most of the transmembrane domain.

The widely expressed anion exchanger polypeptide AE2/SLC4A2 is acutely inhibited by acidic intracellular (pH(i)), by acidic extracellular pH (pH(o)), and by the calmodulin inhibitor, calmidazolium, whereas it is acutely activated by NH(4)(+). The homologous erythroid/kidney AE1/SLC4A1 polypeptide is insensitive to these regulators. Each of these AE2 regulatory responses requires the presence of AE2's C-terminal transmembrane domain (TMD). We have now measured (36)Cl(-) efflux from Xenopus oocytes expressing bi- or tripartite AE2-AE1 chimeras to define TMD subregions in which AE2-specific sequences contribute to acute regulation. The chimeric AE polypeptides were all functional at pH(o) 7.4, with the sole exception of AE2((1-920))/AE1((613-811))/AE2((1120-1237)). Reciprocal exchanges of the large third extracellular loops were without effect. AE2 regulation by pH(i), pH(o) and NH(4)(+) was retained after substitution of C-terminal AE2 amino acids 1120-1237 (including the putative second re-entrant loop, two TM spans and the cytoplasmic tail) with the corresponding AE1 sequence. In contrast, the presence of this AE2 C-terminal sequence was both necessary and sufficient for inhibition by calmidazolium. All other tested TMD substitutions abolished AE2 pH(i) sensitivity, abolished or severely attenuated sensitivity to pH(o) and removed sensitivity to NH(4)(+). Loss of AE2 pH(i) sensitivity was not rescued by co-expression of a complementary AE2 sequence within separate full-length chimeras or AE2 subdomains. Thus, normal regulation of AE2 by pH and other ligands requires AE2-specific sequence from most regions of the AE2 TMD, with the exceptions of the third extracellular loop and a short C-terminal sequence. We conclude that the individual TMD amino acid residues previously identified as influencing acute regulation of AE2 exert that influence within a regulatory structure requiring essential contributions from multiple regions of the AE2 TMD.

Regulation of AE2-mediated Cl- transport by intracellular or by extracellular pH requires highly conserved amino acid residues of the AE2 NH2-terminal cytoplasmic domain.

We reported recently that regulation by intracellular pH (pH(i)) of the murine Cl-/HCO(3)(-) exchanger AE2 requires amino acid residues 310-347 of the polypeptide's NH(2)-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH(o)) with unclamped or clamped pH(i), or during variation of pH(i) at constant pH(o). Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited by acid pH(o), with a value of pH(o50) = 6.87 +/- 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312-347 identified the greatest acid shift in pH(o(50)) value, approximately 0.8 pH units in the mutant (A)6 342-347, but only a modest acid-shift in the mutant (A)6 336-341. Two of the six (A)6 mutants retained normal pH(i) sensitivity of 36Cl- efflux, whereas the (A)6 mutants 318-323, 336-341, and 342-347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH(o) and to pH(i) were found independently and in concert. The E346A mutation acid-shifted the pH(o(0) value to the same extent whether pH(i) was unclamped or held constant during variation of pH(o). Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH(o) as well as pH(i).

A dominant negative mutant of the KCC1 K-Cl cotransporter: both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity.

K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunct strategy for the treatment of sickle cell anemia. However, structure-function relationships among the polypeptide products of the four K-Cl cotransporter (KCC) genes are little understood. We have investigated the importance of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl cotransport function expressed in Xenopus oocytes. Truncation of as few as eight C-terminal amino acids (aa) abolished function despite continued polypeptide accumulation and surface expression. These C-terminal loss-of-function mutants lacked a dominant negative phenotype. Truncation of the N-terminal 46 aa diminished function. Removal of 89 or 117 aa (Delta(N)117) abolished function despite continued polypeptide accumulation and surface expression and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant Delta(N)117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. Delta(N)117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2.

Structure and complex transcription pattern of the mouse SK1 K(Ca) channel gene, KCNN1.

Small conductance calcium-gated K(+) channels (SK channels) are encoded by the three SK genes, SK1, SK2, and SK3. These channels likely contribute to slow synaptic afterhyperpolarizations of apamin-sensitive and apamin-insensitive types. SK channels are also widely expressed outside the nervous system. The mouse SK1 gene comprises at least 12 exons extending across 19.8 kb of genomic DNA. This gene encodes a complex pattern of alternatively spliced SK1 transcripts widely expressed among mouse tissues. These transcripts exhibit at least four distinct 5'-nucleotide sequence variants encoding at least two N-terminal amino acid sequences. Optional inclusion of exons 7 and 9, together with two alternate splice donor sites in exon 8, yields transcripts encoding eight variant C-terminal amino acid sequences for SK1. These include an altered putative S6 transmembrane span, modification of the C-terminal cytoplasmic domain binding site for calmodulin, and generation of two alternate predicted binding sites for PDZ domain-containing proteins. 20 of the 32 predicted mouse SK1 transcripts are expressed in brain at levels sufficient to allow consistent detection, and encode 16 SK1 polypeptide variants. Only four of these 16 polypeptides preserve the ability to bind calmodulin in a Ca(2+)-independent manner. Mouse SK1 also exhibits novel, strain-specific, length polymorphism of a polyglutamate repeat in the N-terminal cytoplasmic domain. The evolutionary conservation of this complex transcription pattern suggests a possible role in the tuning of SK1 channel function.

AE anion exchangers in atrial tumor cells.

Intracellular pH homeostasis and intracellular Cl(-) concentration in cardiac myocytes are regulated by anion exchange mechanisms. In physiological extracellular Cl(-) concentrations, Cl(-)/HCO(3)(-) exchange promotes intracellular acidification and Cl(-) loading sensitive to inhibition by stilbene disulfonates. We investigated the expression of AE anion exchangers in the AT-1 mouse atrial tumor cell line. Cultured AT-1 cells exhibited a substantial basal Na(+)-independent Cl(-)/HCO(3)(-) (but not Cl(-)/OH(-)) exchange activity that was inhibited by DIDS but not by dibenzamidostilbene disulfonic acid (DBDS). AT-1 cell Cl(-)/HCO(3)(-) activity was stimulated two- to threefold by extracellular ATP and ANG II. AE mRNAs detected by RT-PCR in AT-1 cells included brain AE3 (bAE3), cardiac AE3 (cAE3), AE2a, AE2b, AE2c1, AE2c2, and erythroid AE1 (eAE1), but not kidney AE1 (kAE1). Cultured AT-1 cells expressed AE2, cAE3, and bAE3 polypeptides, which were detected by immunoblot and immunocytochemistry. An AE1-like epitope was detected by immunocytochemistry but not by immunoblot. Both bAE3 and cAE3 were present in intact AT-1 tumors. Cultured AT-1 cells provide a useful system for the study of mediators and regulators of Cl(-)/HCO(3)(-) exchange activity in an atrial cell type.

Structure and genetic polymorphism of the mouse KCC1 gene.

The KCC1 K-Cl cotransporter is a major regulator of erythroid and non-erythroid cell volume, and the KCC1 gene is a candidate modifier gene for sickle cell disease and other hemoglobinopathies. We have cloned and sequenced the mouse KCC1 (mKCC1) gene, defined its intron-exon junctions, and analyzed (AC)/(TG) intragenic polymorphisms. A highly polymorphic (AC) repeat of mKCC1 intron 1 was characterized in musculus strains, and used to prove lack of linkage between the mKCC1 gene and the rol (resistant to osmotic lysis) locus. The intron 1 (AC) repeat in CAST/Ei and SPRET/Ei was not only more divergent in length but also underwent additional sequence variation. A dimorphic (TG) repeat in intron 2 distinguished CAST/Ei from other strains, and an intron 17 B1 Alu-like SINE present in all musculus strains was found to be absent from intron 17 in SPRET/Ei. These and additional described strain-specific polymorphisms will be useful mapping and genetic tools in the study of mouse models of sickle cell disease.

Functional and molecular characterization of an anion exchanger in airway serous epithelial cells.

Serous cells secrete Cl(-) and HCO(3)(-) and play an important role in airway function. Recent studies suggest that a Cl(-)/HCO(3)(-) anion exchanger (AE) may contribute to Cl(-) secretion by airway epithelial cells. However, the molecular identity, the cellular location, and the contribution of AEs to Cl(-) secretion in serous epithelial cells in tracheal submucosal glands are unknown. The goal of the present study was to determine the molecular identity, the cellular location, and the role of AEs in the function of serous epithelial cells. To this end, Calu-3 cells, a human airway cell line with a serous-cell phenotype, were studied by RT-PCR, immunoblot, and electrophysiological analysis to examine the role of AEs in Cl(-) secretion. In addition, the subcellular location of AE proteins was examined by immunofluorescence microscopy. Calu-3 cells expressed mRNA and protein for AE2 as determined by RT-PCR and Western blot analysis, respectively. Immunofluorescence microscopy identified AE2 in the basolateral membrane of Calu-3 cells in culture and rat tracheal serous cells in situ. In Cl(-)/HCO(3)(-)/Na(+)-containing media, the 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP)-stimulated short-circuit anion current (I(sc)) was reduced by basolateral but not by apical application of 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (50 microM) and 4, 4'-dinitrostilbene-2,2'-disulfonic acid [DNDS (500 microM)], inhibitors of AEs. In the absence of Na(+) in the bath solutions, to eliminate the contributions of the Na(+)/HCO(3)(-) and Na(+)/K(+)/2Cl(-) cotransporters to I(sc), CPT-cAMP stimulated a small DNDS-sensitive I(sc). Taken together with previous studies, these observations suggest that a small component of cAMP-stimulated I(sc) across serous cells may be referable to Cl(-) secretion and that uptake of Cl(-) across the basolateral membrane may be mediated by AE2.

Short sequence repeat polymorphism in the mouse slc4al gene encoding the AE1 Cl-/HCO3-exchanger.

The human AE1 anion exchanger gene SLC4A1 encodes the Cl-/HCO3-exchangers of the erythrocyte and the Type Acid-secreting intercalated cell basolateral membrane. Mutations in SLC4A1 have been correspondingly linked with autosomal dominant hereditary spherocytotic anemia and with both dominant and recessive forms of distal renal tubular acidosis. Murine knockouts in the slc4a1Ae1 gene have also been generated, and lack erythroid and renal expression. However, intragenic polymorphic markers for the slc4a1 gene have been unavailable. Here we report that a previously identified CA repeat element of intron 13 of the murine Ae1 gene exhibits strain-specific length polymorphism.

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation.

Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endogenous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and mapped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised against recombinant mKCC1 function as immunoblot and immunoprecipitation reagents. The tissue distributions of mKCC1 mRNA and protein are widespread, and mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes of multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticulocyte counts in density-fractionated cells, in bleeding-induced reticulocytosis, in mouse models of sickle cell disease and thalassemia, and in the corresponding human disorders. mKCC1-mediated uptake of (86)Rb into Xenopus oocytes requires extracellular Cl(-), is blocked by the diuretic R(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2, 3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

Expression of AE2 anion exchanger in mouse intestine.

We have characterized expression of anion exchanger 2 (AE2) mRNA and protein in the mouse intestine. AE2 mRNA abundance was higher in colon than in more proximal segments. AE2a mRNA was more abundant than AE2b mRNA throughout the intestine, and AE2c mRNA was expressed at very low levels. This AE2 mRNA pattern contrasted with that in mouse stomach, in which AE2c > AE2b > AE2a. AE2 polypeptide abundance as detected by immunoblot qualitatively paralleled that of mRNA, whereas AE2 immunostaining exhibited a more continuous decrease in intensity from colon to duodenum. AE2 polypeptide was more abundant in colonic surface cells than in crypts, whereas ileal crypts and villi exhibited similar AE2 abundance. AE2 was also observed in mural and vascular smooth muscle. Localization of AE2 epitopes was restricted to the basolateral membranes of epithelial cells throughout the intestine with three exceptions. Under mild fixation conditions, anti-AE2 amino acids (aa) 109-122 detected nonpolarized immunostaining of ileal enterocytes and of Paneth cell granule membranes. An epitope detected by anti-AE2 aa 1224-1237 was also localized to subapical regions of Brunner's gland ducts of duodenum and upper jejunum. These localization studies will aid in the interpretation of anion exchanger function measured in epithelial sheets, isolated cells, and membrane vesicles.

AE2 anion exchanger polypeptide is a homooligomer in pig gastric membranes: a chemical cross-linking study.

Although considerable information is available on the oligomeric states of the AE1 (band 3) anion exchanger, little is known about the physiological state of the polypeptides encoded by the nonerythroid AE genes, AE2 and AE3. We have previously characterized the proteolytic susceptibility of native pig gastric AE2. In the course of studies in which pig gastric membranes were treated with the AE2 transport antagonist, DIDS, we noted evidence for cross-linking of AE2 proteolytic fragments to higher-order oligomeric forms. We have characterized the ability of DIDS and of selected N-hydroxysuccinimide cross-linking agents to increase the proportion of SDS-resistant oligomers of pig gastric AE2 and its proteolytic fragments. Cross-linking exhibited time and concentration dependence. N-Terminal protein sequencing proved that DIDS treatment created AE2 homodimers. Putative homotetramers were also observed. Protomers were cross-linked via residues within the C-terminal 40 kDa of AE2. Prior proteolytic cleavage of AE2 in membranes resulted in decreased yield of subsequently cross-linked products. AE2 cross-linking could not be detected in membranes pretreated by hypotonic wash and freeze-thaw. The results are interpreted in light of the deduced amino acid sequence of the transmembrane domain of pig AE2.

cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation.

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.

Immunolocalization and tissue-specific splicing of AE2 anion exchanger in mouse kidney.

In this study, an epitope-unmasking technique was used to immunolocalize AE2 anion exchanger polypeptide to basolateral plasma membranes of tubular epithelial cells in mouse kidney. Kidney AE2 immunostaining in mouse kidney was less prominent than in rat, consistent with the relative levels of AE2 mRNA and polypeptide in these two species. Glomeruli showed faint but consistent AE2 immunostaining, whereas proximal tubules were generally unstained. Macula densa epithelial cells displayed bright AE2 immunostaining, and cortical thick limbs were stained at a lower intensity. AE2 immunostaining was weak or absent in type B intercalated cells and principal cells of the cortical collecting duct, but increased in intensity in principal cells of the inner stripe of the outer medulla. AE2 staining in medullary thick limbs was also of greater intensity than in cortical thick limbs. AE2 staining was strong and uniform in the epithelial cells of the inner medullary collecting duct, and in epithelial cells of the papillary surface, the ureter, and the urinary bladder. Extratubular and epithelial cells of the inner medulla also showed punctate intracellular AE2 staining in a Golgi-like distribution that, in contrast to cell surface staining, was sodium dodecyl sulfate-sensitive. Golgi localization of AE2 epitope was confirmed by immunoperoxidase electron microscopy. Reverse transcription-PCR analysis of mouse kidney RNA detected AE2a, AE2b, and an AE2c2 transcript, but an AE2c1 transcript was absent. Unlike in rat, the mouse AE2c2 mRNA splice variant encoded a polypeptide with a novel predicted N-terminal amino acid sequence.

Immunolocalization of AE2 anion exchanger in rat kidney.

The cellular and subcellular localizations of the AE2 anion exchanger in rat kidney have remained elusive despite detection of moderately abundant AE2 mRNA and AE2 polypeptide in all kidney regions. In this report a simple epitope unmasking technique has allowed the immunolocalization of AE2 antigenic sites in basolateral membranes of several rat kidney tubular epithelial cells. AE2 immunostaining was faint or absent in the glomerulus and proximal tubule, present in descending and ascending thin limbs, and stronger in the medullary thick ascending limb (MTAL). A lower staining intensity was found in cortical thick ascending limbs and even less in the distal convoluted tubule. In contrast, there was an enhanced staining in the macula densa. In principal cells (PC) of the connecting segment, AE2 was undetectable but gradually increased in intensity along the collecting duct, with strongest staining in inner medullary collecting duct (IMCD) PC. A sodium dodecyl sulfate-sensitive AE2-related Golgi epitope was also detected in some interstitial and endothelial cells of the inner medulla and in epithelial cells of IMCD and MTAL. Colchicine treatment of the intact animal altered the distribution of this Golgi-associated epitope but left plasmalemmal AE2 undisturbed. Reverse transcription-polymerase chain reaction detected AE2a, AE2b, and AE2c2 but not AE2cl transcripts in rat kidney mRNA. The results suggest a widespread occurrence of the AE2 protein in several renal epithelial cell types.

The human rod photoreceptor cGMP phosphodiesterase beta-subunit. Structural studies of its cDNA and gene.

cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase (PDE) were isolated from a human retina library and their sequence was determined. The encoded polypeptide consists of 854 amino acid residues with a calculated molecular mass of 98,416 Da. Alignment of the deduced amino acid sequence with the earlier analysed alpha-, beta- and alpha'-subunits of bovine and mouse PDEs demonstrates a high homology. Two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene were isolated from the genomic library. A total nucleotide sequence of exons 4-22 of the PDE beta-subunit gene was established which completely corresponded to the cDNA structure. According to sequence analysis no potential possibility for alternative splicing of the beta-subunit gene was observed between exons 20 and 21 which led to the formation of the beta'-subunit as described for mouse PDE. Polymerase chain reaction (PCR) experiments also confirm the absence of the PDE beta'-subunit in human retina.

[Detection of expression of a membrane form of the guanylate cyclase type of GC-B in cattle retina]. / Obnaruzhenie ékspressii membrannoi formy guanilattsiklazy tipa GC-B v setchatke byka.

cDNA clones encoding the central and C-terminal parts of a membrane-bound guanylate cyclase (GC) were isolated from the lambda ZAP bovine retinal library. All of the analysed recombinants appeared to carry inserts encoding the guanylate cyclase GC-B. Analysis of the determined nucleotide and deduced amino acid sequences showed extremely high level of homology to the sequences of known GC-B. The results indicate that a mRNA for GC-B is expressed in the bovine retina.

[Structural studies of cDNA and the gene for the beta-subunit of cGMP phosphodiesterase from human retina]. / Strukturnye issledovaniia kDNK i gena beta-sub''edinitsy fosfodiésterazy cGMP iz cetchatki cheloveka.

cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase-(PDE) were isolated from a human retinal library. The encoded polypeptide has 854 amino acid residues with calculated molecular mass of 98416 Da. Alignment of the deduced amino acid sequence with the previously analysed alpha-, beta- and alpha'-subunits of the bovine and mouse PDEs demonstrates highly significant similarities. We have also isolated, from a genomic library, two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene. The cloning of the human gene and the knowledge of its genomic organization will allow the rapid assessment of the role of this gene in the causation of human retinopathies.

[p26--a calcium binding protein from photoreceptor cells in the bovine retina: primary structure and expression in E. coli]. / p26--kal'tsiisviazyvaiushchii belok fotoretseptornykh kletok setchatki byka: pervichnaia struktura i ékspressiia v E. coli.

The primary structure of the bovine retinal calcium binding protein P26 has been determined by the parallel analysis of the protein and the corresponding cDNA. This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. P26 was expressed in E. coli as a fusion protein and, after purification by affinity chromatography on IgG-Sepharose 6, cleaved off with enteropeptidase.

P26-calcium binding protein from bovine retinal photoreceptor cells.

The primary structure of bovine retinal calcium binding protein P26 has been determined by parallel analysis of protein and corresponding cDNA. This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. Preliminary data are presented on expression of P26 as a fusion protein in E. coli.