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Mutations in the human CRALBP gene cause retinal pathology and delayed dark adaptation. Biochemical studies have not identified the primary physiological function of CRALBP. To resolve this, we generated and characterized mice with a non-functional CRALBP gene (Rlbp1(-/-) mice). The photosensitivity of Rlbp1(-/-) mice is normal but rhodopsin regeneration, 11-cis-retinal production, and dark adaptation after illumination are delayed by >10-fold. All-trans-retinyl esters accumulate during the delay indicating that isomerization of all-trans- to 11-cis-retinol is impaired. No evidence of photoreceptor degeneration was observed in animals raised in cyclic light/dark conditions for up to 1 year. Albino Rlbp(-/-) mice are protected from light damage relative to the wild type. These findings support a role for CRALBP as an acceptor of 11-cis-retinol in the isomerization reaction of the visual cycle.

SPACR and SPACRCAN localization in the interphotoreceptor matrix (IPM) of the fovea and peripheral retina of Macaca mulatta was established with antibodies to these core proteins and the chondroitin sulfate epitopes and lectin binding properties of these molecules were defined. The IPM of both rods and cones labeled with anti-SPACR, anti-SPACRCAN, anti-Delta Di6S antibodies and wheat germ agglutinin (WGA). Whereas anti-SPACR and anti-SPACRCAN antibodies labeled rod and cone matrix compartments with similar intensity, the Delta Di6S chondroitin antibody labeling was more intense around cones than rods. Peanut lectin (PNA) labeling was present only around cones. No IPM labeling was observed with Delta Di0S-chondroitin or Delta Di4S-chondroitin antibodies. Western blots of undigested IPM extracts showed anti-SPACR immunoreactivity at 150 kDa, colocalizing with the position of WGA and PNA binding. In Western blots of the chondroitinase ABC digested sample and samples double digested with chondroitinase ABC and AC II, anti-SPACR immunoreactivity, WGA and PNA labeling intensity were virtually identical to that in the undigested sample, with prominent staining of the 150 kDa SPACR band. In contrast, anti-SPACRCAN immunoreactivity was not present in the undigested sample, but was evident in both the chondroitinase ABC and double digested samples as a broad band at approximately 230 kDa. Delta Di6S, Delta Di4S, WGA and PNA labeling colocalized with the anti-SPACRCAN immunoreactivity in the chondroitinase ABC digested sample. These findings indicate that SPACR and SPACRCAN are present around cones in the fovea and both rods and cones in the peripheral retina, but that the specific glycoforms of these molecules are different depending on whether present in the cone or rod associated IPM., (Copyright 2001 Academic Press.)

The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH(2)) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO(2), generating the gamma-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684-1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH(2)) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH(2) preincubation. Amino acid analysis of (14)C- N-ethyl maleimide-modified human carboxylase revealed 1.8-2.3 reactive residues and a specific activity of 7 x 10(8) cpm/hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0. 2% (Cys-99) or 1% (Cys-450), and increased the K(m)s for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH(2) oxygenation.

Mouse SPACR cDNA was cloned by screening a mouse retina cDNA library using a PCR probe derived from human SPACR cDNA. Mouse SPACR cDNA comprises 3675 bp containing an open reading frame coding for 742 amino acids. Multitissue Northern blot analysis and in situ hybridization studies indicate that SPACR expression is restricted to retinal photoreceptors. The SPACR core protein was identified with Western blotting following SDS-PAGE with a SPACR C-terminal peptide polyclonal antibody and a chondroitin-6-sulfate Deltadisaccharide monoclonal antibody. The 150 kD immunopositive band was isolated, digested with trypsin and the peptides analysed by MALDI mass spectroscopy. Peptide mass mapping confirmed the identity of the 150 kD immunopositive band to be mouse SPACR core protein. Alignment comparisons of the deduced amino acid sequence of mouse and human SPACR show 64% homology. Like SPACR in the human interphotoreceptor matrix, the mouse orthologue contains a large central mucin-like domain flanked by consensus sites for N-linked oligosaccharide attachment, one EGF-like domain and four hyaluronan-binding motifs. Unlike human SPACR, which contains no conventional consensus sites for glycosaminoglycan attachment, mouse SPACR contains three. Recent biochemical studies of human and mouse SPACR protein indicate that this novel interphotoreceptor matrix molecule is a glycoprotein in human and a proteoglycan in the mouse. The presence of consensus sites for glycosaminoglycan attachment in the deduced sequence of mouse SPACR and the absence of these sites in human SPACR provide molecular verification of our biochemical results, suggesting that differences in post-translational modifications of SPACR may be important in SPACR function in foveate and non-foveate retinas., (Copyright 2000 Academic Press.)

A family of extremely reactive electrophiles, isolevuglandins (isoLGs), is generated in vivo by free radical-induced lipid oxidation and rearrangement of endoperoxide intermediates of the isoprostane pathway. Protein adducts of two different oxidized lipids, isoLGE(2) and iso[4]LGE(2), and the corresponding autoantibodies are present in human blood. Western blot analysis of a polyacrylamide gel electrophoresis gel detects several immunoreactive plasma proteins. Only a minor fraction of the isoLG-protein modifications is associated with low density lipoprotein since mean levels were decreased only 20-22% by immunoprecipitation of apolipoprotein B (apoB). Mean levels of both isoLGE(2) and iso[4]LGE(2)-protein adducts in plasma from patients with atherosclerosis (AS) (n=16) or end-stage renal disease (RD) (n=8) are about twice those in healthy individuals (n=25). These elevated levels are not related to variations in age, total cholesterol or apoB. A linear correlation (r=0.79) between plasma isoLGE(2) and iso[4]LGE(2)-protein adduct levels in all 49 individuals is consistent with a common free radical-induced mechanism for the production of both oxidized lipids in vivo. The correlation is even stronger (r=0.86) for patients with AS or RD. That isoLG-protein adduct levels are more strongly correlated with disease than are total cholesterol or apoB suggests an independent defect that results in an abnormally high level of oxidative injury associated with AS and RD.

The ABRF amino acid analysis study evaluated the general utility of amino acid analysis (AAA) for identification of proteins after denaturing gel electrophoresis and electroblotting to polyvinylidene difluoride (PVDF) membrane.Thirty-eight participating laboratories analyzed a known control (ovalbumin, 5 microg applied to the gel) and either lysozyme or bovine serum albumin as unknown samples (1-, 5-, and 10-microg amounts applied to the gel). Analyses of the unknowns yielded average compositional errors of approximately 30%, 19%, and 18%, respectively, from the low, intermediate, and higher sample amounts; the ovalbumin control exhibited an approximately 17% average error. Compositional data were submitted to the ExPASy and PROPSEARCH Internet sites for protein identification.Without search parameter adjustments or restrictions, both computer programs provided identification of about 20%, 66%, and 74% of the data from the 1-, 5-, and 10-microg gel samples, respectively. Deleting problematic data (Gly, Met, and Pro) did not always facilitate protein identification. Incorporating control results into the ExPASy search increased identifications 2% to 10%, and restricting search parameters by species, isoelectric pH, and molecular weight increased identifications by more than 80%. Average amounts analyzed for correct identifications were approximately 0.4 microg, 1.8 microg, and 2.9 microg for the 1-, 5-, and 10-microg gel samples, respectively.The results support the efficacy of AAA in the low microgram and nanogram range for the identification of PVDF-immobilized proteins from two-dimensional gels.

Ion channels activated by the binding of cyclic nucleotides first were discovered in retinal rods where they generate the cell's response to light. In other systems, however, it has been difficult to unambiguously determine whether cyclic nucleotide-dependent processes are mediated by protein kinases, their classical effector enzymes, or cyclic nucleotide-gated (CNG) ion channels. Part of this difficulty has been caused by the lack of specific pharmacological tools. Here we report the purification from the venom of the Australian King Brown snake of a peptide toxin that inhibits current through CNG channels. This toxin, which we have named Pseudechetoxin (PsTx), was purified by cation exchange and RP-HPLC and has a molecular mass of about 24 kDa. When applied to the extracellular face of membrane patches containing the alpha-subunit of the rat olfactory CNG channel, PsTx blocked the cGMP-dependent current with a Ki of 5 nM. Block was independent of voltage and required only a single molecule of toxin. PsTx also blocked CNG channels containing the bovine rod alpha-subunit with high affinity (100 nM), but it was less effective on the heteromeric version of the rod channel (Ki approximately 3 microM). We have obtained N-terminal and partial internal sequence data and the amino acid composition of PsTx. These data indicate that PsTx is a basic protein that exhibits some homology with helothermine, a toxin isolated from the venom of the Mexican beaded lizard. PsTx promises to be a valuable pharmacological tool for studies on the structure and physiology of CNG channels.

Light-dependent phosphorylation of rhodopsin (Rho) is a first step in the desensitization of the signaling state of the receptor during vertebrate and invertebrate visual transduction. We found that only 358Ser of the photoactivated octopus Rho (oRho*) was phosphorylated by octopus rhodopsin kinase (oRK). Tryptic truncation of the C-terminal PPQGY repeats of oRho that follow the phosphorylation region did not influence spectral or G-protein activation properties of oRho but abolished phosphorylation. Despite significant structural differences between oRK and mammalian RK, these results provide further evidence of the importance of singly phosphorylated species of Rho* in the generation of arrestin binding sites.

The mammalian renal toxicant tetrafluoroethylcysteine (TFEC) is metabolized to a reactive intermediate that covalently modifies the lysine residues of a select group of mitochondrial proteins, forming difluorothioamidyl lysine protein adducts. Cellular damage is initiated by this process and cell death ensues. NH2-terminal sequence analysis of purified mitochondrial proteins containing difluorothioamidyl lysine adducts identified the lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of the alpha-ketoglutarate dehydrogenase complex (alphaKGDH), a key regulatory component of oxidative metabolism, as targets for TFEC action. Adduct formation resulted in marked inhibition of alphaKGDH enzymatic activity, whereas the related pyruvate dehydrogenase complex was unmodified by TFEC and its activity was not inhibited in vivo. Covalent modification of alphaKGDH subunits also resulted in interactions with mitochondrial chaperonin HSP60 in vivo and with HSP60 and mitochondrial HSP70 in vitro. These observations confirm the role of mammalian stress proteins in the recognition of abnormal proteins and provide supporting evidence for reactive metabolite-induced cell death by modification of critical protein targets.

Purpose: To clone and characterize the mouse gene encoding cellular retinaldehyde-binding protein (CRALBP). CRALBP appears to modulate enzymatic generation and processing of 11-cis-retinol and regeneration of visual pigment in the vertebrate visual cycle. Mutations in human CRALBP segregate with autosomal recessive retinitis pigmentosa., Methods: A genomic clone encompassing the 5' end of the CRALBP gene through exon 6 was isolated from a mouse 129/Sv genomic DNA library. Exons 7 and 8 were PCR amplified from mouse eye cDNA and 129/SvJ genomic DNA. The gene structure was determined by automated DNA sequence analysis., Results: The sequence of 6855 nucleotides was determined, including all 8 exons, 3 introns plus 3932 and 629 bases from the 5'- and 3'-flanking regions, respectively. The lengths of introns 3-6 were determined by PCR amplification. Northern analysis identifies a approximately 2.1 kb transcript in mouse eye; Southern analysis supports a single copy gene., Conclusions: The mouse CRALBP gene is similar to the human gene; the coding sequence is approximately 87% identical, the non-coding sequence approximately 65% identical. In contrast to the human gene, the mouse gene contains a consensus TATA box. One of two photoreceptor consensus elements important for CRALBP expression in human retinal pigment epithelium is also present in the mouse gene. Additional conserved and species-specific consensus sequences are identified. The mouse CRALBP genomic clones and structure provide valuable tools for developing an in vivo model to study protein function and gene regulation.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the retinal pigment epithelium (RPE) and Müller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa. Ligand interactions determine the physiological role of CRALBP in the RPE where the protein is thought to function as a substrate carrier for 11-cis-retinol dehydrogenase in the synthesis of 11-cis-retinal for visual pigment regeneration. However, CRALBP is also present in optic nerve and brain where its natural ligand and function are not yet known. We have characterized the interactions of retinoids with native bovine CRALBP, human recombinant CRALBP (rCRALBP) and five mutant rCRALBPs. Efforts to trap and/or identify a Schiff base in the dark, under a variety of reducing, denaturing, and pH conditions were unsuccessful, suggesting the lack of covalent interactions between CRALBP and retinoid. Buried and solvent-exposed lysine residues were identified in bovine CRALBP by reductive methylation of the holoprotein followed by denaturation and reaction with [3H]acetic anhydride. Radioactive lysine residues were identified by Edman degradation and electrospray mass spectrometry following proteolysis and purification of modified peptides. Human rCRALBP mutants K152A, K221A, and K294A were prepared to investigate possible retinoid interactions with buried or partially buried lysines. Two other rCRALBP mutants, I162V and Q210R, were also prepared to identify substitutions altering the retinoid binding properties of a random mutant. The structures of all the mutants were verified by amino acid and mass spectral analyses and retinoid binding properties evaluated by UV-visible and fluorescence spectroscopy. All of the mutants bound 11-cis-retinal essentially like the wild type protein, indicating that the proteins were not grossly misfolded. Three of the mutants bound 9-cis-retinal like the wild type protein; however, Q210R and K221A bound less than stoichiometric amounts of the 9-cis-isomer and exhibited lower affinity for this retinoid relative to wild type rCRALBP. Residues Gln-210 and Lys-221 are located within a region of CRALBP exhibiting sequence homology with the ligand binding cavity of yeast phosphatidylinositol-transfer protein. The data implicate Gln-210 and Lys-221 as components of the CRALBP retinoid binding cavity and are discussed in the context of ligand interactions in structurally or functionally related proteins with known crystallographic structures.

Cellular retinaldehyde-binding protein (CRALBP) is abundantly expressed in the retinal pigment epithelium (RPE) and Muller cells of the retina, where it is thought to function in retinoid metabolism and visual pigment regeneration. Mutations in human CRALBP that destroy retinoid binding have been linked to autosomal recessive retinitis pigmentosa. To identify the DNA elements that regulate expression of the human CRALBP gene in the RPE, transient transfection studies were carried out with three CRALBP-expressing human RPE cell culture systems. The regions from -2089 to -1539 base pairs and from -243 to +80 base pairs demonstrated positive regulatory activity. Similar activity was not observed with cultured human breast, liver, or skin cells. Since sequence analysis of the -243 to +80 region identified the presence of two photoreceptor consensus element-1 (PCE-1) sites, elements that have been implicated in photoreceptor gene regulation, the role of these sequences in RPE expression was examined. Mutation of either PCE-1 site significantly reduced reporter activity, and mutation or deletion of both sites dramatically reduced activity. Electrophoretic mobility shift analysis with RPE nuclear extracts revealed two complexes that required intact PCE-1 sites. These studies also identified two identical sequences (GCAGGA) flanking PCE-1, termed the binding CRALBP element (BCE), that are also important for complex formation. Southwestern analysis with PCE-1/BCEcontaining probes identified species with apparent masses near 90-100 and 31 kDa. These results begin to identify the regulatory regions required for RPE expression of CRALBP and suggest that PCE-1-binding factor(s) may play a role in regulating RPE as well as photoreceptor gene expression.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.

Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca2+-binding protein, activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca2+-binding proteins from the calmodulin superfamily, the Ca2+-free form of GCAP1 stimulates the effector enzyme. In this study, we analyzed the Ca2+-dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca2+-sensitive modulation of GC1 activity. The change from a Ca2+-bound to a Ca2+-free form of GCAP1 increased susceptibility of Ca2+-free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca2+-bound form, only the N-terminus (myristoylated Gly2-Lys9) and C-terminus (171-205 fragment) of GCAP1 are removed by trypsin, while in the Ca2+-free form, GCAP1 is readily degraded to small fragments. Successive inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca2+-dependent inactivation of GCAP1. GCAP1(E75D,E111D,E155D) mutant did not bind Ca2+ and stimulated GC1 in a [Ca2+]-independent manner. GCAP1 and GCAP2, but not S-100beta, a high [Ca2+]free activator of GC1, competed with the triple mutant at high [Ca2+]free, inhibiting GC1 with similar IC50's. These competition results are consistent with comparable affinities between GC1 and GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca2+ binding and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein from the activator to the inhibitor of GC1.

Purpose: Primary cultures of Müller cells have proven useful in cell biologic, developmental, and electrophysiological studies of Müller cells. However, the limited lifetime of the primary cultures and contamination from non-neural cells have restricted the utility of these cultures. The aim of this study was to obtain an immortalized cell line that exhibits characteristics of Müller cells., Methods: Primary Müller cell cultures were prepared from retinas of rats exposed to 2 weeks of constant light. Cells were immortalized by transfection with simian virus 40. Single clones were obtained by repeatedly passaging cells using cloning wells. Immunocytochemical and immunoblotting studies were carried out with glial fibrillary acidic protein (GFAP)-specific and cellular retinaldehyde-binding protein (CRALBP)-specific antibodies. Transient transfections with CRALBP-luciferase constructs were performed by electroporation., Results: Oncogene transformation resulted in the establishment of a permanent cell line that could be readily propagated. Immunocytochemical and immunoblotting studies demonstrated that the Müller cell line, rMC-1, expressed both GFAP, a marker for reactive gliosis in Müller cells, and CRALBP, a marker for Müller cells in the adult retina. Transient transfection assays showed that promoter-proximal sequences of the CRALBP gene were able to stimulate reporter gene expression in rMC-1., Conclusions: Viral oncogene transformation has been successfully used to isolate a permanent cell line that expresses Müller cell phenotype. The rMC-1 cells continue to express both induced and basal markers found in primary Müller cell cultures as well as in the retina. The availability of rMC-1 should facilitate gene expression studies in Müller cells and improve our understanding of Müller cell-neuron interactions.

The expression of the am (glutamate dehydrogenase) gene is dependent upon two upstream activating sequences, designated URSam(alpha) and URSam(beta). A heteromeric nuclear protein Am Alpha Binding protein (AAB) binds specifically to a CCAAT box within the URSam(alpha) element. AAB appears to be composed of three components. We used polyclonal antiserum raised against the highly purified AAB1 subunit to isolate a partial aab-1 cDNA clone, which was then used to isolate a full-length cDNA and a genomic clone. The full-length cDNA has the potential to encode a 272 amino acid protein with a calculated molecular weight of 30 kD. Amino acid sequence obtained by Edman analysis of the AAB1 protein confirmed that the aab-1 gene had been cloned. AAB-1 shows similarity to the HAP5 protein of yeast and the CBF-C protein of rat. Each of these proteins is an essential subunit of their respective heteromeric CCAAT binding proteins. The aab1 gene maps on linkage group III of Neurospora crassa near the trp-1 locus. Disruption of the aab-1 gene results in pleiotropic effects on growth and development as well as a 50% reduction in glutamate dehydrogenase levels. Transformation of the aab-1 disruption mutant strain with the cloned genomic copy of the aab-1 gene rescued all of the phenotypic alterations associated with the aab-1 mutation.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium and Müller glial cells of the retina, where it forms complexes with endogenous 11-cis-retinoids. We examined the distribution of CRALBP in extraretinal tissues using polyclonal antibodies (pAb) and monoclonal antibodies (mAb). A protein was detected by immunoblot analysis in extracts of bovine and rat brain and optic nerve but not in several other tissues. This protein had electrophoretic, chromatographic, and retinoid-binding properties identical to those of CRALBP from bovine retina. Comparison of the masses of tryptic peptides and of partial amino acid sequences derived from brain and retinal CRALBP indicated that the two proteins are probably identical. Immunoperoxidase cytochemistry and double labeling immunofluorescence revealed CRALBP(+) cells in brain that resembled oligondendrocytes and not astrocytes, microglial cells, or pinealocytes. In 11-day-old rat brain, approximately 11% of the CRALBP(+) cells were labeled with the Rip antibody, a marker for oligodendroglia. In developing rat optic nerve, the temporal appearance of CRALBP(+) cells corresponded to that of oligodendrocytes and not that of astrocytes. In adult rat and mouse optic nerves, the CRALBP(+) somata showed the same distribution as oligodendrocytes. No endogenous retinoids were associated with CRALBP isolated from dark-dissected adult bovine brain. The results suggest that CRALBP has functions in addition to retinoid metabolism and visual pigment regeneration.

Inadequate levels of all-trans-retinol in the blood cause retinal dysfunction; hence, genes implicated in retinal vitamin-A metabolism represent candidates for inherited retinal degenerations. In the current study, molecular genetic analysis of a consanguineous pedigree segregating for non-syndromic autosomal recessive retinitis pigmentosa (arRP) indicated that the affected siblings were homozygous by descent for a G4763A nucleotide substitution in RLBP1, the gene encoding cellular retinaldehyde-binding protein (CRALBP). This substitution is predicted to replace an arginine with glutamine at residue 150. CRALBP is not expressed in photoreceptors but is abundant in the retinal pigment epithelium (RPE) and Müller cells of the neuroretina, where it carries 11-cis-retinol and 11-cis-retinaldehyde. When expressed in bacteria, recombinant CRALBP (rCRALBP) containing the R150Q substitution was less soluble than wild-type rCRALBP. Mutant rCRALBP was purified from the soluble cell lysate and the protein structure was verified by mass spectrometry. The mutant protein lacked the ability to bind 11-cis-retinaldehyde. These findings suggest that arRP in the current pedigree results from a lack of functional CRALBP, presumably leading to disruption of retinal vitamin-A metabolism.

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.

In rod and cone photoreceptor cells, activation of particulate guanylate cyclase (retGC1) is mediated by a Ca2+-binding protein termed GCAP1, that detects changes in [Ca2+]free. In this study, we show that N-acylated GCAP1 restored Ca2+ sensitivity of native and recombinant photoreceptor retGC1. ATP increased the affinity of retGC1 for GCAP1 and accelerated catalysis. Using peptides derived from the GCAP1 sequence, we found that at least three regions, encompassing the N-terminus, the EF-1 motif, and the EF-3 motif, were likely involved in the interaction with retGC1. Mutation of 2Gly to Ala (GCAP1-G2A), which abolished myristoylation and a 25 amino acid truncation at the N-terminus (delta25-GCAP1) reduced retGC1-stimulating activity dramatically, while deletion of 10 amino acids (delta10-GCAP1) reduced the specific activity by only approximately 60% and modified the Ca2+ sensitivity. At 10(-6) M [Ca2+]free, in conditions that inactivated native GCAP1, retGC1 showed significant activity in the presence of delta10-GCAP1. Native and all three mutant forms of GCAP1 had similar affinities for Ca2+ as demonstrated by gel filtration and the changes in tryptophan fluorescence. All mutants bound to ROS membranes in a Ca2+-independent manner, except delta25-GCAP1, which was mostly soluble. These findings suggest that the N-terminal region is important in tethering of GCAP1 to the ROS membranes.

We have purified a 16,000 dalton protein that stimulates growth of human umbilical cord vein-derived endothelial cells (HUV-EC) from mouse submaxillary glands by using heparin-Sepharose affinity and C4 reverse phase chromatography. The purified molecule was identified as an FGF-1 on the basis of its biological activities, its affinity for heparin and its N-terminal amino-acid sequence. The concentrations of FGF-1 in the submaxillary gland of male or testosterone-treated female mice were about 12 times those of untreated females or castrated males. The 2.3 and 4.1 kb FGF-1 mRNAs were expressed in the glands of male mice older than 4 weeks but not in the glands of female mice. These results suggest that FGF-1 may have important functions for growth, differentiation and development of mouse submaxillary glands, and it may act as an endocrine hormone.

Purpose: To evaluate the patterns of expression of beta subunit isoforms of the Na+,K(+)-ATPase and H+,K(+)-ATPase in the human eye and to determine the cell-specific distribution of the beta 2 subunit in the human ciliary epithelium., Methods: Total RNA extracted from human ocular tissues was screened by Northern blot analysis with cDNA probes for the human Na+,K(+)-ATPase subunit isoforms (beta 1 and beta 2) or the H+,K(+)-ATPase (alpha and beta) subunits. Antibodies were raised to the amino and carboxyl terminal regions of the human beta 2 isoform. Polymerase chain reaction was used to verify the expression of beta 2 subunit in nonpigmented ciliary epithelial cells (NPE)., Results: Transcripts for the Na+,K(+)-ATPase beta 1 and beta 2 subunit isoforms were present at different levels in all the ocular tissues except the lens, which expressed only beta 1. No transcripts for the alpha or beta subunits of the H+,K(+)-ATPase were detected in the eye. Isoform beta 2 specific anti-peptide antibodies V15E (N-terminus) and A18R (C-terminus) recognized a 55- to 60-kDa protein in the ciliary epithelium and the core protein of 32 kDa after N-glycanase treatment. Immunocytochemical localization within the ciliary epithelium indicates that the Na+,K(+)-ATPase beta 2 isoform is expressed preferentially in the NPE cells. The expression of Na+,K(+)-ATPase beta 2 isoform in the human NPE cell line, ODM-2, was confirmed by polymerase chain reaction amplification and Southern blot analysis., Conclusions: The Na+,K(+)-ATPase beta 2 subunit isoform, but not H+,K(+)-ATPase, was expressed widely in ocular tissues of the human eye. The restricted cellular distribution of beta 2 isoform within the NPE cells represents an important differential gene marker associated with the multiple alpha subunit isoforms of Na+,K(+)-ATPase.

We previously reported purification of a protein (approximately equal to 57 kDa) from human follicular fluid having FSH binding inhibitory (FSH-BI) activity. Purified hFSH-BI was cleaved with cyanogen bromide and trypsin. The resulting peptide fragments were separated by HPLC and sequence information for individual fragments was obtained. A ten amino acid sequence of hFSH-BI derived from this procedure was identified, and a corresponding peptide amide (BI-10) was synthesized and utilized for further study. A protein database search revealed no significant identity between this decapeptide and other known proteins. We examined the ability of BI-10 to inhibit binding of 125I-hFSH to FSH-receptor enriched bovine testes membranes utilizing a radioligand receptor assay (RRA). BI-10 inhibited binding of 125I-hFSH to its receptor in a concentration-related manner, with an ED50 of 300 microM. BI-10 had no effect on 125I-hCG binding to receptor even at concentrations up to 1000 microM, suggesting that the effect of BI-10 was specific for the interaction between FSH and its receptor. To assess bioactivity of BI-10, we investigated its effect on FSH-stimulated conversion of androstenedione to estradiol by rat Sertoli cells in primary culture in vitro. Inhibition of FSH-stimulated estradiol synthesis (FSH antagonist activity) was significant at a BI-10 concentration of 1000 microM. BI-10 also significantly inhibited FSH-stimulated cAMP accumulation in primary cultures of Sertoli cells when examined at the same concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

An enzyme of bovine retina that catalyzes oxidation of retinaldehyde to retinoic acid was purified to homogeneity and a monoclonal antibody (mAb H-4) was generated. MAb H-4 recognized a single component (Mr = 55,000) in extracts of bovine retina and other bovine tissues. The antibody showed no cross-reactivity with extracts of rat, monkey, or human retinas. A 2067 bp cDNA was selected from a retina cDNA expression library using mAb H-4. The cDNA hybridized with a similarly sized, moderately abundant mRNA prepared from bovine retina. Nucleotide sequence analysis indicated that the cDNA contained a single open reading frame encoding 501 amino acids that have 88% sequence identity with the amino-acid sequence of human hepatic Class 1 aldehyde dehydrogenase. Amino-acid sequence analysis of purified enzyme demonstrated that the cDNA encodes the isolated enzyme. MAb H-4 specifically labeled the somata and processes of a subset of amacrine cells in bovine retinal sections. Labeled amacrine somata were located on both sides of the inner plexiform layer, and their processes ramified into two laminae within the inner plexiform layer. The inner radial processes of Müller (glial) cells were weakly reactive with mAb H-4. Weak immunostaining of amacrine cells was found in monkey retina with mAb H-4, but no signal was detected in rat or human retina. The results provide further evidence for metabolism and function of retinoids within cells of the inner retina and define a novel class of retinal amacrine cells.

Calbindin D-28K is a calcium-binding protein found in the cone but not rod photoreceptor cells in the retinas of a variety of species. Recent studies of the monkey retina indicated that calbindin D-28K may be expressed preferentially in non-foveal regions of the retina. In the current studies of human retinas, immunohistochemical experiments demonstrated that calbindin D-28K is reduced or absent in the fovea and parafovea, but prevalent in the perifovea and periphery. These findings were supported by the quantification of calbindin D-28K in 1-mm trephine punches obtained from different regions of the human retina. The specificity of the anti-calbindin D-28K antibodies used in these studies was confirmed by Western blot analysis using purified calbindin D-28K. The protein was purified from retinal tissue and its identity confirmed by partial amino-acid sequence analysis. The expression of calbindin D-28K did not correlate with the spectral properties of the cones, rather to their position in the retina. The study of spatially expressed genes, like the one encoding calbindin D-28K, may help explain the patterns of retinal degeneration seen in some human cone-rod dystrophies.

Cellular retinaldehyde-binding protein (CRALBP) appears to play a role in the vertebrate visual process as a substrate-routing protein, influencing the enzymatic partitioning of 11-cis-retinol at a key branch point in the visual cycle. Genomic clones spanning 29 kilobases and encompassing the human CRALBP gene have been isolated by screening two human genomic libraries and from polymerase chain reaction amplification of human leukocyte DNA. The sequence of 13,647 contiguous nucleotides has been determined, including 3130 and 516 bases from the 5'- and 3'-flanking regions, respectively. The human CRALBP gene exists as a single copy in the genome based on Southern analyses and localization to a single site on human chromosome 15 (Sparkes, R. S., Heinzmann, C., Goldflam, S., Kojis, T., Saari, J. C., Mohandes, T., Klisak, I., Bateman, J. B., and Crabb, J. W. (1992) Genomics 12, 58-62). The gene is composed of eight exons and seven introns with average lengths of 198 base pairs and 1.2 kilobases, respectively, and which exhibit conventional vertebrate splicing. Alu repetitive sequences exist in introns 4 and 5 as well as in the 5'- and 3'-flanking regions of the gene. RNase protection and primer extension analyses indicate that the human CRALBP gene transcription start site is 922 bases upstream of the initiation codon. The first exon is entirely untranslated and both exon 2 and exon 8 contain untranslated regions. The proximal 5'-flanking region lacks GC boxes and consensus TATA and CCAAT boxes at the usual positions. The 3'-untranslated region of CRALBP exon 8 is essentially identical to a partial cDNA clone reportedly isolated from a human hippocampus cDNA library, suggesting that the protein may be expressed in a wider spectrum of tissues than previously recognized. The human CRALBP genomic clones and structure provide valuable tools for studying the physiological role of the protein in vision and visual disorders.

Guanylyl cyclase-activating protein (GCAP) is thought to mediate Ca(2+)-sensitive regulation of guanylyl cyclase (GC), a key event in recovery of the dark state of rod photoreceptors following light exposure. Here, we characterize GCAP from several vertebrate species by molecular cloning and provide evidence that GCAP contains a heterogeneously acylated N-terminal region that interacts with GC. Vertebrate GCAPs consist of 201-205 amino acids, and sequence analysis indicates the presence fo three EF hand Ca(2+)-binding motifs. These results establish that GCAP is a novel photoreceptor-specific member of a large family of Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-sensitive activation of GC.

A 15kDa-protein (HBp15) was purified from mouse submandibular gland and bovine brain by virtue of its heparin-binding property. The amino acid sequences of mouse and bovine HBp15 showed a high degree of homology to a sea urchin protein encoded by gene called "development specific protein 217." Using reverse transcription-polymerase chain reaction methods, cDNA clones for HBp15 were isolated from submandibular gland mRNA of mouse, human and pig, and sequenced. Database search of HBp15 showed that HBp15 also resembles yeast ribosomal protein YL31 in addition to the 217 protein. Using specific antibodies against HBp15, HBp15 was identified as mammalian ribosomal protein L22, for which no sequence information is available.

Calcium protein placental homolog (Capl) is an S-100-related calcium-binding protein selectively expressed in cell lines that have been induced to grow or differentiate. In addition, the expression of Capl correlates with the induction of the metastatic phenotype in tumor cell lines and the transformation of normal cells by activated oncogenes or chemical carcinogens. Although not previously associated with the nervous system, in this study, Capl was purified from bovine neural retina by a combination of phenyl-Sepharose and organomercurial chromatography. The complete amino acid sequence of bovine Capl was established primarily by Edman degradation of peptides generated by cleavage of methionyl, lysyl, glutamyl, and aspartyl bonds. NH2-terminal methionyl and aspartyl peptides were analyzed by tandem mass spectrometry, which provided the sequence of the first 8 residues and identified the NH2-terminal blocking group as an acetyl moiety. The molecular mass of the intact protein determined by electrospray mass spectrometry (M(r) = 11,716.75 +/- 0.42) and the calculated molecular mass deduced from the amino acid composition (M(r) = 11,718) were in agreement, thus supporting the accuracy of the sequence assignment. Capl isolated from the retina was shown to be indistinguishable by mass and immunochemical properties from its counterpart in the bovine aorta, which previously was the only source of purified Capl. Northern analysis using cloned Capl cDNA revealed that Capl mRNA is present not only in the retina but the choroid as well. Further support for choroidal localization came from immunohistochemical experiments using specific anti-Capl antibodies. The physiological significance of Capl in ocular tissues and the aorta is discussed.

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48-kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44-kDa form of arrestin, called p44, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C-terminal 35 residues (positions 370-404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, p44 blocks Gt activation. The binding characteristics of p44 set it apart from tryptic forms of arrestin (truncated at the N- and C-termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of Gt activation.

Vitamin A (retinol) treatment induces (and/or enhances) mucous cell differentiation and alters keratin gene expression in cultured airway epithelial cells of human and nonhuman primate origin. We observed that retinol greatly reduced the synthesis of keratins 5, 6, 14, 16, and 17, but slightly enhanced keratins 7, 8, 10, 13, 15, 18, and 19. These changes were also reflected at the mRNA level as demonstrated by cell-free translation and by cDNA cloning of human keratin genes based on differential hybridization. One of these cDNA clones, HT27, isolated from the cDNA library of human tracheobronchial epithelial cells and whose expression in cultured cells was greatly suppressed by retinol, had a nucleotide sequence identical to the C-terminus of keratin 16. The identity of this clone was further confirmed by Western blot analysis using an antibody specific to the 15-amino acid synthetic peptide and the C-terminal sequence. Using this cDNA clone and two known keratin clones, pKA1 (keratins 5 and 6) and pKB2 (keratin 14), we found the levels of these corresponding mRNAs in cultured cells to be reduced 10- to 25-fold after treatment of cells with vitamin A. The inhibition was time- and dose-dependent with respect to retinol and was sensitive to prior treatment with cycloheximide. However, nuclear run-on transcriptional assays revealed no significant reduction of the synthesis of these messages in retinol-treated cultures. Furthermore, no change in the half-life of these mRNAs was observed in cells after the retinol treatment. Based on these results, we conclude that vitamin A indirectly controls the synthesis of these keratins at the post-transcriptional level.

The potent and site-selective nephrotoxicity of S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) in vivo has been associated with difluorothioamidyl-L-lysine formation on critical mitochondrial target proteins. Dose-response studies in the Fischer 344 rat indicate that five proteins with apparent molecular masses of 99, 84, 66, 52, and 48 kDa are predominantly adducted in vivo after nephrotoxic doses of TFEC (> 10 mg/kg, intraperitoneally). Microsequence analysis of the major difluorothioamidyl-L-lysine proteins indicated that P66 is identical, over 14 NH2-terminal residues, to mitochondrial P1 protein (HSP60, a chaperonin) and that P84 is identical, over 14 residues, to a recently isolated novel member of the HSP70 family known as mortalin. These studies indicate that mitochondrial heat shock proteins are major targets for modification by reactive metabolites of TFEC. The implications of these data in relation to the nephrotoxicity of cysteine conjugates are discussed.

Differential expression of the cellular retinaldehyde-binding protein (CRALBP), a likely component of the visual cycle, has been observed in the ocular ciliary epithelium, a bilayer of neurepithelial cells involved in the active transport of aqueous humor. Antibody probes raised to bovine ciliary epithelium were used to isolate a cDNA encoding CRALBP from a bovine ciliary epithelium cDNA expression library. Northern analysis of poly A+RNA isolated from three different regions of the neonate ciliary epithelium revealed a three-fold higher level of CRALBP expression in the most anterior portion of the pars plicata region relative to the posterior pars plana region. Western blot analysis using CRALBP-specific antibodies also demonstrated that the level of CRALBP expression was higher in the most anterior portion of the ciliary epithelium and gradually decreases toward the posterior region in both neonate and adult bovine eyes. Using indirect immunofluorescence and frozen-sectioned bovine ciliary epithelium, CRALBP was localized to the pigmented ciliary epithelial cell layer along the entire ciliary epithelium. In the nonpigmented ciliary epithelial cell layer, CRALBP was only detected in the pars plana region. Pigmented ciliary epithelial cells have also been cultured and CRALBP expression demonstrated for several continuous passages by Northern and Western analysis. These results raise questions regarding the physiological role of CRALBP in the ciliary epithelium and the involvement of the protein in the defining regional and functional boundaries with this tissue.

Human and rabbit paraoxonases/arylesterases were purified to homogeneity by chromatographic and gel electrophoretic/isofocusing procedures coupled with activity stains. N-terminal and peptide sequence analysis suggested retention of the secretion signal sequence and allowed design of oligonucleotide probes. The probes were used to isolate a 1294-bp rabbit paraoxonase cDNA clone, which, in turn, was used to isolate three human cDNA clones. Comparison of rabbit and human protein and cDNA sequences indicated a high degree of sequence conservation (approximately 85% identity) and verified that paraoxonase retains its signal sequence (except for the N-terminal Met). The rabbit cDNA encodes a protein of 359 amino acids and the human a protein of 355 amino acids. In situ hybridization demonstrated, as expected, that the paraoxonase gene maps to the long arm of human chromosome 7. Arginine at position 192 specifies high activity paraoxonase and glutamine low activity human paraoxonase. Variation in protein levels explains the variation of enzyme activity observed within a genetic class. Toxicity studies showed that raising rat plasma paraoxonase levels by i.v. administration of partially purified rabbit paraoxonase protected animals against cholinesterase inhibition by paraoxon and chlorpyrifos oxon. Protection correlated with the relative rates of hydrolysis of these two compounds.

PC cell line is a highly tumorigenic insulin-independent variant from the teratoma-derived adipogenic cell line 1246. Culture medium of PC cells contains a growth promoting activity for 3T3 cells and producer cells. PC cell-derived growth factor (PCDGF) was purified to homogeneity from PC cell-conditioned medium as an apparent 88-kDa protein by chromatography on heparin-Sepharose, Sephacryl S-200, and phenyl-Sepharose. Digestion with peptide-N-glycosidase F yielded an apparent 68-kDa protein component indicating that PCDGF is a glycoprotein containing about 20 kDa of carbohydrate. Partial sequence from Edman degradation of peptide fragments obtained by digestion of PCDGF with cyanogen bromide and trypsin demonstrates that PCDGF contains regions of sequence identity to that deduced from the granulin or epithelin precursor cDNAs. Granulins are small polypeptides purified from granulocyte extracts with no apparent biological functions. Epithelins are cell growth modulators purified as small molecular mass 6-kDa polypeptides from kidney extracts. The existence of a large molecular mass precursor for granulin or epithelin has been predicted based upon recently cloned cDNAs encoding these biomolecules within a 63.5-kDa protein with putative glycosylation sites. No biological activity has previously been attributed to the precursor. The present results indicate that PCDGF is a potential precursor for epithelin and/or granulin, that this 88-kDa protein is secreted and glycosylated, and that it can function as a mitogen for 3T3 cells as well as an autocrine growth factor for PC cells.

Heparin or heparin-like heparan sulfate proteoglycans are obligatory for activity of the heparin-binding fibroblast growth factor (FGF) family. Heparin interacts independently of FGF ligand with a specific sequence (K18K) in one of the immunoglobulin-like loops in the extracellular domain of the FGF receptor tyrosine kinase transmembrane glycoprotein. A synthetic peptide corresponding to K18K inhibited heparin and heparin-dependent FGF binding to the receptor. K18K and an antibody to K18K were antagonists of FGF-stimulated cell growth. Point mutations of lysine residues in the K18K sequence abrogated both heparin- and ligand-binding activities of the receptor kinase. The results indicate that the FGF receptor is a ternary complex of heparan sulfate proteoglycan, tyrosine kinase transmembrane glycoprotein, and ligand.

Upon illumination rhodopsin kinase (RK) phosphorylates the visual pigment, rhodopsin, in a reaction that is thought to terminate in part the biochemical events that follow photon absorption. In this paper, RK was studied to assign functional regions to the primary structure of the enzyme. Peptides derived from the sequence of RK were used to prepare site-specific anti-peptide antibodies against: 1) the N-terminal region located between residues 17 and 34, which contains an autophosphorylation site; 2) the Lys/Arg-rich region corresponding to residues 216-237 near the catalytic domain; 3) the region located between residues 483 and 497, which encompasses the major autophosphorylation sites; and 4) the C-terminal region located between residues 539 and 556, close to the isoprenylation site of RK. Antibodies also were raised against purified RK. Application of the antibodies directed against the N-terminal domain blocks RK activity toward Rho*, but has no affect on the phosphorylation of a synthetic peptide substrate. Additionally, a significant portion of the inhibitory effect seen with an antibody directed against whole RK could be reversed by the peptide derived from the N-terminal region. We conclude that the N-terminal region of RK contains a sequence involved in the recognition of photolyzed Rho. Furthermore, the inhibition of RK activity eliminates the effect of ATP during the inactivation of cGMP phosphodiesterase, implying that RK is a necessary component of a cascade of reactions involved in the quenching of phototransduction. Light microscopic immunocytochemistry using these antibodies revealed that RK was localized to the rod and cone outer segments of human and bovine retinas.

We have recently reported that synthetic peptide amides corresponding to regions of human FSH beta-subunit, hFSH-beta-(33-53) and hFSH-beta-(81-95), bind to receptor and stimulate estradiol biosynthesis by cultured rat Sertoli cells. Because of experimental difficulties caused by the presence of free sulfhydryl groups in these peptides, synthetic analogs were prepared in which all Cys residues were replaced with Ser. These analogs, [Ser-51]-hFSH-beta-(33-53) and [Ser-82, 84, 87, 94]-hFSH-beta- (81-95), also bound to receptor but did not stimulate estradiol biosynthesis by cultured rat Sertoli cells. In order to explain this observation, we compared the effects of hFSH-beta-(33-53) and hFSH-beta-(81-95) and their Ser analogs on another recently recognized effect of FSH in Sertoli cells, namely its ability to promote influx of extracellular calcium. We and others have shown that estradiol biosynthesis by these cells is markedly decreased in the presence of high intracellular calcium. Cys-containing hFSH-beta-(33-53) and hFSH-beta-(81-95) did not increase influx of extracellular calcium over basal levels, whereas [Ser-51]-hFSH-beta-(33-53) and [Ser-82, 84, 87, 94]-hFSH-beta-(81-95) induced 2.8- and 1.8-fold increases, respectively. Cellular cAMP and estradiol biosynthesis in response to each Ser-substituted peptide were not significantly different from basal levels. Thus, the explanation for the observed disparate effects of Cys and Ser analog peptides on estradiol biosynthesis may be related to the ability of the Ser peptides to stimulate calcium entry but not cAMP accumulation in cultured rat Sertoli cells.

Purpose: We investigated the hypothesis that visinin, a cone-specific protein first characterized in chicken retina, is a cone homologue of recoverin and may be the cancer-associated retinopathy (CAR) autoantigen in human cone cells., Methods: Visinin was purified from chicken retinas and tested for binding by CAR antisera. In addition, antibodies specific to visinin were used immunocytochemically and for Western analysis to determine whether visinin is present in human or bovine retinas. Anti-peptide antibodies against recoverin were used immunocytochemically to localize recoverin to mammalian cone cells., Results: CAR antisera recognized recoverin but not visinin. Furthermore, visinin could not be detected in mammalian retinas by immunocytochemical methods or by attempts to purify the protein. In contrast to visinin, antibodies specific for different regions of the recoverin molecule stained both rod and cone cells in the human retina., Conclusions: Visinin is not the CAR autoantigen in human cone cells. Differences between recoverin and visinin probably reflect species differences rather than rod-cone differences. Recoverin, or a nearly identical molecule, is present in mammalian cones and likely is the cone cell CAR autoantigen.

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.

The receptor binding surface of human follicle-stimulating hormone (hFSH) is mimicked by synthetic peptides corresponding to the hFSH-beta chain amino acid sequences 33-53 [Santa-Coloma, T. A., Dattatreyamurty, D., and Reichert, L. E., Jr. (1990), Biochemistry 29, 1194-1200], 81-95 [Santa-Coloma, T. A., Reichert, L. E., Jr. (1990), J. Biol. Chem. 265, 5037-5042], and the combined sequence (33-53)-(81-95) [Santa-Coloma, T. A., Crabb, J. W., and Reichert, L. E., Jr. (1991), Mol. Cell. Endocrinol. 78, 197-204]. These peptides have been shown to inhibit binding of hFSH to its receptor. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to determine the structure of the first peptide in this series, the 21 amino acid peptide hFSH-beta-(33-53), H2N-YTRDLVYKDPARPKIQKTCTF-COOH. Analysis of CD data indicated the presence of approximately equal amounts of antiparallel beta-pleated sheet, turns including a beta-turn, "other" structures, and a small amount of alpha-helix. The major characteristics of the structure were found to be relatively stable at acidic pH and the predominant effect of increased solvent polarity was a small increase in alpha-helical content. One- and two-dimensional NMR techniques were used to obtain full proton and carbon signal assignments in aqueous solution at pH 3.1. Analysis of NMR results confirmed the presence of the structural features revealed by CD analysis and provided a detailed picture of the secondary structural elements and global folding pattern in hFSH-beta-(33-53). These features included an antiparallel beta-sheet (residues 38-51 and 46-48), turns within residues 41-46, and 50-52 (a beta-turn) and a small N-terminal helical region comprised of amino acids 34-36. One of the turns is facilitated by prolines 42 and 45. Proline-45 was constrained to the trans conformation, whereas proline-42 favored the trans conformer (approximately 70%) over the cis (approximately 30%). Two resonances were observed for the single alanine residue (A-43) sequentially proximal to P-42, but the rest of the structure was minimally affected by the isomerization at proline-42. The major population of molecules, containing trans-42 and trans-45 prolines, presented 120 NOEs. Distance geometry calculations with 140 distance constraints and energy minimization refinements were used to derive a moderately well-defined model of the peptide's structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Rhodopsin kinase (RK) is a second-messenger-independent protein kinase that is involved in deactivation of photolyzed rhodopsin (Rho*). We have developed a significantly improved method for isolation of RK based on the specific interactions of phosphorylated forms of the enzyme with heparin-Sepharose. Conversion of the dephosphorylated form of RK to the fully phosphorylated enzyme leads to specific elution of the kinase from the resin. Limited proteolysis of RK with endoproteinase Asp-N removes the phosphorylation sites. Peptides containing the autophosphorylation sites were isolated by reverse-phase high performance liquid chromatography and analyzed by Edman degradation and tandem mass spectrometry. The derived amino acid sequence of the peptide containing the major autophosphorylation site yielded the following sequence: DVGAFS488T489VKGVAFEK, where Ser488 and Thr489 are phosphorylated. Additionally, a minor autophosphorylation site was identified at Ser21. A 15-residue peptide (DVGAFSTVKGVAFEK) encompassing the major autophosphorylation site was synthesized and used for phosphorylation and inhibition studies. In contrast to many other protein kinases, the low catalytic activity of RK toward its autophosphorylation site peptide and the poor inhibitory properties of this peptide suggest unique properties of this member of the family of G protein-coupled receptor kinases.

Potentially 96 splice variants among four genes that code for the human heparin-binding fibroblast growth factor receptor family complicate study of structure, metabolism, and function of single isoforms in mammalian cells. As an alternative, we expressed structural subdomains and isoforms of the flg receptor gene in bacteria and baculoviral-infected insect cells. We developed and characterized a panel of 16 isoform and domain-specific polyclonal and monoclonal antibodies. The panel of antibodies was used to distinguish mature glycosylated ligand-binding and kinase-active and -inactive recombinant isoforms in baculoviral insect cells and transfected mammalian cells and natural isoforms in rat prostate and human liver cells. The results revealed a cell type-specific expression of the flg gene and isoforms that result from combinations of splice variations. Reactive epitopes of monoclonal antibodies against both the three (alpha) and two (beta) immunoglobulin-like disulfide loop extracellular domain isoforms were mapped by cross-reactivity with synthetic polypeptide sequences and deletion mutants expressed in bacteria. The native alpha and beta receptor isoforms differed in display of shared epitopes and suggested that the NH2-terminal Loop I and COOH-terminal Loops II and III of the alpha isoform are interactive. Although the common Loops II and III appear qualitatively sufficient for ligand binding, the results suggest that tertiary relationships among loops in the three and two loop isoforms are distinct and, therefore, the two isoforms may have distinct activities. Spatial models for arrangement of immunoglobulin-like loops in the extracellular domain of the two isoforms are presented.

Synthetic peptides corresponding to hFSH-beta-(33-53) and hFSH-beta-(81-95) each contain free sulfhydryl groups, inhibit binding of FSH to receptor and are partial agonists of estradiol synthesis in Sertoli cells. Recently, we have reported that sulfhydryl groups are important in FSH- receptor interaction and that peptides containing free sulfhydryl groups or disulfide bridges, such as glutathione, may nonspecifically inhibit FSH binding to receptor. In the present study, Cys residues of hFSH-beta-(33-53) and hFSH-beta-(81-95) were replaced by Ser residues and the peptides tested for their ability to inhibit binding of FSH to receptor. Results similar to those obtained previously with natural sequence peptides were obtained with the Ser analogs, indicating that Cys residues were not essential for binding inhibition. However, the partial agonist activity of the hFSH-beta-(33-53) and (81-95) in cultured Sertoli cells could not be detected when Cys residues were replaced by Ser. Thus, replacement of Cys residues with Ser does not effect receptor binding activity but is deleterious to the agonist activity of these peptides.

Cellular retinaldehyde-binding protein (CRALBP) has properties that suggest that it is involved in the visual process and, therefore, potentially with retinal diseases. A human cDNA probe has been used to map this gene to human chromosome 15q26 (somatic cell hybrids and in situ hybridization) and to mouse chromosome 7 by somatic cell hybrids.

The catalytic subunit of type-1 protein phosphatase (PP1) was phosphorylated by the tyrosine kinase v-abl as follows: (i) cytosolic PP1 was phosphorylated more (0.73 mol/mol) than PP1 obtained from the glycogen particles (0.076 mol/mol), while free catalytic subunit isolated in the active or inactive form from cytosolic PP1 was phosphorylated even less and catalytic subunit complexed with inhibitor-2 was not phosphorylated; (ii) phosphorylation stoichiometry was dependent on the concentration of PP1 and 3 h incubation at 30 degrees C was required for maximal phosphorylation; (iii) phosphorylation was on a tyrosine residue located in the C-terminal region of PP1 which is lost during proteolysis; (iv) phosphorylation did not affect enzyme activity but allowed conversion from the active to the inactive form upon incubation with inhibitor-2 of a PP1 form that in its dephospho-form did not convert.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.