Purification of a Src family tyrosine protein kinase from bovine retinas.

Since tyrosine phosphorylation appears to play important functions in photoreceptor cells, we searched here for retinal nonreceptor tyrosine kinases of the Src family. We demonstrated that Src family tyrosine kinases were present in the cytosolic fraction of extracted bovine retinas. A Src family tyrosine kinase with an apparent molecular mass of about 62 kDa was purified to homogeneity from the soluble fraction of dark-adapted bovine retinas after three consecutive purification steps: -aminooctyl-agarose hydrophobic chromatography, Cibacron blue 3GA-agarose pseudo-affinity chromatography, and -casein-agarose affinity chromatography.

Light or tyrosine phosphorylation recruits retinal rod outer segment proteins to lipid rafts.

Lipid rafts are localized liquid-ordered regions of the plasma membrane that contain high levels of cholesterol and glycosphingolipids, and are resistant to extraction with nonionic detergents. Retinal photoreceptor cells contain detergent-resistant membrane microdomains (DRM), which were isolated here from bovine rod outer segments (ROS) under dark and light conditions. Rhodopsin (R) was present in both DRM and detergent soluble fractions (DSF), and detergent-insoluble ROS rafts were enriched in caveolin 1 (Cav-1) and c-Src.

Cross-linking of bovine rhodopsin with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate affects its functionality.

Rhodopsin is the photoreceptor protein involved in visual excitation in retinal rods. The functionality of bovine rhodopsin was determined following treatment with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a bifunctional reagent capable of forming covalent cross-links between suitable placed lysines and cysteines. Denaturing polyacrylamide gel electrophoresis showed that rhodopsin incubated with sulfo-SMCC generated intermolecular dimers, trimers, and higher oligomers, although most of the sulfo-SMCC-treated protein remained as a monomer.

Correlation of transducin photoaffinity labeling with the specific formation of intermolecular disulfide linkages in its α-subunit.

Transducin (T) is a heterotrimer of Tα, Tβ, and Tγ subunits. In the presence of light-activated rhodopsin, 8-azidoguanosine triphosphate (8-N3GTP) was covalently incorporated into T in a UV-light photodependent manner, with a low stoichiometry of 0.02 mol of 8-N3GTP per mol of T.

A variant of arrestin-1 binds rod outer segment membranes in a light-independent manner.

A 50-kDa-polypeptide band peripherally bound to retinal rod outer segment (ROS) membranes was purified by anion-exchange chromatography. When the 50-kDa protein was compared with purified arrestin-1, it was observed that: (1) both proteins comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and were recognized by either anti-50-kDa protein polyclonal antibodies or anti-arrestin-1 monoclonal antibodies; (2) protein fragments and peptide fingerprint maps obtained following limited and complete proteolysis with specific proteases were very similar for both molecules; and (3) several chromatographically-purified tryptic peptides from the 50-kDa protein possessed the same amino acid composition as tryptic peptides deduced from the reported arrestin-1 primary structure. Consequently, arrestin-1 and the purified 50-kDa protein must correspond to variants of the same molecule.

85-kDa protein of Trypanosoma cruzi purified by affinity chromatography used in the multiple antigen binding assay (MABA) for the diagnosis of T. cruzi infection in a Venezuelan rural community.

No ideal test exists for Chagas' disease, and better diagnostic strategies are needed. We determined the diagnostic utility of an 85-kDa Trypanosoma cruzi protein in a multiple antigen binding assay (MABA). A standardized MABA test based on concentrated trypomastigote excretory-secretory antigen (TESA) and an 85-kDa purified protein showed 100% sensitivity and specificity.

Chemical modification of transducin with dansyl chloride hinders its binding to light-activated rhodopsin.

Transducin (T), the heterotrimeric guanine nucleotide binding protein in rod outer segments, serves as an intermediary between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase. Labeling of T with dansyl chloride (DnsCl) inhibited its light-dependent guanine nucleotide binding activity. Conversely, DnsCl had no effect on the functionality of rhodopsin.

Distribution of dehydration rates generated by maximal Gardos-channel activation in normal and sickle red blood cells.

The Ca(2+)-activated K+ channels of human red blood cells (RBCs) (Gardos channels, hIK1, hSK4) can mediate rapid cell dehydration, of particular relevance to the pathophysiology of sickle cell disease. Previous investigations gave widely discrepant estimates of the number of Gardos channels per RBC, from as few as 1 to 3 to as many as 300, with large cell-to-cell differences, suggesting that RBCs could differ extensively in their susceptibility to dehydration by elevated Ca2+. Here we investigated the distribution of dehydration rates induced by maximal and uniform Ca2+ loads in normal (AA) and sickle (SS) RBCs by measuring the time-dependent changes in osmotic fragility and RBC volume distributions.

The hydrodynamic properties of dark- and light-activated states of n-dodecyl beta-D-maltoside-solubilized bovine rhodopsin support the dimeric structure of both conformations.

Rhodopsin (Rho) has been extracted in n-dodecyl beta-D-maltoside (DM) from bovine retinal rod outer segments and purified to homogeneity by affinity chromatography on concanavalin A-Sepharose. Because chemical cross-linking of Rho and photoactivated Rho (Rho*) provided initial evidence for the oligomeric nature of the photoreceptor protein, we carried out a hydrodynamic characterization of the native and activated conformations of detergent-solubilized Rho. The molecular weights of the complexes between dark and photoexcited states of Rho and DM were determined by gel filtration chromatography on Sephacryl S-300, in the presence of 0.

Identification of functionally important acidic residues in transducin by group-specific labeling.

Transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells, is a heterotrimer arranged as two units, the alpha-subunit (T alpha) and the beta gamma-complex (T beta gamma). The role of the carboxyl groups in T was evaluated by labeling with N,N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDC). Only a minor effect on the binding of beta, gamma-imido guanosine 5'-triphosphate (GMPpNp) to T was observed in the presence of the hydrophobic carbodiimide, DCCD.

Distribution of plasma membrane Ca2+ pump activity in normal human red blood cells.

The plasma membrane calcium pump (PMCA) is the only active Ca2+ transporter in human red blood cells (RBCs). Previous measurements of maximal Ca2+ extrusion rates (Vmax) reported only mean values in the RBC population. Despite early evidence for differences in Ca2+ extrusion capacity among RBCs, the precise Vmax distribution remained unknown.