Contact sites in interaction between light-activated rhodopsin and transducin (T) have been investigated by using a chemically preactivated crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)-propionate. The 3 propionyl-N-succinimidyl group in the reagent was attached by a disulfide exchange reaction to rhodopsin mutants containing single reactive cysteine groups in the cytoplasmic loops. Complex formation between the derivatized rhodopsin mutants and T was carried out by illumination at > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crosslinking of rhodopsin to the T␣ subunit. Crosslinking to T ␣ was demonstrated for the rhodopsin mutants K141C, S240C, and K248C, and the crosslinked sites in T␣ were identified for the rhodopsin mutant S240C. The peptides carrying the crosslinking moiety were isolated from the trypsin-digested peptide mixture, and their identification was carried out by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The main site of crosslinking is within the peptide sequence, Leu-19 -Arg-28 at the N-terminal region of T ␣. The total results show that both the N and the C termini of T ␣ are in close vicinity to the third cytoplasmic loop of rhodopsin in the complex between rhodopsin and T.cysteine mutants ͉ disulfide exchange ͉ cytoplasmic domain ͉ matrixassisted laser desorption ionization-time of flight ͉ avidin-biotin affinity chromatography I nteractions of rhodopsin with transducin (T) and rhodopsin kinase are the first steps in the two biochemical cascades that are initiated on light activation in visual signal transduction (1). Because both T ␣␤␥ and rhodopsin kinase are known to bind to the cytoplasmic domain of light-activated rhodopsin, it is important to investigate the actual contact sites in the molecular complexes formed between rhodopsin and the above two proteins. With this long-range aim, we have initiated applications of the broad covalent crosslinking approach. We (2) reported on an initial study in which we used a photoactivatable crosslinking agent (Fig. 1 I). Here we report on a study that uses a crosslinking reagent (Fig. 1 II) carrying an activated carboxyl group. An electrophilic attack by the carbonyl group on unprotonated amino groups in suitable proximity will result in crosslinking by formation of amide linkages. Therefore, only a pH change will be required in the crosslinking strategy to deprotonate the relevant amino groups, usually the -amino groups in lysine residues.As in the accompanying paper (2), rhodopsin mutants containing single reactive cysteines at predetermined positions in the cytoplasmic domain (Fig. 2) form the starting points for study of interaction with T. The crosslinking moiety, R in Fig. 1 II, is transferred to the rhodopsin mutants by a disulfide exchange reaction with the reactive pyridyl thio group in Fig. 2 II (step 1 in Fig. 3). Three mutants of rhodopsin containing single reactive cysteine residues, K141C, S240C, and K248C (Fig. 2), were selected, and all were ...

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfideexchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at >495 nm. Subsequent irradiation of the complex at 310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with T ␣. For identification of the sites of crosslinks in T ␣, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the T ␣ species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the Nmaleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting T ␣ derivatives and isolation of T␣ peptides carrying maleimido-butyrylbiocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption͞ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in T ␣ containing the amino acid sequences 310 -313 and 342-345.L ight activation of rhodopsin initiates two biochemical cascades, one leading to sensitization (amplification) and the other to desensitization (quenching). Binding and activation of transducin (T) and rhodopsin kinase are, respectively, the first events in the two cascades (1). Competition between T and rhodopsin kinase for interaction with the light-activated rhodopsin and its phosphorylated form is central to the progression of the two cascades. Therefore, understanding the requirements of the two proteins for binding to rhodopsin and the contact sites in the complexes formed is of much interest. Ideally, three-dimensional structures of the complexes between the proteins are needed, but such structural analysis lies in the future. Alternative approaches have provided some insights into the sites in rhodopsin required for binding of T. Thus, peptides corresponding to the amino acid sequences in the different cytoplasmic loops of rhodopsin inhibit T activation (2), and similarly mutagenic studies (3-5) have indicated that a considerable portion of the cytoplasmic domain of light-activated rhodopsin is involved in binding to T. Some information also has been obtained on the regions of T that contact rhodopsin in the complex. Thus, the effects of a...

Using a photon-ion merged-beam technique, we measured the relative yield spectra of Xe 2+ and Xe 3+ ions created by photoionization of Xe + ions as a function of the photon energy in the 4d ionization region. The Xe 3+ ion production, i.e. the double-electron ionization process is found to be dominant throughout the energy range investigated. A broad structure of 4d excitation ionization around 100 eV photon energy was observed in the Xe 3+ yield spectrum. A spectral shoulder was observed for Xe 3+ at around 85 eV, which was absent in the photoionization of neutral Xe. Some discrete lines were observed for both the Xe 2+ -and Xe 3+yield spectra below 75 eV. The experimental spectra were analysed by a multiconfiguration Dirac-Fock calculation. The largest structure at around 100 eV is attributable to 4d 10 5s 2 5p 5 → 4d 9 4f5s 2 5p 4 np (n = 6, 7) two-electron transitions. The discrete lines were found to be due to 4d → np, nf transitions. A serious 4f-orbital collapse is suspected in the 4fnp two-electron excited states, whereas the collapse is moderate in the singly excited 4f-orbitals.

Using urea-solubilized human fibrin monomer as an immunogen, we raised in mice a battery of monoclonal antibodies that reacted with the immunogen but not with urea-treated or native fibrinogen. Although they all failed to react with acid-solubilized fibrin monomer (acid-FM) alone, an antibody designated as IF-43 was found to recognize acid-FM, which was bound with fibrinogen or its derivatives to form a 1:2 complex of soluble fibrin. The epitope for this antibody, thus, appears to be exposed most probably by conformation changes induced in the acid- FM molecule upon formation of the complex. Because IF-43 was able to recognize fibrin-derived plasmic fragment E treated with urea but not the thrombin- and urea-treated amino-terminal disulfide knot of fibrinogen, the presence of the A alpha (52–78) residue segment seems to be prerequiste for the epitope expression. The antibody was found to react with soluble fibrin monomer spiked to normal plasma dose- dependently up to 200 micrograms/mL. By an aggregation assay using latex beads coated with IF-43, we found that concentrations of soluble fibrin monomer in plasma derived from patients with thrombotic diseases were mostly elevated, but not necessarily correlated with those of the D-dimer, reflecting another aspects of the disease. Furthermore, the soluble fibrin monomer in plasma derived from patients with thrombotic diseases was found to be depleted solely of the A peptides, but not the B peptides, based on its subunit polypeptide compositions lacking the beta-chain on immunoblotting.

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beta(2)-Glycoprotein I (beta(2)GPI) consists of five tandem repeated domains (I, II, III, IV, and V). The nicked form of beta(2)GPI (N-beta(2)GPI ) which was cleaved by plasmin in vitro at Lys 317-Thr 318 in domain V, showed reduced affinity for the negatively charged phospholipids, especially cardiolipin (CL). Recently, the N-beta(2)GPI was detected in the plasma of patients with disseminated intravascular coagulation syndrome (DIC) by an immunological method. In the present study, we prepared monoclonal antibodies for the nicked form, and demonstrated that the concentrations of this form of beta(2)GPI, which were analyzed by a sandwich ELISA using two specially prepared monoclonal antibodies, were significantly increased in the plasma of patients with leukemia (n = 51, mean +/- SD: 162.0 +/- 118.3 ng/ml) and with lupus anticoagulant (LA) (n =40, mean +/- SD: 3,041.5 +/- 16,579.7 ng/ml), compared to the normals (n = 33, mean +/- SD: 1.04 +/- 1.54 ng/ml). We found a significant correlation between the concentrations of N-beta(2)GPI and those of typical molecular markers for a fibrinolytic state such as plasmin-alpha(2) plasmin inhibitor complex (PIC) and D-dimer in patients with leukemia, but not in patients with LA. These results suggested that the generation of N-beta(2)GPI was caused by plasmin in the patients with leukemia, and by unknown proteases in the patients with LA. In the patients with LA, the levels of N-beta(2)GPI tended to be higher in those without thrombosis than in those with thrombosis.

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Photoion spectrometry has been applied to determine the relative photoionization yields of Ba+ ions around the 4d ionization threshold. A ground state Ba+ ion beam was merged with monochromatized synchrotron radiation over a length of 15 cm. Counting the number of doubly and triply charged ions produced in the interaction region, we have obtained partial photoion yields as a function of photon energy. Prominent peaks due to resonance Auger processes have been observed below the threshold. A giant resonance structure above 4d threshold was seen only for the Ba3+ spectrum.