Jun Otomo

Properties and the primary structure of a new halorhodopsin from halobacterial strain mex

A new halorhodopsin-like pigment from the new halobacterial strain mex (Otomo, J., Tomoika, H. and Sasabe, H. (1992) J. Gen. Microbiol. 138, 1027-1037) was partially purified, and its amino acid sequence from helices A to G was determined using PCR technique. Two arginine residues in the A-B interhelix loop segment, a series of six amino acid residues (EMPAGH) in the B-C interhelix segment and most of the residues near the Schiff base of the retinal were found to be conserved in three halorhodopsins (halobium, pharaonis and mex). This result strongly suggests that these residues are essential for anion pumping function in halorhodopsin. The light-induced ion-pump measurements have shown that the selectivity of anion transport between chloride and nitrate in mex halorhodopsin is lower than that of halobium halorhodopsin, but higher than that of pharaonis halorhodopsin. The number of amino acid residues in the B-C interhelix loop segments is different in each halorhodopsin, and it correlates with their anion (chloride and nitrate) selectivity. These results suggest that the length of the B-C segment affects the selectivity of anion transport in halorhodopsin.

Xenopus laevis oocytes expressing human P-glycoprotein: probing trans- and cis-inhibitory effects on [3H]vinblastine and [3H]digoxin efflux.

P-glycoprotein (P-gp; MDR1) recognizes and actively transports many structurally diverse compounds (hydrophobic neutral and cationic). We studied MDR1-mediated drug transport using a high-throughput (96-well) oocyte expression system. MDR1-expressing oocytes contained sufficient ATP levels to conduct fundamental efflux studies; the optimal experimental temperature was 25 degrees C. [(3)H]Vinblastine efflux by MDR1-expressing oocytes was detectable and afforded a K(m) of 145.5+/-25.4microM. [(3)H]Vinblastine (5.6+/-0.3microM) and [(3)H]digoxin (1.0+/-0.1microM) were individually injected into MDR1-expressing oocytes and their efflux monitored. Quinidine and verapamil, known MDR1 substrates/inhibitors, showed trans-inhibition on MDR1-mediated [(3)H]vinblastine and [(3)H]digoxin efflux. Conversely, doxorubicin demonstrated cis-inhibition without trans-inhibition on MDR1-mediated [(3)H]vinblastine efflux. The MDR1-expressing oocyte system offers researchers with an alternative in vitro method to screen compounds and may allow one to probe P-gp drug-drug and/or drug-inhibitor interactions.

Dynamic light scattering study of suspensions of purple membrane

Purple membrane from Halobacterium halobium in suspensions has been studied by quasielastic light scattering. The intensity correlation functions of polarized scattered light were measured at various K2 values (K being the magnitude of the scattering vector), and the first cumulant Gamma of the field correlation function G1(tau) was obtained by a cumulant expansion method. The apparent diffusion coefficient Gamma /K2 did not increase monotonically with K2 values and showed a distinct anomaly in an intermediate range of K. A theoretical formulation of G1(tau) for a disc and an extremely oblate ellipsoidal shell of revolution (S. Fujime and K. Kubota, Biophys. Chem. 23 (1985) 1) was applied to the analysis of the spectra, and characteristic features of experimental spectra were well reproduced. It was suggested that a strong interference effect between scattered rays on Gamma /K2 should be attributed to a slight noncircular shape of the purple membrane and that a contribution to Gamma /K2 from membrane flexibility should be taken into account. This study will provide experimental evidence of the feasibility of membrane studies by dynamic light scattering.

Polarization properties in phase conjugation with bacteriorhodopsin

Polarization properties of the phase conjugate beam in biological photochrome bacteriorhodopsin with photoinduced anisotropic nonlinearity are studied. We derived the polarization state and reflectivity of the phase conjugate beam in degenerate four-wave mixing (DFWM) where two pump beams have the same or orthogonal polarization and a probe beam has arbitrary polarization. Theoretical results are proved by experiments.

Anion selectivity and pumping mechanism of halorhodopsin

Comparison of the amino acid sequences in the A-B and B-C interhelical loop segments in all bacteriorhodopsins and halorhodopsins has shed light on the anion selectivity and pumping mechanism of halorhodopsin. The nucleotide sequences of two haloopsins from two new halobacterial strains, shark and port, have been determined, and shark halorhodopsin was functionally overexpressed in Halobacterium halobium. Although a series of six amino acid residues (EMPAGH) in the B-C interhelical loop segment was substituted by QMPPGH, all putative charged residues were conserved. It was also shown that His-95 mutants had lower pumping activity in low chloride concentrations. These results further support the hypothesis that His-95 is important in the halorhodopsin function.

Expression of the histamine receptor in Xenopus oocyte and its application to the histamine sensor

Abstract We developed a receptor-mediated biological sensor for real time detection of histamine derived from granules of mast cell and basophils. A human histamine receptor gene was cloned by polymerase chain reaction and its mRNA was injected into Xenopus oocyte. After incubation for 2–3 days, the histamine receptor was expressed in the cell membrane of the oocyte. When the oocyte was activated by histamine, a calcium-activated chloride channel was opened, and the chloride ions in the oocyte flowed to the outside. The chloride ion current intensity was correlated with the histamine concentration. The histamine sensitivity of the oocyte was sufficient to allow comparison with other methods histamine analyses, such as enzyme immunoassay or HPLC analysis. The oocyte sensor system described here is a new chemical sensor system using receptor-mediated biological reactions.

OVER-EXPRESSION OF A NEW PHOTO-ACTIVE HALORHODOPSIN IN HALOBACTERIUM SALINARIUM

The gene of haloopsin (hop) from halobacterial strain shark was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of shark halorhodopsin (HR) showed that its homology with halobium HR was 62%. The gene product seems to be HR having several positively charged residues that are conserved in all known HRs. The gene encoding shark hop as well as that encoding halobium hop were successfully expressed in Halobacterium salinarium (halobium) by using a plasmid shuttle vector containing the bacterioopsin (bop) promoter. The expression level of shark HR is almost the same as that for halobium HR with the same shuttle vector containing the bop promoter. Under the physiological conditions, the anion pumping activity of the shark HR expressed in H. salinarium was almost the same as that for halobium HR; however, the anion selectivity and half-maximal anion transport were different. Furthermore, its absorption maximum in the absence of chloride shifted to approx. 596 nm in contrast to that for halobium HR. The half-lifetimes of HR520 formation for shark HR and halobium HR were almost the same; however, the half-lifetime of its decay was approx. 6-times faster for shark HR than it was for halobium HR at a high chloride concentration (1000 mM). Even at a low chloride concentration (50 mM), HR520 and HR640 intermediates could be detected for shark HR, and the half-lifetime of HR640 decay was found to be approx. 25 ms. In the presence of nitrate, the half-lifetime of HR565 recovery for shark HR was approx. 10-times slower than that for halobium HR. Some of amino acid substitutions between shark HR and halobium HR may affect the anion selectivity and the photoreaction of HR.

Evaluating Protein Sequence Signatures Inferred from Protein-Protein Interaction Data by Gene Ontology Annotations

We propose a systematic method to find sequence signatures of proteins which share a common interacting partner, using a protein similarity measure based on gene ontology (GO) annotations. In a computational experiment on our original human data set of protein-protein interactions determined by Y2H assays, we have succeeded in discovering convincing candidates for interacting sites and other functional regions.