Isolation of photochemically active archaebacterial photoreceptor, pharaonis phoborhodopsin from Natronobacterium pharaonis.

A photoreceptor, pharaonis phoborhodopsin for the negative phototaxis of extremely halophilic and alkalophilic archaebacterium, Natronobacterium pharaonis was isolated in a photochemically active state. A detailed examination of the chromatographic separation made it possible to separate contaminating proteins, such as cytochromes. The procedure resulted in a 2938-fold enrichment with a yield of 15.5%. The isolated pharaonis phoborhodopsin had an absorption maximum at 498 nm, an A280/A498 ratio of 1.27 and a single band near 24 kDa on the SDS-polyacrylamide gels. The isolated pharaonis phoborhodopsin underwent a photochemical reaction after flash excitation. The photocyclic reaction closely resembled that of the membrane-bound pharaonis phoborhodopsin.

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.

Voltage-dependent absorbance change of carotenoids in halophilic archaebacteria.

Membrane vesicles of wild-type Halobacterium sp. mex strain show a wavy absorbance change which has not been so far reported in halophilic archaebacteria. A white mutant strain lacking carotenoids did not show the wavy absorbance change. The wavy absorbance change in the range of 440-590 nm was induced by a red flash (600-640 nm), which photoexcited electrogenic ion pumps, mex bacteriorhodopsin and mex halorhodopsin but not carotenoids. The wavy change was also caused by K+ diffusion potentials without light. These results suggest that the wavy absorbance change in the membrane vesicles is the voltage-dependent absorbance change of the carotenoids.

Two-dimensional crystallization of catalase on a monolayer film of poly(1-benzyl-L-histidine) spread at the air/water interface.

Two-dimensional (2D) crystals of beef liver catalase were prepared by adsorption to a film of synthetic polypeptide, poly(1-benzyl-L-histidine) (PBLH), spread at the air/water interface. The crystallization experiments were carried out in the pH range of 4.8-6.4 for catalase solutions at low concentration (10 micrograms/ml). The pH-dependence suggested an electrostatic interaction in the binding of catalase to the PBLH film. At lower pH, small crystals were formed at a low binding rate, and at higher pH the binding was rapid and densely-packed 2D arrays with poor crystallinity were formed. To stimulate crystal growth, a thermal treatment was applied. One-shot heating of the interfacial catalase-PBLH film to 35-40 degrees C was remarkably effective to form larger 2D crystals. The structure of catalase 2D crystals has been analyzed by Fourier filtering of the transmission electron micrographs. The crystal form is a new one, containing four catalase molecules in the unit cell with lattice parameters of alpha = 187 A, b = 225 A and gamma = 92.8 degrees.

Folding intermediate binds to the bottom of bullet-shaped holo-chaperonin and is readily accessible to antibody.

Holo-chaperonin from Thermus thermophilus (Thermus holo-cpn) is a bullet-shaped particle where chaperonin-10 heptamer locates at one axial end of the cylindrical body of chaperonin-60 tetradecamer. Thermus holo-cpn promotes in-vitro folding of denatured 3-isopropylmalate dehydrogenase (IPMDH) from the same bacterium. We observed the complexes of Thermus holo-cpn and folding intermediates of IPMDH by immuno-electron microscopy after decoration by single layer labeling with anti-IPMDH IgG or by double layer labeling with anti-IPMDH IgG as first layer and antibodies against IgG as second layer. Images of the electron microscope showed that anti-IPMDH IgG was bound to the bottom end of the bullet-shaped Thermus holo-cpn. This result provides direct evidence that the folding intermediate binds to the axial end, which is opposite to the end where chaperonin-10 heptamer resides, of the cylindrical body of chaperonin-60 tetradecamer, and that bound folding intermediate in the complex is sufficiently exposed to the outside to be accessible by antibody.

Crystallization of a photosensitive nitrile hydratase from Rhodococcus sp. N-771.

A photosensitive nitrile hydratase from Rhodococcus sp. N-771 has been crystallized in two different crystal forms in its inactive form. One crystal form belongs to an orthorhombic space group P2(1)2(1)2 with unit cell dimensions of a = 117.4 A, b = 145.7 A and c = 52.1 A, and the other form belongs to a hexagonal space group P6(3)22 with unit cell dimensions of a = 110.2 A and c = 412.1 A.

Equatorial split of holo-chaperonin from Thermus thermophilus by ATP and K+.

Holo-chaperonin molecule from Thermus thermophilus is a bullet-shaped particle whose cylinder part and round top are composed of two stacked rings of the cpn60 heptamer and a single ring of the cpn10 heptamer, respectively. We found that it splits at the plane between two cpn60 rings into two halves under physiological conditions, that is, in the presence of ATP (but not AMP-PNP, ADP) + K+ (but not Na+) at 60 degrees C. This equatorial split could be functionally important although it has not been considered in any current mechanistic model of chaperonin functioning.

Light-induced oxidation of iron atoms in a photosensitive nitrile hydratase.

The photoactivation process of a photosensitive nitrile hydratase (NHase) from Rhodococcus sp. N-771 has been investigated by 57Fe Mössbauer spectroscopy and magnetic susceptibility measurements in order to clarify the behavior of iron atoms in the enzyme. Mössbauer spectra of inactive NHase gave two symmetric-doublet components indicating the presence of two iron species, while that of the active NHase gave a single symmetric doublet indicating the presence of a single iron species. Magnetic susceptibility measurements of the inactive and active HNase both showed small effective magnetic moments. These results led us to conclude that one of the two iron atoms incorporated in the NHase is oxidized during photoactivation, namely from a low spin ferrous to a low spin ferric state. This is the first observation of an intramolecular photooxidation phenomena involving iron in a single protein molecule.

Photosensitive nitrile hydratase intrinsically possesses nitric oxide bound to the non-heme iron center: evidence by Fourier transform infrared spectroscopy.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photosensitive enzyme that catalyzes hydration of nitriles to the corresponding amides. Light-induced Fourier transform infrared difference spectra between the inactive and active forms of NHase were measured with both the natural (14N) and 15N-labeled NHases. The results showed, for the first time, that NHase intrinsically possesses nitric oxide (NO) molecules bound to the non-heme iron center. The possible role of NO in the photoactivation process of NHase is discussed.

In vitro rheological evaluation of antithrombogenicity or anticoagulability of styrene derivative polymer.

In vitro evaluation of antithrombogenicity or anticoagulability of styrene derivative polymer with functional silyl groups, the polymer treated with acid, polystyrene and glass as a reference was attempted using a rheological method. The results were compared with those obtained from measurements of platelet adhesion. The coagulation process of blood in polymer-coated tubes was monitored using a damped oscillation-type rheometer recently developed. The change of fluidity during coagulation of blood was dependent on the polymers. Poly((4-vinylphenyl)dimethyl-2-propoxysilane) (poly(1)) was more antithrombogenic than polystyrene and acid-treated poly(1). The evaluation of antithrombogenicity or anticoagulability for the polymers and glass by the rheological method coincided well with that obtained from other methods. Furthermore, it was confirmed that the rheological method would be useful for elucidating mechanisms of coagulation of blood as well as thrombus formation in developing artificial blood-compatible materials.

Selective adhesion and proliferation of cells on ion-implanted polymer domains.

We have found that the adhesion and proliferation of endothelial cells can be drastically improved when cultivated on an ion-implanted polymer surface. When the surface of segmented polyurethane, where endothelial cells are not capable of proliferating, is modified by Ne+ or Na+ ion implantation with a fluence of 1 x 10(15) ions/cm2 at an energy of 150 keV, cell adhesion and proliferation occurred selectively on the ion-implanted region irrespective of the ion species. The cells did not proliferate at ion fluences below 1 x 10(14) ions/cm2. Most cells migrated into the ion-implanted domain within 1-2 h, but some of the cells attached outside of the region and then slowly migrated into the region. Ion implantation of polystyrene, on which cells are capable of proliferating, further promoted cell spreading and proliferation, and increased resistance to detachment when the cells were exposed to trypsin.

Scanning electron microscopy of negatively stained catalase on a silicon wafer.

A high-resolution scanning electron microscope capable of 7 A spatial resolution at 30-kV accelerating voltage was used to observe negatively stained protein molecules. Thin platelet crystals, densely packed monolayers, and low-density deposits of beef liver catalase were prepared on the surface of silicon wafers and negatively stained with phosphotungstic acid. The tetrameric structure of the catalase molecule was observed for the first time by scanning electron microscopy on the surface of the smooth silicon wafer.

Suppression of the development of experimentally induced uterine adenomyosis by a novel matrix metalloproteinase inhibitor, ONO-4817, in mice.

The inhibitory effects of a novel, orally active matrix metalloproteinase (MMP) inhibitor, ONO-4817, on the development of uterine adenomyosis induced experimentally by pituitary grafting were examined in mice. Mice were given transplants of isologous anterior pituitary glands (PGs) into the right uterine lumen at 7 weeks of age and were fed chow containing 0.1% to 1.0% ONO-4817 from 8 to 14 weeks of age. Mice treated with 0.3% or 1.0% ONO-4817 showed a significantly lower incidence of the development of adenomyosis than vehicle-treated mice. To evaluate the inhibitory effects of ONO-4817 on the progression of the invasion of the adenomyotic tissues, mice receiving PG grafts at 7 weeks of age were treated with 1.0% ONO-4817 from 13 to 17 weeks of age. The degree of pathological progression of adenomyosis was graded from 1 to 5 in increments of 1. The degree of the progression of the lesion was less in the uteri exposed to ONO-4817 (2.71 +/- 0.93) than in the uteri not exposed to the inhibitor (4.33 +/- 0.75). Finally, the invasiveness of endometrial stromal cells obtained from adenomyotic uteri into Matrigel consisting mainly of type IV collagen and laminin was examined using an invasion assay. The assay showed that the treatment with ONO-4817 markedly suppressed the invasion of the stromal cells of the adenomyotic uteri into the gel. These results indicate that ONO-4817 may be an effective inhibitor of the development of adenomyosis.

Mechanism of protein folding. IV. Forming and breaking of disulfide bonds in bovine pancreatic tripsin inhibitor.

The folding mechanism of bovine pancreatic tripsin inhibitor (BPTI) is explained theoretically on the basis of the island model, where the driving force of folding is hydrophobic interaction. For this purpose, we take a look at the formation and breaking of disulfide bonds during the folding process of BPTI. The intermediate conformations and the native one are successfully obtained, which satisfy the so-called "lampshade" geometrical criterion for the formation of the disulfide bonds. The folding pathway is consistent with the renaturation experiment by Creighton. In addition, an elaborate treatment of side chains of amino acid residues by the software programme CHARMm confirms quantitatively the formation of disulfide bridges.

Chemisorption of proteins and their thiol derivatives onto gold surfaces: characterization based on electrochemical nonlinearity.

This study was conducted to monitor the electrochemical responses of two proteins (bovine serum albumin (BSA) and gelatin) and their thiol derivatives adsorbed onto gold (Au) electrodes, which were analyzed by a "nonlinear" impedance method. A sinusoidal voltage is applied to a protein-containing aqueous solution and the waveform of the output current is analyzed by fast Fourier transformation (FFT). The intensities of the higher harmonics in the FFT varied with the species of protein and their thiol derivatives, and with time. From the higher harmonics, voltage-dependent capacitance and conductance were quantitatively evaluated to differentiate the state of adsorbed protein. Adsorption and desorption characteristics of BSA and its thiol derivative on the Au surface were continuously measured by a quartz crystal microbalance (QCM) in situ. The microscopic state of thiol-derivatized BSA adsorbed onto the Au surface was imaged by atomic force microscopy (AFM). In general, thiol-derivatized proteins were tightly adsorbed on the Au surface and showed no desorption. The present electrochemical measurements clearly differentiated adsorption characteristics of physically adsorbed (physisorbed) and chemically adsorbed (chemisorbed) proteins on Au surfaces.

Imaging two-dimensional arrays of soluble proteins by atomic force microscopy in contact mode using a sharp supertip.

A sharp tip with high aspect ratio is required for imaging biological macromolecules by atomic force microscopy (AFM). A tip with the end radius of curvature less than 3 nm has been reproducibly fabricated by means of electron beam deposition (EBD) in a field-emission scanning electron microscope. Two-dimensional protein arrays of ferritin and catalase, prepared at air/water interface and transferred onto silicon wafer, could be imaged both in air and in water by AFM using this sharp EBD-tip in contact mode. The negative staining preparation conventionally used in the transmission electron microscopy of protein was applied and shown to be quite effective in fixing the protein arrays for the AFM imaging in air. Individual molecules of ferritin and catalase were visible in the two-dimensional arrays. Also, imaging in water of these protein arrays presented molecular images clearer than in air, due probably to the absence of the adhesion force and the resulting weak lateral force during scanning. These images convince us of the capability of this supertip for AFM studies of biological molecules under aqueous conditions.

Two-dimensional crystallization of streptavidin by nonspecific binding to a surface film: study with a scanning electron microscope.

A two-dimensional (2D) crystal of streptavidin has been obtained by a nonspecific binding method. The protein molecules were bound and formed a dense packing on the film of poly(1-benzyl-L-histidine) spread at the surface of protein solution. The surface film was moderately heated to stimulate crystallization of bound streptavidin. A potential of this method for obtaining 2D crystals of soluble proteins is demonstrated. The present 2D crystal structure of streptavidin resembles that previously obtained by specific binding to biotinylated lipid. We show in addition that the 2D array of protein with usual size approximately 50 A can be imaged using a high resolution scanning electron microscope (HR-SEM) and subject to structural analysis at low resolution. Various limitations in HR-SEM degrade considerably the image quality. However, the usability of a bulk plate as specimen support would make HR-SEM a convenient tool, when such a substrate must be considered in application of protein arrays, and if an intrinsic low resolution is acceptable.

Pharmacokinetics of a new positive inotropic agent, 3, 4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-qu inolinone (OPC-8212), in the rat, rabbit, beagle dog and rhesus monkey.

The pharmacokinetics of 3, 4-dihydro-6-[4-(3,4- dimethoxybenzoyl )-1-piperazinyl]-2(1H)- quin olinone ( OPC -8212) were studied after the administration of 14C- OPC -8212 or OPC -8212 to animals of different species. After oral doses of 10 mg/kg of 14C- OPC -8212 to rats and beagle dogs, the Tmax, Cmax and T1/2 values of OPC -8212 were 4 h, 2995 ng eq/ml, and 3-4 h in rats and 1 h, 2244 ng eq/ml and 5-6 h in beagle dogs, respectively. After oral doses of 10 mg/kg of 14C- OPC -8212 to rats, the radioactivity was distributed comparatively widely in the tissues. However, there was no evidence of accumulation of radioactivity in the tissues due to repeated oral doses of 10 mg/kg of 14C- OPC -8212 once a day for 21 days. After oral doses of 10 mg/kg of 14C- OPC -8212, the amounts of radioactivity excreted in the urine and feces in the first 72 h accounted for 29.25% and 60.24% of the dose in rats and 35.53% and 63.18% of the dose in beagle dogs, respectively. There were no apparent changes in the urinary and fecal excretions of radioactivity due to repeated oral doses of 10 mg/kg of 14C- OPC -8212 once a day for 21 days in rats. Biliary excretion of radioactivity was 22.41% of the dose after oral doses of 10 mg/kg 14C- OPC -8212 in rats. Enterohepatic circulation was 22.04% of the dose after an intraduodenal dose in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly sensitive method for the determination of 5-fluorouracil in biological samples in the presence of 2'-deoxy-5-fluorouridine by gas chromatography-mass spectrometry.

A highly sensitive and convenient gas chromatographic-mass spectrometric (GC-MS) method is described for the determination of 5-fluorouracil in the presence of 2'-deoxy-5-fluorouridine (which breaks down into 5-fluorouracil during ordinary GC derivatization) in biological samples such as plasma and urine. After extraction with ethyl acetate, 5-fluorouracil and 5-chlorouracil, the latter being used as an internal standard, were converted into their tert.-butyldimethylsilyl derivatives by allowing the mixture to stand for 30 min at room temperature and were assayed by electron-impact ionization GC-MS. Under these conditions, 2'-deoxy-5-fluorouridine did not decompose or interfere with the determination of 5-fluorouracil. The assay method, including the extraction and tert.-butyldimethylsilyl derivatization of 5-fluorouracil, showed good linearity in the range 0-100 ng/ml for 5-fluorouracil in plasma (detection limit 0.5 ng/ml) and urine (detection limit 1 ng/ml). The usefulness of this method was demonstrated by determining plasma concentrations of 5-fluorouracil in rats treated intravenously with 5-fluorouracil and 2'-deoxy-5-fluorouridine.