Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis.

To delineate domains essential for G-protein coupling in angiotensin II type 1 receptor (AT1), we mutated the receptor cDNA in the putative cytosolic regions and determined consequent changes in the effect of GTP analogs on angiotensin II (Ang II) binding and in inositol trisphosphate production in response to Ang II. Polar residues in targeted areas were replaced by small neutral residues. Mutations in the second cytosolic loop, carboxy terminal region of the third cytosolic loop or deletional mutation in the carboxyl terminal tail simultaneously abolished both the GTP-induced shift to the low affinity form and Ang II-induced stimulation of inositol trisphosphate production. These results suggest that polar residues in the second cytosolic loop, the carboxy terminal region of the third cytosolic loop, and the carboxy terminal cytosolic tail are important for G-protein coupling of AT1 receptor.     

Induction and Repair of Damage to DNA in Cucumber Cotyledons Irradiated with UV-B

Photoinduced lesions in DNA, namely, cyclobutane pyrimidinedimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts[(6-4)photoproducts], in cucumber cotyledons that had been irradiatedwith naturally occurring levels of UV-B (290–320 nm) werequantitated by enzyme-linked immunosorbent assays with monoclonalantibodies specific to each type of photolesion. Induction ofthese photolesions was dependent on temperature and their extentwas reduced by simultaneous irradiation with white light. Thedark repair of both types of photolesion was undetectable. Light-dependentremoval of (6-4)photoproducts was very slow, with 50% removalin 4 h. By contrast, 50% of initial CPDs were removed within15 min. Both photorepair processes were dependent on the intensityof white light and were sensitive to temperature. These resultsindicate that high photolyase activity is present in cucumbercotyledons and that repair activities in cucumber cotyledonsare different from those reported in Arabidopsis, in which (6-4)photoproductsare photorepaired more rapidly than CPDs. (Received October 13, 1995; Accepted December 28, 1995)     

Lipopolysaccharide Isolated from a New O-Antigenic Form (O13) of Vibrio parahaemolyticus

The chemical properties of a lipopolysaccharide (LPS) isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L -glycero-D -manno-heptose and glucosamine. 2-Keto-3-deoxy-octonate (KDO) was not detected in the LPS by the periodate-thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong-acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4-phosphate in its inner-core region.     

Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G

Affinity tag AG consisting of immunoglobulin G (lgG)-binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of beta-galactosidase (betagal) from Escherichia coli. Poly(methylmethacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human gamma-globulin (HgammaGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGbetagal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGbetagal per unit amount of immobilized HgammaGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HgammaGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HgammaGb-P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HgammaGb molecules. Immobilized AGbetagal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. (c) 1995 John Wiley & Sons, Inc.     

Rhythm in potassium uptake by a duckweed, Lemna gibba G3

The potassium uptake activity of the "flow-medium culture" ofa long-day duckweed, Lemna gibba G3, followed a circadian rhythmwhich persisted for more than 5 days under continuous light.The period of the rhythm was about 25 hr under 3000 lux at 26?Cand was slightly over-compensated against temperature, Q₁₀ beinga little less than 1.0. The amplitude of the rhythm was dependenton light intensity, and there was no potassium uptake in thedark. Magnesium uptake was affected by the potassium movementand showed circadian rhythmicity with a small amplitude underconditions where the potassium uptake was already saturated.Calcium uptake did not show any obvious rhythm. In Contrastto L. gibba, a short-day duckweed L. perpusilla 6746 displayedcircadian rhythm of potassium uptake only in the dark and notin the light. This rhythm did not persist beyond the secondcycle. (Received June 13, 1978; )     

Demonstration of acid phosphatase activity in antigenic glycoprotein fractions obtained from the culture filtrate of Pseudomonas pseudomallei.

Pseudomonas pseudomallei, the causative microorganism of melioidosis, was grown in Mueller-Hinton liquid medium, and glycoprotein fractions were separated from the culture filtrate by ammonium sulfate precipitation, gel-filtration with Sephadex G-75, and column chromatography with DEAE-cellulose. The fractions revealed acid phosphatase activity, and reacted to the sera from melioidosis patient in gel-diffusion precipitation assay.     

Alpha-elastin coacervate as a protein liquid membrane: effect of pH on transmembrane potential responses.

A protein liquid membrane composed of coacervated alpha-elastin, a chemical fragmentation product of the biological elastic fiber protein, functioned as an amphoteric liquid ion-exchange membrane. Ionic permselectivities of the alpha-elastin coacervate membrane to a series of metal chlorides were investigated for the concentration-cell systems by the ordinary electrochemical measurements. Effects of pH on the transmembrane potential responses for NaCl, CaCl2, and MgCl2 systems were examined. Only in the Ca(2+)-containing system did potential responses stay at constant levels against the pH changes, whereas in the other systems, increasing pH caused potential changes, indicating an improvement of cationic permselectivity across the alpha-elastin coacervate membrane. It was suggested that the characteristic Ca2+ transport mechanisms across the alpha-elastin coacervate membrane are related in some way to the polypeptide backbone interactions specific and selective to Ca2+ ions.