Purification of a K(+)-channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system

A K(+)-channel protein of the sarcoplasmic reticulum (SR) was purified by assaying the channel activity in a planar lipid bilayer system. The light fraction of SR vesicles was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and fractionated by an anion-exchange chromatography and followed by gel filtration chromatography and affinity chromatography with concanavalin A. All fractions in each steps were mixed with asolectin solubilized in CHAPS and reconstituted into vesicles by dialysis. The channel activity of each fraction was assayed after the reconstituted vesicles had been fused into a planar lipid bilayer. The final fraction which showed the K(+)-channel activity contained only 100 kDa protein in a silver-stained gel after SDS-PAGE and an anti-Ca(2+)-ATPase antibody did not recognize the protein. The characteristics of the K(+)-channel were identical to those observed in native SR vesicles when using the same method. The channel showed a single-channel conductance of 120 pS in 0.1 M KCl and marked voltage dependence. The channel did not permeate Ca2+ and Cl- and was blocked by neomycin B.     

Studies on the substrate specificity of Taka-amylase A. XII. Investigation of the active site of Taka-amylase A by examining the properties of p-phenylazobenzoyl Taka-amylase A.

1. When p-phenylazobenzoyl Taka-amylase A (PhAB-TAA) was incubated at pH 6.5 with hydroxylamine for 3 hr at 20degrees, some of the p-phenylazobenzoyl residues that had been introduced into Taka-amylase A (TAA) [1, 4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1, Aspergillus oryzae] were liberated as a hydroxamic acid, and the activity pattern of PhAB-TAA changed to that of intact TAA. This result suggested that the p-phenylazobenzoyl residues liberated had been bound to the tyrosyl residue located near the active site in the enzyme. 2. The transferase action of TAA or PhAB-TAA was studied using phenyl alpha-maltoside as a substrate and maltotritol as an acceptor. Unlike intact TAA, PhAB-TAA was not able to transfer the maltose residue to maltotritol, and this suggested that the p-phenylazobenzoyl residue was located near one of the aglycone-binding subsites, causing steric hindrance.     

Clinical study on early changes in thyroid function of hyperthyroidism treated with propylthiouracil and a relatively small dose of iodide.

In order to compare the acute effects of three kinds of antithyroid agents of iodide (I-), propylthiouracil (PTU) and PTU combined with iodide (PTU+I-) on thyroid function in hyperthyroid patients with diffuse goiter, serum concentrations of thyroxine (T4), triiodothyronine (T3), T3-resin sponge uptake (T3-RU) and free thyroxine index (FT4I) were employed as thyroid function parameters. In the group given iodine (1 mg/day) as iodinated-lecithine, the initial values of T4, T3, T3-RU and FT4I were 20.9 +/- 1.6 microng/100 ml (T4), greater than 740 ng/100 ml (T3), 49.5 +/- 2.3% (T3-RU) and 14.7 +/- 1.8 (FT4I). At the end of one week of therapy, they decreased clearly to 15.6 +/- 2.2 microng/100 ml, 457 +/- 87 ng/100 ml, 42.2 +/- 4.0% and 9.7 +/- 2.4. The so-called "escape phenomenon" from iodide inhibition was observed in serum T4, T3-RU and FT4I values at the end of two weeks of iodide therapy, while serum T3 continued to decrease but the value of T3 was far outside of the normal range. In the PTU group (300 mg/day), thyroid function parameters were 22.5 +/- 0.8 microng/100 ml (T4), greater than 592 ng/100 ml (T3), 54.9 +/- 1.0% (T3-RU) and 18.7 +/- 1.0 (FT4I) before treatment. They decreased continually week by week. At the end of four-week treatment with PTU, the value of each thyroid function parameter was 11.1 +/- 1.9 microng/100 ml, 229 +/- 56 ng/100 ml, 36.6 +/- 4.4% and 5.7 +/- 1.7. In the group of hyperthyroidism simultaneously given both PTU and iodide (300 mg/PTU and 1 mg/iodine), these thyroid function parameters decreased as well as in the group treated with PTU alone for more than two weeks. More rapid or significant decrease of T4, T3, T3-RU and ft4i in PTU+I- group than in PTU group was observed in the present study. These results suggested strongly that iodide alone was not an adequate therapy for hyperthyroidism as well known and they were also compatible with the idea that the concomitant administration of PTU and iodide was more effective in the early phase of therapy of hyperthyroidism than PTU alone.     

Hydroxylations of substituted naphthalenes by Escherichia coli expressing aromatic dihydroxylating dioxygenase genes from polycyclic aromatic hydrocarbon-utilizing marine bacteria

Bioconversion experiments of various mono- or di-substituted naphthalenes such as dimethylnaphthalenes were carried out using the cells of Escherichia coli that expressed aromatic dihydroxylating dioxygenase genes (phnA1A2A3A4 and phdABCD) from polycyclic aromatic hydrocarbon-utilizing marine bacteria, Nocardioides sp. KP7 and Cycloclasticus sp. A5, respectively. We found that the former dioxygenase PhnA1A2A3A4 had broad substrate preference for these compounds and often was able to hydroxylate their methyl groups. Specifically, 1,4-dimethylnaphthalene was predominantly bioconverted into 1,4-dihydroxymethylnaphthalene.     

Human placenta beta-galactoside-binding lectin. Purification and some properties

A beta-galactoside-binding lectin was extracted from human placenta homogenate with lactose solution and purified to apparent homogeneity by affinity chromatography on asialofetuin-Sepharose. The apparent subunit molecular weight of the lectin was 13,800 and its isoelectric point was about 5. Several saccharides containing D-galactose inhibited the hemagglutinating activity. The lectin resembles other vertebrate beta-galactoside-binding lectins in various biochemical characteristics.     

The mutations induced by oxidatively damaged nucleotides, 5-formyl-dUTP and 5-hydroxy-dCTP,in Escherichia coli.

The mutational properties of 5-formyl-2'-deoxyuridine 5'-triphosphate (5-CHO-dUTP) and 5-hydroxy-2'-deoxycytidine 5'-triphosphate (5-OH-dCTP), the major oxidatively damaged pyrimidine nucleotides derived from dTTP and dCTP, respectively, were analyzed by an in vivo assay. 5-CHO-dUTP and 5-OH-dCTP were directly incorporated into Escherichia coli , and their mutagenicities were evaluated by the chromosomal lacI forward mutation assay. The mutation frequencies increased, depending on the dose of these damaged nucleotides, indicating that these nucleotides were incorporated into E.coli and acted as mutagens in vivo . The mutagenicities of 5-CHO-dUTP and 5-OH-dCTP were comparable to that of 8-hydroxy-2'-deoxyguanosine 5'-triphosphate, a major form of dGTP oxidative damage. 5-CHO-dUTP induced G.C to A.T, A.T to G.C and G.C to T.A mutations, and 5-OH-dCTP elicited G.C to A.T, A.T to C.G and G.C to T.A mutations.     

Essential role of the PSI–LHCII supercomplex in photosystem acclimation to light and/or heat conditions by state transitions

Light and temperature affect state transitions through changes in the plastoquinone (PQ) redox state in photosynthetic organisms. We demonstrated that light and/or heat treatment induced preferential photosystem (PS) I excitation by binding light-harvesting complex II (LHCII) proteins. The photosystem of wheat was in state 1 after dark overnight treatment, wherein PQ was oxidized and most of LHCII was not bound to PSI. At the onset of the light treatment [25 °C in the light (100 µmol photons m^?2 s^?1)], two major LHCIIs, Lhcb1 and Lhcb2 were phosphorylated, and the PSI–LHCII supercomplex formed within 5 min, which coincided with an increase in the PQ oxidation rate. Heat treatment at 40 °C of light-adapted wheat led to further LHCII protein phosphorylation of, resultant cyclic electron flow promotion, which was accompanied by ultrafast excitation of PSI and structural changes of thylakoid membranes, thereby protecting PSII from heat damage. These results suggest that LHCIIs are required for the functionality of wheat plant PSI, as it keeps PQ oxidized by regulating photochemical electron flow, thereby helping acclimation to environmental changes.