Resonance Raman investigation of CO-ligated monomeric insect hemoglobins. Direct evidence for reciprocal changes in iron-axial ligand bonds induced by allosteric transitions

The resonance Raman spectra of the two affinity states of the CO-ligated monomeric insect hemoglobins, Chironomus thummi thummi (CTT) III ad IV, have been investigated. We have identified (via 54Fe/57Fe and 13C18O/12C16O isotope exchange) the Fe-N epsilon(His) stretching mode at approximately 317 cm-1. This stretching mode changes from 329 (pH 5.5) to 317 cm-1 (pH 9.5) reflecting the pH-induced t in equilibrium with r conformational transition. The Fe-CO stretching mode is also pH-sensitive changing from 483 (pH 5.2) to 485 cm-1 (pH 9.2) in 57Fe CTT III . 13C18O complex. However the C-O stretching mode is pH-insensitive. The nonallosteric monomeric insect hemoglobin CTT I does not exhibit a pH-dependence of these vibrational modes. pH-Induced effects were also observed for a vinyl bending mode at 379 cm-1 (pH 9.5) in CTT III deuterated at the beta-carbons of the vinyls in position 2 and 4. It shifts to 390 cm-1 at pH 5.5. The other vinyl vibration at 573 cm-1 exhibits intensity enhancement via through-space coupling with the Fe-C-O bending mode. Our resonance Raman data provide the first direct evidence that the trans-effect is operative as a trigger mechanism for ligand-binding in monomeric allosteric insect hemoglobins. In going from the low-affinity to the high-affinity state, the Fe-N epsilon(His) bond becomes weaker, whereas the Fe-CO bond becomes stronger.     

High-Resolution Proton Magnetic Resonance Analysis of Human Cerebrospinal Fluid

High-resolution proton magnetic resonance spectroscopy was used to analyze human cerebrospinal fluid obtained from patients with several neurological problems. The major metabolites measured included glucose, lactate, glutamine, citrate, inositol, acetate, creatine, creatinine, beta-hydroxybutyrate, alanine, and pyruvate. A drug vehicle, propylene glycol, was also measured. Alterations in the cerebrospinal fluid of these metabolites provided information concerning metabolism of the brain. Magnetic resonance spectroscopy offered a simple and rapid means of assessing these and other exogenous and endogenous compounds in diseases affecting the nervous system.     

利用单克隆抗体鉴定人甲胎蛋白(HAFP)抗原结构可分性

本文利用两株针对HAFP分子不同抗原决定簇的单克隆抗体,鉴定HAFP酶解片断的抗原抗体反应性质,并同完整HAFP分子进行比较。结果表明,酶解片断上失去了一株单克隆抗体所对应的分子部份,完整保留着另一株单克隆抗体所识别的抗原决定簇,从而证实HAFP分子某些抗原结构之间具有可分割性。     

Ventilatory response during CO2 inhalation after airway anesthesia with lidocaine

Airway anesthesia with inhaled aerosolized lidocaine has been associated with increases in minute ventilation (VE) and mean inspiratory flow rate (VT/TI) during CO2 inhalation. However, it is unclear whether these increases are local effects of the anesthesia or systemic effects of absorbed and circulating lidocaine. To evaluate this 20 normal subjects were treated on separate days with aerosolized lidocaine, intravenous lidocaine, aerosolized control solution, or intravenous control solution, and the effects of each treatment on VE and VT/TI were determined and compared during room-air breathing and inhalation of 5% CO2-95% O2. None of the treatments altered VE or VT/TI during room-air breathing. Aerosolized lidocaine produced small (5.9-6.0%) increases in VE and VT/TI during CO2 inhalation, but these effects were not present after intravenous lidocaine despite equivalent lidocaine blood levels. We concluded that the increases in VE and VT/TI after aerosolized lidocaine were local effects of airway anesthesia rather than systemic effects of absorbed and circulating lidocaine.     

Distribution of mass in relaxed frog skeletal muscle and its redistribution upon activation.

Five orders of equatorial reflection were recorded from both relaxed and fully activated intact frog sartorius muscle using synchrotron x-ray radiation. Electron density maps of the myofilament lattice in axial projection were calculated from the integrated intensities by Fourier synthesis, using all possible phase combinations. These maps were evaluated systematically in terms of their compatibility with electron microscopically and biochemically derived properties of the lattice structure and with the minimum wavelength principle. For the relaxed state, one phase combination emerged as most consistent with these constraints: it shows a thick filament with a compact core surrounded by an annular shell of density. The distribution of mass suggests that the S-2 moiety of the myosin molecule is an integral part of the thick-filament backbone and the S-1 moiety makes up the shell and is tilted or slewed around the backbone. For the active state, there are two feasible maps, which differ according to whether or not the activation process is associated with phase inversion in two of the reflections. Both maps represent patterns of redistribution of mass upon activation in which the thick-filament backbone is practically unaffected and there is movement of density from the annular shell towards the thin filaments. In addition to this outward radial flux of density from the thick-filament periphery, the pattern of net mass transfer involves a pronounced azimuthal component in both cases. The total net mass transfer is equivalent to approximately 20% (no phase change) or approximately 40% (with phase change) of the S-1 mass. From the observed systematic increase in peak widths of the higher orders, the size of the crystalline domain in the myofilament lattice in the relaxed sartorius is estimated to be greater than 650 nm and the variations in myofilament lattice spacing among different myofibrils to be about +/- 3%. Furthermore, in the activated state, the equilibrium positions of the myofilaments are no longer well ordered, but are distributed statistically about the lattice points with a standard deviation of approximately 3 nm.     

Interaction of histones with estrogens. Covalent adduct formation with 16 alpha-hydroxyestrone

Disturbed estrogen metabolism leading to increased 16 alpha-hydroxyestrone (16 alpha-OHE) has been described in patients with systemic lupus erythematosus and mammary carcinoma. Previous studies showed the formation of covalent complexes between 16 alpha-OHE and nonspecific cellular membrane proteins. The present study is concerned with the interaction of 16 alpha-OHE and histones. Covalent adduct formation between 16 alpha-OHE and individual histones was maximal with H1 histone. Other endogenous estrogens such as estrone, estradiol, and estriol did not interact with histones and form covalent adducts, nor did they interfere with the interaction of 16 alpha-OHE with these nuclear proteins. The evidence supports that the adduct formation between 16 alpha-OHE and histones proceeds via a stabilized Schiff base and subsequent rearrangement. This adduct formation which may have in vivo analogues may represent a mechanism for cellular transformation by this estrogen metabolite.     

A Yersinia enterocolitica serotype 0:3 lipopolysaccharide-specific monoclonal antibody reacts more strongly with bacteria cultured at room temperature than those cultured at 37 degrees C

It has been suggested that the O-side chains of the lipopolysaccharide (LPS) of serotype 0:3 strains of Yersinia enterocolitica vary quantitatively and qualitatively depending on whether they are cultured at 37 degrees C or 25 degrees C. It is uncertain whether this affects the expression of the serotype-specific antigens that are probably carried on the LPS. We studied this question with a serotype 0:3-specific monoclonal antibody, 2C1. This monoclonal antibody immunoprecipitated a 39K major protein from detergent-solubilized 125I-labeled Yersinia preparation. However, results of Western blot experiments demonstrated that the antigens reactive with 2C1 were not actually the 39K protein but the O-side chains of the LPS. Significantly, reactivity could be detected whether the bacteria were cultured at room temperature or at 37 degrees C. However, absorption experiments confirmed that there were less accessible antigenic determinants on the 37 degrees C-LPS. The LPS preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. These SDS-PAGE profiles showed that less O-side chains were present in the 37 degrees C-LPS. Hence, the most likely explanation for our observation is that the 37 degrees C incubation condition induced a partial smooth to rough transition of the Yersinia LPS with a decrease in the amount of 2C1-reactive antigen.     

Antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens

Simple methods for the generation, purification, and assay of antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens have been described. Chicken antibodies against the alpha-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the beta-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen.     

Interaction and identification of ubiquinone-binding proteins in ubiquinol-cytochrome c reductase by azido-ubiquinone derivatives

Various azido-ubiquinone derivatives were synthesized and characterized. 3-Azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone was found to be suitable for the study of specific interaction between ubiquinone (Q) and protein. It was synthesized with high specific radioactivity and used to identify the Q-binding proteins in purified ubiquinol-cytochrome c reductase. This azido-Q derivative showed partial efficiency in restoring activity to the Q- and phospholipids-depleted ubiquinol-cytochrome c reductase in the absence of light. Azido-Q derivative treated samples, however, became completely inactivated upon photolysis, and the inactivation was not reversed by addition of Q derivatives. The redox state of the azido-Q derivative has little effect on the Q-binding affinity. Two protein subunits with Mr = 37,000 and 17,000 were found to be heavily labeled when depleted ubiquinol-cytochrome c reductase was treated with [3H] azido-Q derivative followed by photolysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of radioactive labeling of the Mr = 17,000 protein was proportional to the degree of inactivation and affected by the presence of phospholipids. The radioactive labeling of the Mr = 37,000 protein subunit, however, showed no correlation with degree of inactivation and was not affected by phospholipids. Since the radiolabeling at the Mr = 17,000 protein subunit was affected by phospholipids and correlated with the enzymatic activity, this subunit is probably the Q-binding protein in this enzyme complex (QPc). The inhibition of enzymatic activity by n-heptyl-4-hydroxyquinoline-N-oxide was easily reversed by addition of the azido-Q derivative. The distribution of radioactivity among the subunits of ubiquinol-cytochrome c reductase was not affected by the presence of antimycin A, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole or n-heptyl-4-hydroxyquinoline-N-oxide, suggesting that the binding site(s) of these inhibitors are not the Q-binding site.     

Effects of proteinase K on the energy transfer between phycobiliproteins in phycobilisomes

Spectral changes in fluorescence of phycobilisomes (PBS) of A. variabilis treated with proteinase K were studied at room and liquid nitrogen temperature. In control PBS, the relative yield of 77 K fluorescence of F686 was very high, and those of F655 and F666 were low. In PBS treated with proteinase K for less than 1 h, F686 decreased, and F655 and F666 increased. In PBS treated with proteinase K for 2 h, F655 was the main peak of fluorescence emission, F686 was the second peak, the fluorescence emission peak of F666 disappeared. In PBS treated with proteinase K for more than 8 h, F655 showed only one fluorescence emission peak.We suggested that phycobiliporteins in the PBS of A. variabilis constitute an energy transfer chain, shown as follows:{fx91-1}The linkages between APC and APC-B, C-PC and APC, and C-PC and APC-B had different sensitivity towards proteinase K.