Mass-spectrometric determination of O2 and CO 2 gas exchange in illuminated higher-plant cells : Evidence for light-inhibition of substrate decarboxylations.

In order to estimate photosynthetic and respiratory rates in illuminated photoautotrophic cells of carnation (Dianthus caryophyllus L.), simultaneous measurements of CO2 and O2 gas exchange were performed using (18)O2, (13)CO2 and a mass-spectrometry technique. This method allowed the determination, and thus the comparison, of unidirectional fluxes of O2 and CO2. In optimum photosynthetic conditions (i.e. in the presence of high light and a saturating level of CO2), the rate of CO2 influx represented 75±5% of the rate of gross O2 evolution. After a dark-to-light transition, the rate of CO2 efflux was inhibited by 50% whereas the O2-uptake rate was little affected. The effect of a recycling of respiratory CO2 through photosynthesis on the exchange of CO2 gas was investigated using a mathematical model. The confliction of the experimental data with the simulated gas-exchange rates strongly supported the view that CO2 recycling was a minor event in these cells and could not be responsible for the observed inhibition of CO2 efflux. On the basis of this assumption it was concluded that illumination of carnation cells resulted in a decrease of substrate decarboxylations, and that CO2 efflux and O2 uptake were not as tightly coupled in the light as in the dark. Furthermore, it could be calculated from the rate of gross photosynthesis that the chloroplastic electron-transport chain produced enough ATP in the light to account for the measured CO2-uptake rate without involving cyclic transfer of electrons around PS I or mitochondrial supplementation.

Molecular dynamics of two homologous neurotoxins revealed by 1H-2H exchange: an infrared spectrometry study.

Temperature effects on the hydrogen exchange kinetics and the infrared spectra of two homologous snake neurotoxins (Laticauda semifasciata erabutoxin b and Naja nigricollis toxin alpha) were investigated between 10 and 40 degrees C, at their isoionic pH. (1) Erabutoxin b is more accessible to the solvent than toxin alpha. (2) With increasing temperature, both toxin molecules undergo a global transition affecting the most accessible as well as the most buried hydrogens: the overall accessibility changes are more important for erabutoxin b than for toxin alpha. The different conformational stabilities of the toxins are also qualitatively supported by the temperature-induced shifts which affect the infrared amide I band of toxin alpha only. The existence of two conformer families could be responsible for the different conformational stability of these proteins.

Freeze-fracture studies on the plasma membranes of normal human, thrombasthenic, and Bernard-Soulier platelets.

The description of severe molecular deficiencies of different membrane glycoproteins in thrombasthenic and Bernard-Soulier platelets has led us to investigate the intramembrane organization of their plasma membranes using freeze-fracture electron microscopy. An initial examination of the cleaved plasma membranes of freeze-fractured normal human platelets revealed randomly distributed MAPs on the fracture faces of both the outer and inner phospholipid leaflets of the bilayer. Particle densities of 925 +/- 52/micrometer22 on the EF and 427 +/- 29/micrometer2 on the PF were calculated with a computer-linked picture analyzer. The particle size was heterogeneous on both fracture faces, and the number of particles decreased exponentially in the 5 to 13 nm size range. Examination of the platelets of three thrombasthenic patients revealed a low particle density (36% to 69% of the normal range) on the PF of the cleaved plasma membrane and a reduced particle coefficient between the two fracture faces (PF/EF). This abnormality was accompanied by a preferential decrease in the larger sized particles. In marked contrast 8 to 13 nm particles predominated on both fracture faces of the plasma membranes of the platelets of a Bernard-Soulier patient, and a greater concentration of particles on the PF rather than the EF was uniquely observed. The results further define the modified structure of thrombasthenic and Bernard-Soulier platelet plasma membranes and suggest a structural heterogeneity within the total MAP population of the membranes of normal human platelets.

Conformation of corticotropin. An infrared spectrometry study of hydrogen exchange kinetics.

1H--2H exchange kinetics of the peptide hydrogens in corticotropin have been examined in 2H2O and CF3C2H2O2H solutions by means of infrared absorption measurements. In aqueous solution, around pH 3, the experimental data suggest a partially ordered structure, since in the two corticotropins 1--24 and 1--32 about 6 slowly exchanging peptide protons are numbered. These might belong to the N-terminal part of the molecule. The C-terminal 25--32 octapeptide segment appears to be unordered and slightly destabilizes the overall hormone conformation. For corticotropin1--24 in CF3C2H2O2H, the qualitative interpretation of infrared spectra and the quantitative analysis of exchange data give evidence of a strong stabilization: a predominantly alpha-helical structure is induced by trifluoroethanol.

PMR-relaxation and steric computations give unequivocal nucleoside conformations.

The configuration and the conformation of alpha and beta anomers of pyrazomycin, cytidine and pseudouridine in aqueous solution have been investigated by 1H-NMR at 250 MHz. T1 proton relaxation measurements are an excellent method to determine the conformation of the base around the glycosidic linkage. Frequently, steric hindrance considerations can help to decide which conformations are possible in nucleoside anomer pairs. The proton-proton coupling constants indicate that the N conformer is largely predominant in the alpha anomers while the S conformer is particularly abundant in beta-pyrazomycin. The steric hindrance is much larger for alpha than for beta-nucleosides and change of a C-C to a C-N glycosidic bond reduces considerably the rotational possibilities of the base. The relaxation data show that alpha-cytidine adopts the anti conformation with gamma = 200 degrees in good agreement with the crystal structure and with the sterical computations. In the other case, when the syn and anti conformations are sterically accessible, the orientation of the base may be completely different from one nucleoside to the other. It can be predicted neither from the crystal structure nor from comparisons with similar compounds. For alpha-pseudo-uridine the predominant orientation of the base (gamma = 120 degrees) is in the boundary of the syn-anti regions; for beta-cytidine the syn (gamma = 65 degrees) and anti (gamma = 215 degrees) conformations are equiprobable at room temperature while beta-pseudouridine shows the syn conformation with gamma = 40 degrees, the smallest angle observed until now. There is no correlation between the N/S and syn-anti ratios.

Quantitative 250 MHz proton magnetic resonance study of hydrogen-deuterium exchange. Angiotensin II hormone in trifluoroethanol.

Hydrogen-deuterium exchange kinetics of (Asn1-Val5) angiotensin II has been investigated by proton magnetic resonance at 250 MHz in deuterated trifluoroethanol, as an approach to the "in situ" hormone conformation. An interactive program was specially developed to perform the data analysis on a computer similar to those used for spectroscopic data acquisition. Nine exchange sites are evidenced and characterized by their individual kinetic parameters. Three of them are assigned to peptide NH hydrogens, and the six remaining to slowly exchanging side chain protons. At 11 degrees C, more than three peptide hydrogens, sterically hindered or involved in hydrogen bonds, do not exchange. These results corroborate previous circular dichroism and infrared investigations performed in the same solvent, and suggest a family of well-folded conformations, stabilized in trifluoroethanol by internal hydrogen bonds, involving both the backbone and the side chain hydrogens.

Hydrogen-isotope exchange of oxidized and reduced cytochrome c. A comparison of mass spectrometry and infrared methods.

Hydrogen-deuterium exchange in 2H20 solutions of the two redox states of horse heart cytochrome c was investigated at 20 degrees C, pH 7, by mass spectrometry and infrared spectroscopy. Mass spectrometry indicates that ferricytochrome has 20 hydrogens unexchanged after 24 h, 28 hydrogens exchanging between 10 min and 24 h, and 156 hydrogens exchanging within 10 min; comparative values for ferrocytochrome are 45, 19 and 140. The displacement of the exchange curves obtained by infrared corresponds to 8 to 9 peptide hydrogens. These combined methods show many non-peptide hydrogens exchanging rapidly (87 and 79 for ferricytochrome c and ferrocytochrome c respectively), whereas others, probably buried inside the molecule and involved in hydrogen bonds, are not exchanged, even after 24 h (14 and 30 hydrogens respectively, which is relatively large for a small protein). Infrared results are given in terms of changes of standard free energy for the transconformational reaction which exposes the peptide hydrogens to solvent: in ferricytochrome c and ferrycoytochrome c, 30% and 40% respectively of the peptide hydrogens are protected by conformational transitions stabilized by more than 5 kcal/mol (21 kJ/mol), which implies a large increase in rigidity for the reduced form.