Investigations of the rhodopsin/Meta I and rhodopsin/Meta II transitions of bovine rod outer segments by means of kinetic infrared spectroscopy

We have applied our recently developed technique of flash induced kinetic infrared spectroscopy to the rhodopsin/Meta I and rhodopsin/Meta II transitions. Features of the infrared spectrum reflecting the C=C-vibration and the isomeric form of the chromophore are in agreement with resonant Raman experiments. Different results are obtained for the C=N-vibration of the Schiff base retinal opsin link. They are interpreted in terms of a Schiff base protonated via an hydrogen bond. A proton transfer in the excited state is suggested to explain the deviating results. In addition we have obtained spectral changes which cannot be attributed to molecular changes in the chromophore. We assume that these spectral features reflect molecular events in the protein part of rhodopsin.     

Asp85 is the only internal aspartic acid that gets protonated in the M intermediate and the purple-to-blue transition of bacteriorhodopsin. A solid-state 13C CP-MAS NMR investigation.

High-resolution solid-state 13C NMR spectra of the ground state and M intermediate of the bacteriorhodopsin mutant D96N with the isotope label at [4-13C]Asp and [11-13C]Trp were recorded. The NMR spectra show that Asp85 is protonated in the M intermediate. The environment of Asp85 is quite hydrophobic. On the other hand, Asp212 remains deprotonated and a slight shift to lower field indicates a more hydrophilic environment. Asp85 also protonates in the purple-to-blue transition of bacteriorhodopsin in the deionized membrane, where it experiences a similar environment to M. The shift of Trp resonances in M reflect a conformational change of the protein in forming the M intermediate.     

Structural investigation of bacteriorhodopsin and some of its photoproducts by polarized Fourier transform infrared spectroscopic methods-difference spectroscopy and photoselection

The direction of selected IR-transition moments of the retinal chromophore of bacteriorhodopsin (BR) and functional active amino acid residues are determined for light- and dark-adapted BR and for the intermediates K and L of the photocycle. Torsions around single bonds of the chromophore are found to be present in all the investigated BR states. The number of twisted single bonds and the magnitude of these torsions decreases in the order K, L, light-adapted BR, dark-adapted BR. In the last, only the C₁₄—C₁₅ single bond is twisted. The orientation of molecular planes and chemical bonds of such protein side chains, which are perturbed during the transition of light-adapted BR to the respective intermediates, are deduced and the results compared with the current three dimensional model of BR. Trp 86 and Trp 185 are found to form a rigid part of the protein, whereas Asp 96 and Asp 115 perform molecular rearrangements upon formation of the L-intermediate.     

Enzymatic cleavage of the epsilon-peptide bond in alpha- and epsilon-substituted glycyl- and phenylalanyl-lysine peptides

Lysine peptides, X-Lys-OH (Formula: see text) were synthesized, following classic or non-classic routes. Some bacterial and mammalian enzymes, endo- and exo-peptide hydrolases of the enzyme nomenclature type EC 3.4., were tested for their ability to split the epsilon-peptide bond in the above substrates. Kinetic constants (Km,kcat) were evaluated with leucine aminopeptidase from hog kidney and eye lens with aminopeptidase I from yeast. Aminopeptidase M (hog pancreas) and hog intestinal aminopeptidase were additionally examined for their Ki values with the above substrates in comparison to the classic protease substrate leucine p-nitroanilide. Especially the intestinal mucosa hydrolases are shown to be efficient in cleaving epsilon-peptide bonds.     

A brief comparative study of four disposable bubble oxygenators

Four widely used bubble oxygenators-the Optiflo I, the Bentley Q 200 A, the Harvey 200, and the Shiley 100 A-were tested and compared in 182 patients undergoing cardiac valve surgery. Fifty-six cases were performed with normothermia and 126 cases incorporated mild hypothermia (28-30 degrees C). There was no significant difference in the average age of the patients (51 yrs) or the perfusion time (60 min). All components of the extracorporeal circuit were identical, and anesthetic regimens and surgical techniques were also similar. In this study, the Shiley 100 A oxygenator was found to be the most suitable for cases requiring mild hypothermia and was generally considered to be the oxygenator of choice.     

Substrate affinity in PGM1, PGM2, and PGM3 isozymes

Summary An easy method for routine detection of PGM₁, PGM₂, and PGM₃ isozymes is given. Differences in substrate affinity are discussed. Gene products pgm₁ can be differentiated from gene products pgm₃ by cofactor requirement.