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H Otto

Publications and source records attributed to H Otto.

At least 37 records · Page 2Linked to original sources
PubMed1992

GTP-binding proteins in bovine brain nuclear membranes.

Nuclear membranes and other subcellular fractions derived from bovine brain cortex were investigated for the existence of GTP-binding proteins. By using photolytic labeling with [alpha-32P]GTP a 29 kDa GTP-binding protein was shown to be present in nuclear membranes which was not present in the plasma membranes nor in microsomal or cytosolic fractions. Two-dimensional gel electrophoresis revealed that this protein is rather acidic with a pI lower than 4.5. Members of the heterotrimeric Gi/o family are not present in the nuclear envelope: a 39 kDa protein, ADP ribosylated by pertussis toxin, was shown to originate from plasma membrane contamination.

Adenosine Diphosphate Ribose↗
PubMed1991

Between the ground- and M-state of bacteriorhodopsin the retinal transition dipole moment tilts out of the plane of the membrane by only 3 degrees.

The orientation of the transition dipole moments in the ground state and the M-intermediate of bacteriorhodopsin were determined by time-resolved and steady-state polarized absorption spectroscopy on samples of oriented immobilized purple membranes. The angle between the transition dipole moment and the membrane normal decreases from 66.8 +/- 0.5 degrees in the all-trans ground state to 64.1 +/- 0.8 degrees in the 13-cis M-state. The light-induced isomerization of the chromophore is thus accompanied by an orientational change of only about 3 degrees out of the plane of the membrane. The absorption anisotropy at 410 nm remains constant over more than 4 decades of time covering both the rise and decay of M. Conformational changes accompanying a sequential M1----M2 transition thus do not affect the chromophore orientation.

Bacteriorhodopsins↗
PubMed1991

The retinylidene Schiff base counterion in bacteriorhodopsin.

Previous studies of bacteriorhodopsin have indicated interactions between Asp-85, Asp-212, Arg-82, and the retinylidene Schiff base. The counterion environment of the Schiff base has now been further investigated by using single and double mutants of the above amino acids. Chromophore regeneration from bacterioopsin proceeds to a normal extent in the presence of a single aspartate or glutamate residue at position 85 or 212, whereas replacement of both charged amino acids in the mutant Asp-85----Asn/Asp-212----Asn abolishes the binding of retinal. This indicates that a carboxylate group at either residue 85 or 212 is required as counterion for formation and for stabilization of the protonated Schiff base. Measurements of the pKa of the Schiff base reveal reductions of greater than 3.5 units for neutral single mutants of Asp-85 but only decreases of less than 1.2 units for corresponding substitutions of Asp-212, relative to the wild type. Substitutions of Asp-85 show large red shifts in the absorption spectrum that are partially reversible upon addition of anions, whereas mutants of Asp-212 display minor red shifts or blue shifts. We conclude, therefore, that Asp-85 is the retinylidene Schiff base counterion in wild-type bacteriorhodopsin. In the mutant Asp-85----Asn/Asp-212----Asn formation of a protonated Schiff base chromophore is restored in the presence of salts. The spectral properties of the double mutant are similar to those of the acid-purple form of bacteriorhodopsin. Upon addition of salts the folded structure of wild-type and mutant proteins can be stabilized at low pH in lipid/detergent micelles. The data indicate that exogenous anions serve as surrogate counterions to the protonated Schiff base, when the intrinsic counterions have been neutralized by mutation or by protonation.

Bacteriorhodopsins↗
PubMed1991

Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation.

To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.

Amino Acid Sequence↗
PubMed1990

Transmembrane location of retinal in bacteriorhodopsin by neutron diffraction.

The transmembrane location of the chromophore of bacteriorhodopsin was obtained by neutron diffraction on oriented stacks of purple membranes. Two selectively deuterated retinals were synthesized and incorporated in bacteriorhodopsin by using the retinal- mutant JW5: retinal-d11 (D11) contained 11 deuterons in the cyclohexene ring, and retinal-d5 (D5) had 5 deuterons as close as possible to the Schiff base end of the chromophore. The membrane stacks had a lamellar spacing of 53.1 A at 86% relative humidity. Five orders were observed in the lamellar diffraction pattern of the D11, D5, and nondeuterated reference samples. The reflections were phased by D2O-H2O exchange. The absolute values of the structure factors were nonlinear functions of the D2O content, suggesting that the coherently scattering domains consisted of asymmetric membrane stacks. The centers of deuteration were determined from the observed intensity differences between labeled and unlabeled samples by using model calculations and Fourier difference methods. With the origin of the coordinate system defined midway between consecutive intermembrane water layers, the coordinates of the center of deuteration of the D11 and D5 label are 10.5 +/- 1.2 and 3.8 +/- 1.5 A, respectively. Alternatively, the label distance may be measured from the nearest membrane surface as defined by the maximum in the neutron scattering length density at the water/membrane interface. With respect to this point, the D11 and D5 labels are located at a depth of 9.9 +/- 1.2 and 16.6 +/- 1.5 A, respectively. The chromophore is tilted with the Schiff base near the middle of the membrane and the ring closer to the membrane surface. The vector connecting the two label positions in the chromophore makes an angle of 40 +/- 12 degrees with the plane of the membrane. Of the two possible orientations of the plane of the chromophore, which is perpendicular to the membrane plane, only the one in which the N----H bond of the Schiff base points toward the same membrane surface as the vector from the Schiff base to the cyclohexene ring is compatible with the known tilt angle of the polyene chain.

Bacteriorhodopsins↗
PubMed1990

Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.

Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early (less than 0.2 microseconds) reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82----Gln mutant, no net transient proton release was observed, whereas, in the Arg-82----Ala mutant, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85----Glu, Arg-82----Ala, and Arg-82----Gln mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85----Ala, Asp-85----Asn, and Asp-212----Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.

Arginine↗
PubMed1989

The purple to blue transition of bacteriorhodopsin is accompanied by a loss of the hexagonal lattice and a conformational change.

X-ray diffraction measurements show that in contrast to the purple membrane, the bacteriorhodopsin molecules are not organized in a hexagonal lattice in the deionized blue membrane. Addition of Ca2+ restores both the purple color and the normal (63 A) hexagonal protein lattice. In the blue state, the circular dichroism spectrum in the visible has the typical exciton features indicating that a trimeric structure is retained. Time-resolved linear dichroism measurements show that the blue patch rotates in aqueous suspension with a mean correlation time of 11 ms and provide no evidence for rotational mobility of bacteriorhodopsin within the membrane. The circular dichroism spectra of the blue and the Ca2+-regenerated purple state in the far-UV are different, indicating a small change in secondary structure. The thermal stability of the blue membrane is much smaller than that of the purple membrane. At pH 5.0, the irreversible denaturation transition of the blue form has a midpoint at 61 degrees C. The photocycle of the blue membrane (lambda ex 590 nm) has an L intermediate around 540 nm whose decay is slowed down into the millisecond time range (5 ms). Light-dark adaptation in the blue membrane is rapid with an exponential decay time of 38 s at 25 degrees C. The purple to blue transition apparently involves a conformational change in the protein leading to a change in the aggregation state from a highly ordered and stable hexagonal lattice to a disordered array of thermally more labile trimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗
PubMed1989

delta pH-induced fluorescence quenching of 9-aminoacridine in lipid vesicles is due to excimer formation at the membrane.

The fluorescence of 9-aminoacridine (9-AA) is quenched in vesicular suspensions containing negatively charged lipid headgroups (e.g., phosphatidylserine) upon imposition of a transmembrane (inside acidic) pH-gradient. It is shown that this fluorescence loss is accompanied by the formation of 9-AA dimers that undergo a transition in the dimer excited state to a dimer-excimer state. This result has been obtained on the basis of the specific dimer fluorescence excitation and hypochromic absorbance spectra that are redshifted by maximally 275 cm-1 (4.4 nm) with respect to the corresponding monomer spectra, as well as by the detection of the characteristic broad excimer emission band, centered at 560 nm. The existence of the spectrally distinct dimer-excimer is further corroborated by fluorescence life-time measurements that indicate an increased lifetime of up to 24 ns for this complex as compared with the normal monomer fluorescence lifetime of 16 ns. The formation of this dimer-excimer complex from the monomers can be reversed completely and the original monomeric spectral properties restored after the abolishment of the electrochemical proton gradient. In addition to the delta pH-induced dimer redshift in absorbance and fluorescence excitation, a further small redshift in monomer absorbance, fluorescence excitation, and emission spectra is observed due solely to the presence of the negatively charged phospholipid headgroups.

Aminacrine↗
PubMed1989

Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin.

Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, [H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition. The decay rate of N is proportional to [H+]. By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (tau)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.

Aspartic Acid↗
PubMed1989

Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement.

The photocycle, electrical charge translocation, and release and uptake of protons from the aqueous phase and release and uptake of protons from the aqueous phase were investigated for bacteriorhodopsin mutants with aspartic acid-96 replaced by asparagine or glutamic acid. At neutral pH the main effect of the Asp-96----Asn mutation is to slow by 2 orders of magnitude the decay of the M intermediate and the concomitant charge displacement associated with the reprotonation of the Schiff base from the cytoplasmic side of the membrane. The proton uptake measured with the indicator dye pyranine is likewise slowed without affecting the stoichiometry of proton pumping. The corresponding results for the Asp-96----Glu mutant, on the other hand, are very close to those for the wild-type protein. These results provide a kinetic explanation for the fact that at pH 7 and saturating light intensities the steady-state proton pumping is almost abolished in the Asp-96----Asn mutant but is close to normal in the Asp-96----Glu mutant. Thus, the pump is simply turning over much more slowly in the Asp-96----Asn mutant. The time constants of the decay of M and the associated charge translocation increase strongly with increasing pH for the Asp-96----Asn mutant but are virtually pH-independent for the Asp-96----Glu mutant and wild-type bacteriorhodopsin. At pH 5 the M decay of the Asp-96----Asn mutant is as fast as for wild type. These results suggest that Asp-96 serves as an internal proton donor in the proton-uptake pathway from the cytoplasm to the Schiff base.

Asparagine↗
PubMed1988

Heat-resistant inhibitors of protein kinase C from bovine brain.

Bovine brain cytosol is shown to contain two heat-resistant inhibitors of protein kinase C, with the following characteristics: 1. One protein kinase C inhibitor can be easily purified to homogeneity. Evidence is presented that this polypeptide of Mr 19,000 is calmodulin. It inhibits protein kinase C with an EC50 of about 2.5 microM and the inhibition is Ca2+-independent. It inhibits only intact protein kinase C. Removal of the regulatory domain of protein kinase C, by limited proteolysis with trypsin, abolishes the inhibition. 2. Another protein kinase C inhibitory activity has been partially purified. Its Mr is low (Mr 600-700, as estimated by gel chromatography). It is not digested by proteases, is hydrophilic, acid- and alkali-resistant, acts Ca2+-independently, and, in contrast to calmodulin, inhibits even the catalytic fragment of protein kinase C after removal of the regulatory domain by limited proteolysis. This inhibition is, at least partially, due to a competition with ATP. Besides protein kinase C, calcium/calmodulin-dependent protein kinase II is inhibited to a similar extent. cAMP-dependent protein kinase is not affected.

Amino Acid Sequence↗
PubMed1988

[The so-called pituitary snuff-taker's lung. A case contribution].

A 37-year-old woman who had developed diabetes insipidus after an abortion, requiring nasal substitution treatment with desmopressin (Minirin), began to suffer from fatigue, nocturnal sweating, cough and dyspnoea on exertion. Exogenous-allergic alveolitis was demonstrated by chest x-ray, lung function tests, blood gas analysis, broncho-alveolar lavage and transbronchial lung biopsy. After changing the treatment to an intramuscularly administered preparation and starting steroid therapy the clinical, radiological and lung function findings rapidly improved.

Administration, Intranasal↗