Biomedical subjects

J Heberle

Publications and source records attributed to J Heberle.

26 records · Page 2Linked to original sources

PubMed1994

Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe.

The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium salinarium has been genetically transferred into the inner mitochondrial membrane (IM) of the eukaryotic cell Schizosaccharomyces pombe, where the archaebacterial proton pump replaces or increases the proton gradient usually formed by the respiratory chain. For targeting and integration, as well as for the correct orientation of bR in the IM, the bacterioopsin gene (bop) was fused to signal sequences of IM proteins. Northern and Western blot analysis proved that all hybrid gene constructs containing the bop gene and a mitochondrial signal sequence were expressed and processed to mature bR. Fast transient absorption spectroscopy showed photocycle activity of bR integrated in the IM by formation of the M intermediate. Experiments with the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein revealed bR-mediated proton pumping from the mitochondrial matrix into the intermembrane space. Glucose uptake measurements under anaerobic conditions showed that yeast cells containing photoactive mitochondria need less sugar under illumination. In summary, our experiments demonstrate the functional genetic transfer of a light energy converter to a naturally nonphotoactive eukaryotic organism.

Bacteriorhodopsins↗

PubMed1994

Proton migration along the membrane surface and retarded surface to bulk transfer.

Since the proposal of the chemiosmotic theory there has been a continuing debate about how protons that have been pumped across membranes reach another membrane protein that utilizes the established pH gradient. Evidence has been gathered in favour of a 'delocalized' theory, in which the pumped protons equilibrate with the aqueous bulk phase before being consumed, and a 'localized' one, in which protons move exclusively along the membrane surface. We report here that after proton release by an integral membrane protein, long-range proton transfer along the membrane surface is faster than proton exchange with the bulk water phase. The rate of lateral proton diffusion can be calculated by considering the buffer capacity of the membrane surface. Our results suggest that protons can efficiently diffuse along the membrane surface between a source and a sink (for example H(+)-ATP synthase) without dissipation losses into the aqueous bulk.

Arylsulfonates↗

PubMed1993

Bacteriorhodopsin expressed in Schizosaccharomyces pombe pumps protons through the plasma membrane.

Bacterioopsin (bO) from Halobacterium salinarium ("Halobacterium halobium") has been functionally expressed in a heterologous system, the fission yeast Schizosaccharomyces pombe. Regeneration of bO to bacteriorhodopsin (bR) in S. pombe has been achieved in vivo by addition of the chromophore retinal to the culture medium, as shown for a retinal-negative mutant of H. salinarium (JW5). Western blot analysis revealed that bR is more stable than bO against proteolysis in fission yeast and also in JW5. The light-driven proton pump is expressed in the eukaryotic organism and incorporated into the plasma membrane. Illumination of intact yeast cells leads to acidification of the external medium due to the translocation of H+ from inside to outside of the cell, indicating the same orientation of bR in the yeast plasma membrane as in H. salinarium. The kinetics of proton release into the water phase was observed with the optical pH indicator pyranine. Time-resolved absorbance changes of isolated plasma membrane measured by flash spectroscopy showed rise and decay of the M intermediate during the photocycle similar to those in the homologous system. Photocurrents and photovoltages were recorded with yeast plasma membrane attached to a planar lipid membrane and to a polytetrafluoroethylene (Teflon) film, respectively. Stationary currents measured in the presence of a protonophore showed continuous pumping activity of bR. The action spectrum of the photocurrent and the kinetics of the photovoltage were analyzed and compared with signals obtained from purple membranes. From all these different investigations we conclude that the integral membrane protein bR is correctly folded in vivo into the cytoplasmic membrane of the fission yeast S. pombe.

Bacteriorhodopsins↗

PubMed1993

Decoupling of photo- and proton cycle in the Asp85-->Glu mutant of bacteriorhodopsin.

Surface bound pH indicators were applied to study the proton transfer reactions in the mutant Asp85-->Glu of bacteriorhodopsin in the native membrane. The amino acid replacement induces a drastic acceleration of the overall rise of the M intermediate. Instead of following this acceleration, proton ejection to the extracellular membrane surface is not only two orders of magnitude slower than M formation, it is also delayed as compared with the wild-type. This demonstrates that Asp85 not only accepts the proton released by the Schiff's base but also regulates very efficiently proton transfer within the proton release chain. Furthermore, Asp85 might be the primary but is not the only proton acceptor/donor group in the release pathway. The Asp85-->Glu substitution also affects the proton reuptake reaction at the cytoplasmic side, although Asp85 is located in the proton release pathway. Proton uptake is slower in the mutant than in the wild-type and occurs during the lifetime of the O intermediate. This demonstrates a feed-back mechanism between Asp85 and the proton uptake pathway in bacteriorhodopsin.

Asparagine↗

PubMed1992

Surface-bound optical probes monitor protein translocation and surface potential changes during the bacteriorhodopsin photocycle.

Light-induced H+ release and reuptake as well as surface potential changes inherent in the bacterio-rhodopsin reaction cycle were measured between 10 degrees C and 50 degrees C. Signals of optical pH indicators covalently bound to Lys-129 at the extracellular surface of bacteriorhodopsin were compared with absorbance changes of probes residing in the aqueous bulk phase. Only surface-bound indicators monitor the kinetics of H+ ejection from bacteriorhodopsin and allow the correlation of the photocycle with the pumping cycle. During the L550----M412 transition the H+ appears at the extracellular surface of bacteriorhodopsin. Surface potential changes detected by bound fluorescein or by the potentiometric probe 4-[2-(di-n-butylamino)-6-naphthyl]vinyl-1-(3-sulfopropyl)pyridinium betaine (di-4-ANEPPS) occur in milliseconds concomitantly with the formation and decay of the N intermediate. pH indicators residing in the aqueous bulk phase reflect the transfer of H+ from the membrane surface into the bulk but do not probe the early events of H+ pumping. The observed retardation of H+ at the membrane surface for several hundred microseconds is of relevance for energy conversion of biological membranes powered by electrochemical H+ gradients.

Bacteriorhodopsins↗

PubMed1991

Long-term follow-up after heterotopic splenic autotransplantation for traumatic splenic rupture.

The trapping function of the heterotopic splenic autotransplants (HSA) in 13 polytraumatized patients, aged 5-38 yr, was evaluated using heat damaged technetium-99m-labeled autologous red blood cells in early (1-7 mo) and late (3-4.5 yr) period after heterotopic autotransplantation to the omentum. The intensity of tracer accumulation was graded in comparison to the liver uptake. The splenic tissue surface was calculated on anterior projection each time. The shapes of the transplants were compared and new uptake foci suggesting spontaneous splenosis were looked for on both scans. The average surface of HSA was 28.2 (+/- 14.7) cm2 on early and 44.1 (+/- 14.3) cm2 on late examination (p less than 0.003) and the increase in intensity of tracer accumulation on both occasions was significant as well (p less than 0.0001). In three patients, some additional splenotic foci were found on follow-up scans. Howell-Jolly bodies in peripheral blood were detected in six of eight patients in early and remained detectable in lower number in three of eight patients on follow-up. No serious infection was noticed in our group of patients. Our work confirmed the excellent survival rates of HSA with improving trapping function and no important spread from original implantation site on long-term follow-up.

Adolescent↗

PubMed1990

Bacteriorhodopsin in ice. Accelerated proton transfer from the purple membrane surface.

The photocycle and the proton pumping kinetics of bacteriorhodopsin, as well as the transfer rate of protons from the membrane surface into the aqueous bulk phase were examined for purple membranes in water and ice. In water, the optical pH indicator pyranine residing in the aqueous bulk phase monitors the H(+)-release later than the pH indicator fluorescein covalently linked to the extracellular surface of BR. In the frozen state, however, pyranine responds to the ejected H+ as fast as fluorescein attached to BR, demonstrating that the surface/bulk transfer is in ice no longer rate limiting. The pumped H+ appears at the extracellular surface during the transition of the photocycle intermediate L550 to the intermediate M412. The Arrhenius plot of the M formation rate suggests that the proton is translocated through the protein via an ice-like structure.

Bacteriorhodopsins↗

PubMed1988

Creativity and schizotypal traits. Creativity test scores and perceptual aberration, magical ideation, and impulsive nonconformity.

Using a nonclinical and noneminent population, this study demonstrates an overlap in creative and schizotypal traits in the areas of perceptual functioning, behavioral and personality styles, and interests. No such overlap is observed in the area of divergent thinking. A battery containing five creativity measures was administered to a group of college student subjects scoring high on either the Perceptual Aberration Scale or the Magical Ideation Scale (N = 52) and to a group of control subjects (N = 65). A multivariate analysis of variance indicated that subjects high on the schizotypal traits of Perceptual Aberration or Magical Ideation (Per-Mag subjects) differed significantly from control subjects on the five creativity tests. Per-Mag subjects scored significantly higher than control subjects on the Barron-Welsh Revised Art Scale, a measure of preferences for figures, and the How Do You Think, a biographical and personality measure. There was a tendency for female Per-Mag subjects to score higher than female control subjects on the Domino Creativity Scale of the Adjective Check List. Per-Mag and control subjects did not differ on the Gough Creative Personality Scale of the Adjective Check List or on the Alternate Uses test, a test of divergent thinking. Per-Mag subjects who scored above the median for their group and gender on the Impulsive Nonconformity Scale received the highest creativity scores on the Barron-Welsh Revised Art Scale and the How Do You Think, although these results only approached significance. These findings argue for the specificity of areas of similarity and difference in schizotypy and creativity.

Adult↗