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Biomedical subjects

C R Hackenbrock

Publications and source records attributed to C R Hackenbrock.

At least 37 records · Page 2Linked to original sources
PubMed1985

Calcium-mediated fusion to produce ultra large osmotically active mitochondrial inner membranes of controlled protein density.

We have developed a new membrane fusion method which produces ultra large, spherical mitochondrial inner membranes attached to microscope slides. The fused inner membranes measured up to 200 microns in diameter. The technique fuses native inner membranes as well as inner membranes in which the protein density has been varied by enriching with exogenous phospholipid. The fusion process is accomplished through the use of calcium, low pH and elevated temperature. Characterization of the fused membranes was carried out using phase, fluorescence, and freeze-fracture electron microscopy. These ultra large, fused inner membranes were found to model the inner membranes from which they were formed. The fused inner membranes were found to be osmotically active and are large enough for measuring the lateral diffusion of membrane components by fluorescence recovery after photobleaching and are large enough for microelectrode impalement.

Animals↗
PubMed1985

Independent lateral diffusion of cytochrome bc1 complex and cytochrome oxidase in the mitochondrial inner membrane.

Distinct fluorophores have been conjugated to antibodies for cytochrome bc1 complex and cytochrome oxidase, two integral electron transferring proteins in the mitochondrial inner membrane. Addition of these fluorescent antibodies to preparations of mitochondrial inner membranes followed by appropriate secondary antibodies causes distinct and independent aggregation of the two cytochrome proteins. These results reveal that both cytochrome bc1 complex and cytochrome oxidase diffuse laterally in the membrane plane independent of one another consistent with the random collision model for electron transport in the mitochondrial inner membrane.

Animals↗
PubMed1984

Relationship between lateral diffusion, collision frequency, and electron transfer of mitochondrial inner membrane oxidation-reduction components.

Fluorescence recovery after photobleaching was used to determine the diffusion coefficients of the oxidation-reduction (redox) components ubiquinone, complex III (cytochromes b-c1), cytochrome c, and complex IV (cytochrome oxidase) of the mitochondrial inner membrane. All redox components diffuse in two dimensions as common-pool electron carriers. Cytochrome c diffuses in two and three dimensions concomitantly, and its diffusion rate, unlike that of all other redox components, is modulated along with its activity by ionic strength. The diffusion coefficients established in this study reveal that the theoretical diffusion-controlled collision frequencies of all redox components are greater than their experimental maximum (uncoupled) turnover numbers. Since electron transport is slower than the theoretical limit set by the lateral diffusion of the redox components, ordered chains, assemblies, or aggregates of redox components are not necessary to account for electron transport. Rather, mitochondrial electron transport is diffusion coupled, consistent with a "random-collision model" for electron transport.

Animals↗
PubMed1982

Lateral diffusion of ubiquinone during electron transfer in phospholipid- and ubiquinone-enriched mitochondrial membranes.

After fusion of small unilamellar phospholipid liposomes with mitochondrial inner membranes, the rate of electron transfer between membrane dehydrogenases and cytochrome c decreases as the average distance between integral membrane proteins increases, suggesting that electron transfer is mediated through a diffusional process in the membrane plane (Schneider, H., Lemasters, J. J., Höchli, M., and Hackenbrock, C. R. (1980)., J. Biol. Chem. 255, 3748-3756). The role of ubiquinone in this process was evaluated by fusing liposomes containing ubiquinone-10 or ubiquinone-6, with inner membranes. In control membranes enriched with phospholipid only, ubiquinol-cytochrome c reductase and NADH- and succinate-cytochrome c reductase activities decreased proportionally to the increase in bilayer lipid. These decreases were restored substantially in phospholipid plus ubiquinone-supplemented membranes. The degree to which restoration occurred was dependent upon the length of the isoprenoid side chain of the ubiquinone with the shorter chain length ubiquinone-6, always giving greater restoration than ubiquinone-10. It is concluded that electron transfer between flavin-linked dehydrogenases (Complexes I and II) and cytochrome bc1 (Complex III) occurs by independent, lateral diffusion of ubiquinone as well as independent, lateral diffusion of ubiquinone as well as the protein complexes within the plane of the membrane.

Animals↗
PubMed1982

Relationship between the density distribution of intramembrane particles and electron transfer in the mitochondrial inner membrane as revealed by cholesterol incorporation.

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to-cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron-transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.

Animals↗
PubMed1981

Rate of lateral diffusion of intramembrane particles: measurement by electrophoretic displacement and rerandomization.

A method combining electrophoresis and freeze-fracture electron microscopy is described; the method was used to determine the lateral diffusion coefficient of intramembrane particles (integral proteins) in the mitochondrial inner membrane. An electric current was passed through microsuspensions of purified, spherical inner membranes at pH 7.4, which caused an electrophoretic migration of intramembrane particles in the membrane plane into a single, crowded patch facing the positive electrode. The membrane microsuspensions were quick-frozen at specified times after the packed particles were released from the electrophoretic force and while the particles were diffusing back to a random distribution. Observed concentration gradients of intramembrane particles during this time were quantitatively compared with and found to follow a mathematical model for Fickian diffusion of particles on a spherical membrane. The results determine the kinetics of free diffusion of integral proteins at the resolution of individual proteins. The diffusion coefficient of the integral proteins in the mitochondrial inner membrane was determined to be 8.3 X 10(-10) cm2/sec at 20 degrees C, from which a root-mean-square displacement of 57 nm in 10 msec is predicted.

Animals↗
PubMed1981

Alterations in density and size distribution of intramembrane particles in the inner membrane of mitochondria from chloramphenicol-fed mice.

The diameters and number per unit area (density) of intramembrane particles in mitochondrial inner membranes isolated from livers of chloramphenicol-fed and control mice were assessed by freeze-fracture electron microscopy. In addition, the levels of cytochrome hemes aa3, b, and c + c1 of the mitochondria were measured spectrophotometrically. The results show that CAP caused (1) a portion of the mitochondria to develop much larger than normal matrix volumes and inner membrane surface areas, (2) a decrease in the average diameter of intramembrane particles on both the convex and concave fracture faces of all membranes regardless of the membrane surface area, (3) a decrease in intramembrane particle density on only those membranes with a greater than normal surface area, and (4) a 75% decrease in heme aa3, a 25% decrease in heme b, and no change in heme c + c1 levels. It is concluded that intramembrane particles represent membrane integral proteins and that the components of integral proteins which originate from mitochondrial translation are located in the hydrophobic region of the bilayer and occupy volumes shared equally by each bilayer leaflet. In addition the mechanism by which CAP induces a portion of the mitochondria to develop larger than normal sizes most likely includes a net lipid insertion into the bilayer which exceeds the net protein insertion during the CAP-feeding interval.

Animals↗
PubMed1980

Relationships between bilayer lipid, motional freedom of oxidoreductase components, and electron transfer in the mitochondrial inner membrane.

The relationships between bilayer lipid, diffusional and conformational activities of oxidoreduction components, and electron transfer activity in the mitochondrial inner membrane are considered. Using a new, low pH method to fuse liposome phospholipid (asolectin) with the isolated mitochondrial inner membrane, the membrane bilayer is enriched up to 700% with exogenous phospholipid. During such enrichment, ultrastructural analysis reveals that integral proteins diffuse freely and randomly into the expanding bilayer. Kinetic analysis reveals that a diffusion limited step occurs between succinate- and NADH dehydrogenase and cytochromes bc1, and that the dehydrogenases, ubiquinone, and cytochromes bc1 are free to diffuse independently of one another in the membrane plane. Whether cytochromes bc1 and cytochrome c oxidase codiffuse in the membrane plane, or diffuse independently of one another remains unclear. The specific activities of succinate- and NADH-dehydrogenase as well as cytochrome c oxidase are affected by bilayer enrichment. This most likely occurs through the direct modulation by the newly incorporated phospholipid on conformational activity required in the oxidoreductases for electron transfer.

Animals↗
PubMed1980

Fusion of liposomes with mitochondrial inner membranes.

A procedure is outlined for the fusion of mixed phospholipid liposomes (small unilamellar vesicles) with the mitochondrial inner membrane, which enriches the membrane lipid bilayer 30-700% in a controlled fashion. Fusion was initiated by manipulation of the pH of a mixture of freshly sonicated liposomes and the functional inner membrane/matrix fraction of rat liver mitochondria. During the pH fusion procedure, liposomes became closely apposed with and sequestered by the inner membranes as revealed by freeze-fracture electron microscopy. After the pH fusion procedure, a number of ultrastructural, compositional, and functional characteristics were found to be proportionally related: the membrane surface area increased; the lateral density distribution of intramembrane particles (integral proteins) in the plane of the membrane decreased whereas the particles remained random; the membrane became more buoyant; the ratio of membrane lipid phosphorus to total membrane protein increased; the ratio of membrane lipid phosphorus to heme a of cytochrome c oxidase increased; and the rate of electron transfer between some interacting membrane oxidoreduction proteins decreased. These data reveal that liposomal phospholipid was incorporated into the membrane bilayer (not simply adsorbed to the membrane surface) and that integral membrane proteins diffused freely into the laterally expanding bilayer. Furthermore, the data suggest that the rate of electron transfer may be limited by the rate of lateral diffusion of oxidoreduction components in the bilayer of the mitochondrial inner membrane.

Animals↗
PubMed1979

Lateral translational diffusion of cytochrome c oxidase in the mitochondrial energy-transducing membrane.

The degree of freedom for lateral translational diffusion by cytochrome c oxidase and other integral proteins in the energy-transducing membrane of the mitochondrion was determined by combining the use of an immunoglobulin probe monospecific for the oxidase with thermotropic lipid phase transitions. Lateral mobility of the oxidase was monitored by observing the distribution of the immunoglobulin probe on the membrane surface by deep-etch electron microscopy and by observing the distribution of intramembrane particles (integral proteins) in the hydrophobic interior of the membrane by freeze-fracture electron microscopy. Incubation of the membrane with the immunoglobulin resulted in a time-dependent clustering of predominantly large intramembrane particles. Low temperature-induced lipid phase transitions resulted in the close packing of all intramembrane particles and cytochrome c oxidase by lateral exclusion from domains of gel-state bilayer lipid and was completely reversible. However, when cytochrome c oxidase was crosslinked through an immunoglobulin lattice prior to returning the membrane to above the lipid phase transition temperature, small intramembrane particles rerandomized while the large oxidase-related particles remained clustered. These observations reveal that cytochrome c oxidase can diffuse laterally in the energy-transducing membrane, either independently of all other integral proteins or in physical union with one or more other integral proteins. In addition, many other as yet unidentified smaller integral proteins can diffuse independently of the oxidase.

Animals↗
PubMed1977

Kinetics of product inhibition during firefly luciferase luminescence.

A theoretical and experimental analysis is made of the kinetics of product inhibition during firefly luciferase luminescence. Equations for competitive, noncompetitive, and uncompetitive inhibition are derived which are useful in determining inhibitory mechanism when the product inhibitor, or its concentration, is unknown and not subject to direct experimental manipulation. Comparisons of experimental data with predictions based upon the three inhibitory models show that product inhibition during luciferase luminescence is noncompetitive with respect to both luciferin and ATP as substrates. The competitive and uncompetitive models are inconsistent with experimental data. These findings provide the basis for using luminescence to measure ATP concentration continuously in in vitro biological systems such as isolated mitochondria.

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