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Biomedical subjects

L Ernster

Publications and source records attributed to L Ernster.

At least 109 records · Page 6Linked to original sources
PubMed1980

Reconstitution of mitochondrial oligomycin and dicyclohexylcarbodiimide-sensitive ATPase.

1. Oligomycin and dicyclohexylcarbodiimide-sensitive ATPase was isolated from beef-heart mitochondria and treated with 3.5 M NaBr in order to remove F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecylsulfate-polyacrylamide-gel electrophoresis. Two of them correspond to the oligomycin-sensitivity-conferring protein and coupling factor F6, with apparent molecular weights of 21,000 and 9,400, respectively. Three additional polypeptides of molecular weights 23,000, 10,500 and 8,600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide-binding protein. 2. F0 induced an oligomycin and dicyclohexylcarbodiimide-sensitive enhancement of K+ + valinomycin-driven proton translocation across the membrane of artificial phospholipid vesicles. 3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1-ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 in both an oligomycin-sensitive and an oligomycin-insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F0/F1 ratios favoured in oligomycin-sensitive type of binding, indicating that F1 binds preferentially to oligomycin-sensitivity-conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin-sensitivity-conferring binding sites and a diminished ability to lower the specific activity an cold lability of F1. 4. Reconstitution of F0 treated with trypsin and F1, oligomycin-sensitivity-conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin-sensitivity-conferring protein and F6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity. 5. The effect of the order of addition of F1, oligomycin-sensitivity-conferring protein and F6 to F0 on the reconstitution of oligomycin-sensitive ATPase activity, and of F1 and oligomycin-sensitivity-conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components.

Adenosine Triphosphatases↗
PubMed1979

Inactive to active transitions of the mitochondrial ATPase complex as controlled by the ATPase inhibitor.

The hydrolytic and phosphorylation activities of the ATPase complex of bovine heart mitochondria are regulated by the ATPase inhibitor of Pullman and Monroy [1]. The inhibiting action of the peptide on ATPase activity can be overcome by a proton-motive force. Submitochondrial particles that contain the inhibitor, either intrinsically or externally added, show a lag that precedes phosphorylation. Particles devoid of the inhibitor, of particles that are in an 'active' state fail to present the lag. Accordingly, the data indicate that, prior to the onset of phosphorylation, the ATPase complex undergoes a transition to an active state through a process that involves the inhibitor. The transition depends on the concentration of ATP, 50 microM ATP giving 50% inhibition of the proton-motive force-induced transition.

Adenosine Triphosphatases↗
PubMed1978

The mechanism of oxidation of reduced nicotinamide dinucleotide phosphate by submitochondrial particles from beef heart.

1. Oxidation of NADPH by various acceptors catalyzed by submitochondrial particles and a partially purified NADH dehydrogenase from beef heart was investigated. Submitochondrial particles devoid of nicotinamide nucleotide transhydrogenase activity catalyze an oxidation of NADPH by oxygen. The partially purified NADH dehydrogenase prepared from these particles catalyzes an oxidation of NADPH by acetylpyridine-NAD. In both cases the rates of oxidation are about two orders of magnitude lower than those obtained with NADH as electron donor. 2. The kinetic characteristics of the NADPH oxidase reaction and reduction of acetylpyridine-NAD by NADPH are similar with regard to pH dependences and affinities for NADPH, indicating that both reactions involve the same binding site for NADPH. The binding of NADPH to this site appears to be rate limiting for the overall reactions. 3. At redox equilibrium NADPH and NADH reduce FMN and iron-sulphur center 1 of NADH dehydrogenase to the same extents. The rate of reduction of FMN by NADPH is at least two orders of magnitude lower than with NADH. 4. It is concluded that NADPH is a substrate of NADH dehydrogenase and that the nicotinamide nucleotide is oxidized by submitochondrial particles via the NADH--binding site of the enzyme.

Animals↗
PubMed1977

Funiculosin: an antibiotic with antimycin-like inhibitory properties.

The antibiotic funiculosin mimics the action of antimycin in several ways. It inhibits the oxidation of NADH and succinate, but not TMPD+ascorbate. The titer for maximal inhibition in Mg2+-ATP particles (0.4-0.6 nmol/mg protein) is close to the concentrations of cytochromes b and cc1. Funiculosin also induces the oxidation of cytochromes cc1 and an extra reduction of cytochrome b in the aerobic steady state, and it inhibits duroquinol-cytochrome c reductase activity in isolated Complex III. The location of the funiculosin binding site is clearly similar to that of antimycin. In addition, funiculosin, like antimycin, prevents electron transport from duroquinol to cytochrome b in isolated Complex III if the complex is pre-reduced with ascorbate. Funiculosin and antimycin differ, however, in the manner in which they modulate the reduction of cytochrome b by ascorbate+TMPD.

Aerobiosis↗
PubMed1977

Preparation and characterization of total, rough and smooth microsomes from the lung of control and methylcholanthrene-treated rats.

Optimal conditions for the preparation of relatively pure microsomes and microsomal subfractions from rat lung have been determined. The most importnat of these conditions is homogenization of a 20% (w/v) suspension of lung tissue in 0.44 M sucrose/1% (w/v) bovine serum albumin with four up-and-down strokes at 440 rev./min in a Potter-Elvehjem homogenizer. The 10000 X g supernatant prepared from this homogenate can be centrifuged at 105000 X g to obtain total microsomes or subfractionated into rough and smooth microsomes on a Cs+-containing discontinuous sucrose gradient. The total, rough and smooth microsomes have been characterized in terms of their chemical composition, enzymatic activity, and morphology. These preparations should prove useful in studies of various enzymes in lung (e.g. benzpyrene monooxygenase, epoxide hydrase, enzymes of phospholipid and ascorbic acid synthesis) and in subfractionations designed to reveal heterogeneites in the lateral plane of the lung endoplasmic reticulum.

Animals↗
PubMed1977

Characterization of rat lung epoxide (styrene oxide) hydrase with a modified radioactive assay of improved sensitivity.

The epoxide hydrase assay developed by Oesch et al. (Biochim. Biophys. Acta, 227: 685-691, 1971) using [3H]styrene oxide as substrate was modified in three ways for use with rat lung microsomes: the substrate was purified before use, the volume of the incubation mixture was scaled down 4-fold, and the incubation time was extended to 45 min (activity was found to be linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was found to be linear with lung microsomal protein up to at least 1.8 mg protein per incubation mixture. This modified assay for epoxide hydrase was used to characterize the enzyme in rat lung. Its apparent vmax is 0.5 nmole of styrene glycol formed per min per mg microsomal protein, and its apparent Km was 0.11 to 0.25 mM. The pH optimum is around 9.7. Upon subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase) and in a different way from markers for the nuclei, mitochondria, concentric lamellar organelles, lysosomes, Golgi membranes, plasma membrane and soluble cytoplasm. The specific activity of epoxide hydrase in rough and smooth lung microsomes is aobut the same. Treatment i.p. of rats with methylcholanthrene (3 injections of 20 mg/kg), phenobarbital (5 daily injections of 80 mg/kg) or styrene oxide (5 daily injections of 40 mg/kg), did not induce lung microsomal epoxide hydrase activity. 1,1,1-Trichloropropene 2,3-oxide was shown to be an uncompetitive inhibitor, and cyclohexene oxide was a noncompetitive inhibitor of this enzyme. Ethanol and butanol activate the epoxide hydrase of lung microsomes at low concentrations and inhibit it at higher concentrations.

Alcohols↗