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Biomedical subjects

B Kadenbach

Publications and source records attributed to B Kadenbach.

At least 145 records · Page 8Linked to original sources
PubMed1981

Immunological and chemical characterization of rat liver cytochrome c oxidase.

Rat liver cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase; EC 1.9.3.1) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into 12 different polypeptide chains. Specific antisera against the holoenzyme and against purified subunits IV and VIII were used to characterize the enzyme complex. The antiserum against subunit IV precipitates from sodium dodecyl sulfate-dissociated mitochondria only subunit IV and from Triton X-100-dissolved mitochondria all 12 polypeptide chains, indicating their integral location within the enzyme complex. Different antisera against the holoenzyme only precipitate subunits IV, V and VIb from sodium dodecyl sulfate-dissociated mitochondria, suggesting the location of these subunits on the surface layer of the complex. Subunit VIII is thought to be located within the complex, since a specific antiserum does not precipitate the complex. The amino acid composition of all 12 protein subunits is different, thus excluding their origin from proteolytic degradation. The proteolytic degradation of subunit IV into IV during isolation of the enzyme was corroborated by the very similar amino acid composition of both proteins.

Amino Acids↗
PubMed1981

Reaction of mersalyl with mitochondrial proteins under native and denaturing conditions as exemplified by the identification and isolation of the phosphate carrier.

Pig heart mitochondria were incubated with [203Hg]mersalyl and the radioactive pattern was analyzed by fluorography after dodecyl sulfate gel electrophoresis. No differences in the radioactivity distribution were found after labeling with various mersalyl concentrations, at different pH and after labeling in the native or dodecyl sulfate-dissociated state of mitochondria. A redistribution of [203Hg]mersalyl between various proteins in the presence of dodecyl sulfate could directly be demonstrated by mixing labeled membranes with unlabeled matrix proteins, as well as by comparison of the radioactivity patterns of whole mitochondria labeled with irreversibly reacting N-([2-3H]ethyl)maleimide and reversibly binding [203Hg]mersalyl. From these data it is concluded that under native conditions mersalyl is principally bound to the phosphate carrier protein, whereas during dissociation in dodecyl sulfate the organomercurial is redistributed and mainly attached to the ADP/ATP-carrier protein.

Animals↗
PubMed1980

The subunit composition of mammalian cytochrome c oxidase.

Cytochrome c oxidase from rat liver mitochondria was separated into 12 different protein subunits by application of a highly resolving sodium dodecylsulfate/gel electrophoretic system of different compositions. The 12 protein subunits are shown to represent integral components of mammalian type cytochrome c oxidase for the following reasons. 1. All 12 subunits copurify through various purification procedures. 2. The subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. 3. All 12 subunits are present in the complex at one to one stoichiometric amounts. 4. A similar composition of 12 subunits was also found for cytochrome c oxidase from rat kidney, pig heart, rabbit liver and stone-marten liver. The difference between our results and all other published data on the subunit composition of mammalian-type cytochrome c oxidase, based on gel electrophoretic analysis, is due to the insufficient resolving power of previously used gel systems and the very similar molecular weight of subunits VIa, b, c, and VIIa, b, c.

Animals↗
PubMed1980

Characterization of two high molecular weight proteins immunoprecipitated with an antibody against rat liver cytochrome c oxidase.

Isolated rat hepatocytes were labelled with [35S]methionine, dissolved in Triton X-100-containing buffer, and incubated with antibodies against rat liver cytochrome c oxidase. After separation by dodecyl sulfate-gel electrophoresis the fluorogram of immunoprecipitated proteins showed two labelled bands with apparent molecular weights of 52000 and 182000. The immunological relationship of the two proteins to cytochrome c oxidase was demonstrated by immunocompetition with the isolated enzyme and with purified subunits IV-VIII. Although the precursor nature of the two described proteins for cytoplasmically synthesized subunits of cytochrome c oxidase cannot be excluded, the following observations do not support this assumption: 1) The amount of incorporated radioactivity is too high; 2) they are exclusively located with the microsomal fraction; 3) the turnover is rather slow, compared to that of known precursor proteins.

Animals↗
PubMed1979

Specific labeling of a phosphate-transporting protein from rat-liver mitochondria by [203Hg]mersalyl.

1. A highly specific labeling of a phosphate-transporting protein from rat-liver mitochondria was obtained with [203Hg]mersalyl under the following conditions: (a) labeling of mitoplasts, isolated by the French-Press procedure; (b) washing of the isolated inner membranes with NaCl/octylglucoside. 2. The concentration-dependent labeling of the protein with mersalyl showed a biphasic saturation which correlated well with the protection and inhibition of phosphate transport. 3. The molecular weight (Mr 31000) and the amount of labeled carrier protein (24 nmol/g mitochondrial protein) at 100% protection correlated well with published data from the literature. 4. Gel filtration of the labeled protein in the presence of non-ionic detergents showed a molecular weight of at least twice of that obtained by gel electrophoresis in the presence of sodium dodecylsulfate.

Animals↗
PubMed1978

Immunoprecipitation of a cytoplasmic precursor of rat-liver cytochrome oxidase.

A simple method for the isolation of rat liver cells is described. The cells are shown, by an isotope dilution method, to maintain a constant rate of protein synthesis for 8 h of incubation. Antibodies to purified rat liver cytochrome oxidase were raised in rabbits and used to investigate the labeling of cytochrome oxidase in isolated rat liver cells and in vivo. The data demonstrate the occurrence of a precursor of the subunits of cytochrome oxidase that are synthesized in the cytoplasm. 1. Dodecylsulfate gel electrophoresis of the immunoprecipitates from isolated rat liver cells that had been labeled with [35S]methionine for 1 h showed a single radioactive peak with a molecular weight of 50000. 2. Judged by the effects of cycloheximide and chloramphenicol the labeled protein is synthesized on cytoplasmic ribosomes. 3. After labeling for 1 h in vivo with [3H]leucine the labeled protein appears to be exclusively associated with the hepatic microsomal fraction. 4. Ouchterlony double-diffusion analysis demonstrated immunological relationship between the precipitates from microsomes and cytochrome oxidase. In addition to the precipitates derived from mitochondria and microsomes immunoprecipitates were also obtained from the cytosol in comparable amounts; these again were immunologically related. The occurrence of large amounts of precursor(s) (or degradation products) of cytochrome oxidase in rat liver fractions is interpreted in terms of a regulatory pool for amino acid homeostasis in the organism.

Animals↗
PubMed1978

Transport of phosphate analogues in rat-liver mitochondria.

The swelling of rat liver mitochondria has been studied in isotonic ammonium salts of phosphate, thiophosphate, monofluorphosphate, difluorphosphate or in isotonic ammonium malate induced by low concentrations of the mentioned anions. The data lead to the following conclusions. 1. Phosphate, thiophosphate, and monofluorphosphate are taken up by the mitochondria, whereas difluorphosphate is not, indicating that the substrate of the transporter represents the divalent anion HPO24- and not H2PO4-. 2. Thiophosphate, monofluorphosphate and phosphate, but not difluorphosphate induce the swelling of mitochondria in isotonic ammonium malate, only after a lag phase. The length of the lag phase depends on the concentration of inducing anion and pH. This phenomena cannot be explained by the current concept of two independent phosphate-transporting systems in mitochondria.

Animals↗
PubMed1977

On the molecular weight of mitochondrially synthesized subunits of rat liver cytochrome oxidase.

The subunit composition of cytochrome c oxidase from rat liver mitochondria was studied by dodecylsulfate polyacrylamide gel electrophoresis. The apparent molecular weight of the seven subunits are in reasonable agreement with published data on cytochrome c oxidase subunits from other sources. Two additional subunits were found if the electrophoresis was performed with 8m urea, due to splitting of the smallest subunit. Performic acid oxidation of the isolated subunits I and II increased the apparent molecular weights from 38000 to 48000 and from 24500 to 29000, respectively, accompained by a normalization of the anomalous behaviour of subunit I in the Ferguson plot. It is suggested that performic acid, by splitting extremely inaccessible disulfide bridges, mediates full complexing of the subunits by dodecylsulfate, thus permitting the determination of the real molecular weights by dodecylsulfate polyacrylamide gel electrophoresis.

Animals↗
PubMed1976

Identification of a membrane protein involved in mitochondrial phosphate transport.

A specific labeling by radioactive N-ethylmaleimide of a protein involved in phosphate transport was obtained by protecting one of the two-SH-groups of the transport system with low concentrations of mersalyl. Subsequently, the other free-SH groups were blocked with excess N-ethylmaleimide. Removal of mersalyl by cysteine and subsequent inbucation with labeled N-ethylmaleimide results in a "specific" binding of N-ethylmaleimide to one-SH group functionally involved in phosphate transport. The isolated inner membrane fraction of the labeled mitochondria was subjected to dodecylsulfate gel electrophoresis. The followin results were obtained. 1. The difference of the radioactivity pattern on the dodecylsulfate-polyacrylamide gel of inner membrane proteins, labeled with N-[14C]ethylmaleimide in the absence and with N-[3H-A1-ethylmaleimide in the presence of mersalyl during preincubation of mitochondria, shows only one main labeled peak. The same labeled peak is obtained from the difference of labeling after preincubation with a constant low concentration of mersalyl at 32 degrees C and at 0 degrees C. 2. The position of the labeled peak on the dodecylsulfate-polyacrylamide gel corresponds to a protein of molecular weight of 26500 +/- 800. 3. The amount of one of the two-SH groups, involved in phosphate transport, was estimated to be 30 nmol per g of mitochondrial protein.

Animals↗