Search PubMed⌕ Search

Biomedical subjects

B D Nelson

Publications and source records attributed to B D Nelson.

At least 91 records · Page 5Linked to original sources
PubMed1983

Evidence for the sequential assembly of cytochrome oxidase subunits in rat liver mitochondria.

The assembly of cytochrome oxidase was studied in isolated rat liver mitochondria and isolated rat hepatocytes labelled in vitro with L-[35S]methionine. This was achieved by studying the temporal association of radioactive subunits which are immunoabsorbed with antibodies against subunits I, II and the holoenzyme. Antibodies against the holoenzyme were shown to be highly specific for subunit V. The results show that subunit I appears in the holoenzyme late in the assembly process. No radioactive subunit I is absorbed with antiserum against subunit II or the holoenzyme (subunit V) after a 30 min pulse in either isolated mitochondria or hepatocytes. However, both antisera absorb radioactive subunits I after a 150 min chase in isolated hepatocytes. This was confirmed using antibodies against subunit I, which absorbed only radioactive subunit I after a 30 min pulse but absorbed radioactive subunits I-III and VI after a 150 min chase. Thus, the late assembly of radioactive subunit I is explained by a temporal sequence in the assembly process and not by the presence of a large, non-radioactive pool of subunit I. Using the above approach and the three specific antisera, the following temporal sequence in the assembly of cytochrome oxidase was established. Subunits II and III assemble rapidly with each other or with cytoplasmically translated subunit VI. This complex of three peptides in turn assembles slowly with subunit I or with the other cytoplasmically translated subunits. The early association of subunit VI with the mitochondrially translated subunits II and III suggests a possible role of the former in integration of the holoenzyme.

Animals↗
PubMed1983

Studies on beef heart ubiquinol-cytochrome c reductase. Topological studies on the core proteins using proteolytic digestion and immunoreplication.

The topology of beef heart Complex III has been studied by tryptic and chymotryptic digestion of isolated Complex III, Mg2+-ATP submitochondrial particles, and mitoplasts. Degradation products were detected by the immunoreplication technique using specific antibodies against core protein 1 (50 K) and core protein 2 (47 K). It can be shown that both peptides are digested from the matrix side of the inner membrane. However, no evidence was found that these peptides were digested by trypsin or chymotrypsin from the cytoplasmic side. It is concluded that the beef heart core proteins are membrane-bound peptides containing tryptic and chymotryptic digestion sites only on the matrix surface of the inner membrane. The data also suggest that beef heart core protein 2 contains multiple domains which are inserted into the membrane from the matrix surface. Proteolytic treatment of submitochondrial particles under conditions which digested at least 50% of the core proteins from the matrix surface did not, however, influence NADH oxidation rates or the respiratory control ratios.

Animals↗
PubMed1983

Going the distance.

Explore the source record for details and available documents.

Economic Competition↗
PubMed1982

Purification of a protein having pore forming activity from the rat liver mitochondrial outer membrane.

A protein with pore-forming activity has been isolated from the outer membrane of rat liver mitochondria. The purification involves sucrose gradient centrifugation, differential centrifugation in the presence of Triton X-100, and DEAE-Sepharose and CM-Sepharose chromatography. The yield of the purified protein was approx. 2% of the total outer membrane proteins. The protein, when inserted into soya bean phospholipid vesicles, increases the [3H]sucrose permeability of the vesicles but had no effect on the permeability of high-molecular-weight [14C]dextran (Mr 70 000). The protein is very active, since as little as 3-4 micrograms of protein per mg of phospholipid is required for the complete release of [3H]sucrose from the vesicles. Sucrose diffusion channels could not be reconstituted with other membrane proteins such as rat liver cytochrome oxidase or cytochrome b5. Purified pore protein revealed a single band of apparent Mr 30000 when resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This polypeptide could be further resolved by isoelectric focusing into a major (pI7.9) and two relatively minor (pI7.6 and 7.2) components. Proteolytic mapping with V8 proteinase from Staphylococcus aureus suggests that these probably represent a single component showing charge heterogeneity. The reason for the charge heterogeneity is not known. The amino acid composition of the protein revealed 47.8% polar amino acids with a relatively high lysine content.

Amino Acids↗
PubMed1982

Studies on the assembly of cytochrome oxidase in isolated rat hepatocytes.

1. The assembly of rat liver cytochrome oxidase was studied in isolated hepatocytes and isolated liver mitochondria labelled with L-[35S]methionine. 2. Labelled subunits II and III appeared in the immunoabsorbed holoenzyme within minutes after the initiation of a pulse label. In contrast, labelled subunit I appeared in immunoabsorbed holoenzyme only after a subsequent 2 h chase or after an additional 2 h of labelling. Subunit I was heavily labelled, however, in intact mitochondria after 10 min. 3. A similar pattern of labelling was observed in holo-cytochrome oxidase which was chemically isolated by a small scale procedure adapted for this purpose. The appearance of subunit I in the holoenzyme was delayed for 1.5-2 h after a 60 min pulse with labelled methionine. 4. Incubation of hepatocytes for 4 h in the presence of cycloheximide had no effect on the labelling pattern described above. 5. Methods were developed in which newly translated, presumably unassembled, subunits of cytochrome oxidase could be separated from the holoenzyme by fractionation in Triton X-114. Short-term pulse experiments indicate that subunits II and III are associated with the holoenzyme fraction immediately after their completion, whereas subunit I is not. 6. The data are consistent with a model in which cytochrome oxidase assembly is viewed as an ordered and sequential event.

Animals↗
PubMed1981

Studies on beef heart ubiquinol-cytochrome c reductase. Quantification and distribution of the core proteins as determined by radioimmunoassay.

Core proteins I (Mr 50 000) and II (Mr 47 000) were isolated from beef heart ubiquinol-cytochrome c reductase, and radioimmunoassays were developed for both. Immunoreplica experiments show that antisera against each protein react with a single peptide in both isolated Complex III and in mitochondria. Thus, core proteins are not aggregated forms of smaller peptides as suggested for the yeast protein (Jeffrey, A., Power, S. and Palmer, G., Biochem. Biophys. Res. Commun. (1979) 86, 271-277). Core proteins were quantitated in Complex III and in mitochondria using radioimmunoassay. Approx. 2 mol core protein II per mol core protein I were found. A molar ratio of 1 : 2 : 2 : 1 is suggested for core protein I : core protein II : cytochrome b : cytochrome c1. Radioimmunoassay shows that the antibodies react as extensively with Complex III-bound core protein as with the isolated core proteins. In spite of this, the antibodies do not inhibit electron transport in submitochondrial particles or isolated Complex III, and they have no oligomycin- or uncoupler-like effects on submitochondrial particles oxidizing NADH. The combined results from radioimmunoassay and immunoreplica experiments strongly suggest, however, that core proteins are specifically associated with Complex III in the mitochondria, implying a specific role there.

Animals↗
PubMed1981

Phospholipid requirements for the reconstitution of complex-III vesicles exhibiting controlled electron transport.

Phospholipid requirements for the reconstitution of Complex-III vesicles exhibiting respiratory control (electron-transport control) were studied. Vesicles prepared from pure phosphatidylethanolamine gave maximal control ratios. Phosphatidylcholine alone did not support respiratory control, although these vesicles were capable of maintaining stable K+-diffusion gradients. Apparently Complex III cannot insert into a bilayer of phosphatidylcholine. Formation of mixed phosphatidylcholine/phosphatidylethanolamine (6:1, w/w) vesicles was sufficient, however, to allow Complex-III insertion and to restore respiratory control. Mixtures of acidic phospholipids with either phosphatidylethanolamine or phosphatidylcholine did not improve respiratory control over that obtained with pure phosphatidylethanolamine. Phosphatidylethanolamine from bovine heart mitochondria, soya beans or Escherichia coli was equally effective in reconstituting respiratory control, suggesting that the specificity is referable to the head group and not to the fatty-acid moiety.

Animals↗
PubMed1981

Synthesis of cytochrome oxidase in isolated rat hepatocytes.

1. The synthesis of cytochrome oxidase was studied in isolated rat hepatocytes labeled in vitro. Labeled whole cells, isolated mitochondria, microsomes and the post microsomal supernatant were treated with antisera to rat liver holo-cytochrome oxidase, and the subunits were adsorbed onto Sepharose-protein A. 2. Seven peptides, corresponding to subunits of rat liver cytochrome oxidase, were immunoabsorbed from mitochondria isolated from cells labeled in the absence of inhibitors. Two peptides, corresponding to subunits I (45 500 daltons) and II (26 000 daltons), were labeled in mitochondria isolated from cycloheximide-treated cells. Labeling of these peptides was inhibited by chloramphenicol. Peptides I and II correspond to the two most heavily labeled mitochondrial translation products found in submitochondrial particles. Possible explanations for the lack of labeling of a third mitochondrially translated subunit are discussed. Labeling of the five smallest peptides was inhibited by cyclohexamide but not by chloramphenicol. 3. Peptide I appears in the holoenzyme later than the other six peptides after a pulse-chase. It is not labeled in the immunoabsorbed cytochrome oxidase after a 30 min pulse with [35S]-methionine, but appears after a 3 h chase with unlabeled methionine. Labeling of the other subunits showed no further increase after the chase.

Animals↗
PubMed1981

Isolation of the cytochrome-bc1 complex from rat-liver mitochondria.

The cytochrome bc1 complex has been isolated from rat-liver mitochondria by two different procedures. The enzyme isolated by either procedure exhibits a specific cytochrome b and cytochrome c1 heme content of approximately 8 and 4 nmol/mg protein respectively. Both preparations contain only seven polypeptides on sodium dodecylsulfate gel electrophoresis, with the following apparent molecular weights: I, 50000; II, 46000; III, 33000; IV, 25000; V, 12500; VI, 10000; VII, 5600. The polypeptide composition is identical to that of the beef-heart enzyme isolated by cholate/ammonium sulfate fractionation. Furthermore, with the exception of subunit II (core protein 2), the apparent molecular weights of the subunits are identical in the rat-liver and beef-heart enzymes.

Animals↗
PubMed1980

The effects of tri-iodothyronine on the synthesis of mitochondrial proteins in isolated rat hepatocytes.

The effects of tri-iodothyronine on mitochondrial protein synthesis have been studied in in vitro labeled, isolated rat hepatocytes. Hepatocytes were isolated from hypothyroid rats or from hypothyroid rats 24 h after injecting a single, low dose of hormone (20-30 microgram/180-230 g body weight). Tri-iodothyronine increased translation on mitochondrial ribosomes by 2-3-fold, but, under our conditions, appears to have little or not effect on the general synthesis of cytoplasmically-translated mitochondrial proteins. Electrophoretic and fluorographic analysis indicated that tri-iodothyronine stimultes labeling of the four major mitochondrially-translated peptides. The hormone appears to act by inducing a general increase in translation/transcription of mitochondrially-synthesized peptides.

Animals↗