Translation of mitochondrial proteins in digitonin‐treated rat hepatocytes

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.

Section: Assembly Of Cytochrome Oxidase In Isolated Mitochondriamentioning

confidence: 99%

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

Wielburski¹,

Nelson²

1983

“…The samples were incubated for 30s at 30°C and then centrifuged for 30s in an Eppendorf centrifuge. The supernatant and the pellet [containing mitochondria (Fiskum et al, 1980;Kuzela et al, 1981)] were separated, and the pellet was rinsed once in 1 ml of buffer containing 25mM-Hepes, pH7.5, 0.25M-sucrose and 5mM-MgCl2. The pellet was then dissolved in 200,uI of the same buffer containing 0.5% Triton X-100.…”

Section: Methodsmentioning

confidence: 99%

Altered metabolic states do not change the intracellular distribution of hexokinase in Zajdela hepatoma ascites cells

Nelson¹,

Kabir²,

Muchiri³

1984

The distribution of hexokinase between bound and soluble forms was studied by digitonin fractionation of Zajdela hepatoma ascites cells maintained under various metabolic conditions. Addition of glucose to Zajdela cells respiring on endogenous substrates induces an immediate inhibition of respiration by 50-60% ( Crabtree effect), and a production of acid due to glycolysis. Acid production decreases abruptly after 60s to 50% of the initial rate. The ATP/ADP ratio is not altered by the addition of glucose or by different rates of glycolysis. The uncoupling agent carbonyl cyanide m-chlorophenylhydrazone decreases the ATP/ADP ratio by 10-fold in cells respiring on endogenous substrate, but has little effect on cells oxidizing glucose. Rapid fractionation of the cells under these various metabolic conditions revealed no change in the distribution of hexokinase. Approx. 75% of hexokinase is bound in all cases, in contrast with lactate dehydrogenase, 95% of which was in the soluble form. Longer-term incubations (to 20 min) revealed only slight (10-15%) increases in soluble hexokinase in cells incubated with glucose. Various metabolic inhibitors had little additional affect on the subcellular distribution of hexokinase. Thus a rapid release of hexokinase from mitochondrial membrane is not a mechanism by which glycolysis is regulated in rapidly growing Zajdela hepatoma.

“…Labelling of the cytoplasmic subunits of cytochrome oxidase was performed in the presence S. Parimoo, N. Rao and G. Padmanaban of chloramphenicol (200,g/ml). Labelling of the mitochondrial subunits was performed in digitonintreated hepatocytes in the presence of cycloheximide (1 mM) as described by Kuzela et al (1981). After labelling of the cells for 1 h at 370C, the mitochondrial and RERM fractions were isolated, homogenized with 0.25 M-sucrose containing 0.5 mM-EDTA to strip the mitochondria of the associated RER (Meier et al, 1981), and mitochondria were sedimented by centrifugation at 80)0g for 10min.…”

Section: Methodsmentioning

confidence: 99%

Cytochrome c oxidase is preferentially synthesized in the rough endoplasmic reticulum-mitochondrion complex in rat liver

Parimoo¹,

Rao²,

Padmanaban³

1982

The specific activity and content of cytochrome oxidase in the rough endoplasmic reticulum--mitochondrion complex are higher than in the mitochondrial fraction. Radiolabelling studies with the use of hepatocytes and isolated microsomal and rough endoplasmic reticulum--mitochondrion fractions, followed by immunoprecipitation with anti-(cytochrome oxidase) antibody, reveal that the nuclear-coded cytoplasmic subunits of cytochrome oxidase are preferentially synthesized in the latter fraction. The results have a bearing on the mechanism of transport of these subunits into mitochondria.