Electron microscopy and subunit-subunit interaction studies reveal a first architecture of COP9 signalosome.

The COP9 signalosome is involved in signal transduction, whereas the 26 S proteasome lid is a regulatory subcomplex of the 26 S proteasome responsible for degradation of ubiquitinated proteins. COP9 signalosome and lid possess significant sequence homologies among their eight core subunits and are likely derived from a common ancestor. Surprisingly, from our two-dimensional electron microscopy data, a common architectural plan for the two complexes could not be deduced. None-the-less, the two particles have structural features in common. Both COP9 signalosome and lid lack any symmetry in subunit arrangement and exhibit a central groove, possibly qualified for scaffolding functions.Filter-binding assays with recombinant COP9 signalosome components revealed a multitude of subunit-subunit interactions, supporting the asymmetrical appearance of the complex in electron microscopy. On the basis of two-dimensional images and subunit interaction studies, a first architectural model of COP9 signalosome was created. The fact that four distinct classes of particle views were identified and that only 50 % of the selected particles could be classified indicates a high degree of heterogeneity in electron microscopic images. Different orientations with respect to the viewing axis and conformational variety, presumably due to different grades of phosphorylation, are possible reasons for the heterogeneous appearance of the complex. Our biochemical data show that recombinant COP9 signalosome subunits 2 and 7 are phosphorylated by the associated kinase activity. The modification of COP9 signalosome subunit 2 might be essential for c-Jun phosphorylation. Dephosphorylation does not inactivate the associated kinase activity. Although substrate phosphorylation by COP9 signalosome is significantly decreased by lambda protein phosphatase treatment, "autophosphorylation" is increased.

A novel protein complex involved in signal transduction possessing similarities to 26S proteasome subunits.

A novel protein complex has been identified in human cells that has a molecular mass of approximately 450 kDa. It consists of at least eight different subunits including JAB1, the Jun activation-domain binding protein 1, and Trip15, the thyroid hormone receptor-interacting protein 15. The purified complex contains COP9 and COP11 protein homologs and is very similar, if not identical, to the plant COP9 complex involved in light-mediated signal transduction. The isolated JAB1-containing particle has kinase activity that phosphorylates IkappaBalpha, the carboxy terminus of p105, and Ser63 and/or Ser73 of the amino-terminal activation domain of c-Jun. The phosphorylation of c-Jun requires the carboxy terminus of the protein containing the DNA binding and dimerization domains. Three subunits of the new complex--Sgn3, Sgn5/JAB1, and Sgn6--exhibit sequence similarities to regulatory components of the 26S proteasome, which could indicate the existence of common substrate binding sites. Immunofluorescence staining reveals that the new complex shows a subcellular distribution similar to that of the 26S proteasome. The functional relationship of the two particles in regulating transcriptional activity is discussed. Considering the putative role of the complex in signal transduction and its widespread occurrence, we suggest the name JAB1-containing signalosome.

Molecular cloning and expression of subunit 12: a non-MCP and non-ATPase subunit of the 26 S protease.

A cDNA encoding subunit 12 (S12) of human erythrocyte 26 S protease has been isolated, sequenced and expressed. The cDNA contains an open reading frame that encodes a 36.6 kDA protein 96% identical to mouse Mov-34 and 67% identical to its Drosophila melanogaster homolog. Based on the high degree of sequence identity between human S12, mouse and Drosophila Mov-34 proteins, we conclude that the Mov-34 gene product is a component of the 26 S protease. Antibodies produced against two S12 fragments, Met1-Tyr95 (S12f95) and Met1-Leu205 (S12f205), react with S12 transferred to nitrocellulose from SDS-PAGE. In contrast, after transfer from native gels, the epitope(s) recognized by anti-S12f205 is exposed in the regulatory complex but appears to be masked when the regulatory complex associates with the multicatalytic protease.

ATP-producing and consuming processes of Ehrlich mouse ascites tumor cells in proliferating and resting phases.

The extents of ATP-yielding and consuming processes in Ehrlich mouse ascites tumor cells during the proliferating and resting growth phase were compared. In the resting phase the total ATP production was decreased by one-third. The ATP supply by oxidative phosphorylation was drastically reduced, whereas the rate of glycolysis stayed nearly constant. All ATP-consuming processes investigated, i.e., protein turnover, Na+/K(+)-ATPase, Ca2(+)-ATPase, and RNA synthesis, were decreased proportionally with the total ATP consumption.

Pyruvate utilization of rabbit reticulocytes--a compartmental study.

The utilization of [2-14C] pyruvate via the citric acid cycle of rabbit reticulocytes was studied to elucidate the importance of carboxylation- and decarboxylation reactions in this cell type. Unknown flux rates were determined by fitting the observed time-dependent labeling of CO2, alanine, glutamate and aspartate (all carbon atoms and C alpha) to the corresponding set of differential equations. The results reveal a high activity of the shuttle rate between mitochondrial pyruvate and the C4 pool catalysed by the pyruvate carboxylase (v = 19.1 nM/ml cells/min) and the malic enzyme (v = 57.5 mM/ml cells/min) which is comparable with the flux rate within the citrate cycle (v2 = 46.2 nM/ml cells/min). Moreover, our studies provide strong indications for the existence of a pyruvate utilizing metabolic pathway which has not been described so far.

The action of N,N-diethyldithiocarbamate(DDC) on red blood cells.

Glutathione exerts a pro-oxidative effect by increasing H2O2-formation in the presence of methemoglobin and a radical-generating substance (e.g. DDC). This effect might contribute to the loss of glutathione in cells in which formation of methemoglobin is provoked by radicals.

Balancing of mitochondrial and glycolytic ATP production and of the ATP-consuming processes of Ehrlich mouse ascites tumour cells in a high phosphate medium.

A balance of energy budgeting of Ehrlich mouse ascites tumour cells including mitochondrial and glycolytic ATP production and about 80% of ATP consumption in a high phosphate medium is presented. In the share of glycolysis was about one-third of the total ATP production, more than twice that found in a low phosphate medium. The extent of a single energy reaction was assessed from the decrease of coupled oxygen consumption and lactate formation following the specific inhibition of this process. The inhibitory effects on coupled respiration and glycolysis were identical for the energy consuming processes measured: protein turnover, Na+/K(+)-ATPase, Ca2(+)-transport and RNA synthesis.

Quantification of ATP-producing and consuming processes of Ehrlich ascites tumour cells.

ATP production of Ehrlich ascites tumour cells was estimated on the basis of their coupled respiration and lactate formation. ATP-consuming processes were assessed from the effects of selective inhibitors of RNA synthesis, protein synthesis and proteolysis, Na+/K+-ATPase on respiration. The extent of protein synthesis and proteolysis were also determined directly. From these values and those of the inhibition of respiration by selective inhibitors, a P/O ratio of 2.2 was calculated. About 75% of the total ATP consumption could be assigned to specific processes. The major ATP-consuming processes of tumour cells in an amino-acid-enriched medium, in which they are in an approximate steady state, are protein synthesis with about 30% of total ATP consumption, and Na+/K+-ATPase with about 20%, while RNA synthesis, ATP-dependent proteolysis and Ca2+-ATPase contribute about 10% each. In an amino-acid-free glucose medium, protein synthesis is reduced to a third, with a corresponding decrease of respiration, whereas the rate of the other ATP-consuming processes is unchanged.

ATP production and consumption of rabbit reticulocytes increase in an amino-acid-enriched medium.

The ATP production and the main processes of ATP consumption, globin synthesis, Na+, K+-ATPase, and proteolysis of rabbit reticulocytes in two different media (1. complete medium with amino acids and glucose and 2. glucose containing electrolyte medium) were measured. The ATP formation in the complete medium amounts to 157 mmol/l X h which is 28% higher than in the incomplete medium. The increased ATP formation rate is due to increased ATP consumption by the more than doubled protein synthesis in the complete medium, whereas proteolysis and Na+,K+-ATPase are nearly unchanged. The increase in the glucose consumption in the complete medium is not fully accounted for by the increased oxidative degradation of glucose in the citric acid cycle and by lactate accumulation.

Accounting for the ATP-consuming processes in rabbit reticulocytes.

The report deals with a detailed balance of ATP production and consumption of the rabbit reticulocyte. The sum-total of ATP produced amounts to 135 mmol . 1-1 . h-1. About 70% of the ATP consumption has been accounted for by specific processes. The main contributing processes are the globin synthesis with about 28%, the Na+, K+-ATPase with 23% and the proteolysis with more than 15%. 30% of ATP consumption has not been accounted for. Cycloheximide (20 microM) leads to a dissociation between synthesis and degradation of proteins, which argues against any obligatory connection between these processes. More than 90% of the lysine liberated from mitochondria by proteolysis were reutilized for the globin synthesis demonstrating the high nitrogen economy of reticulocytes. Each of the ATP-consuming processes studied appears to control ATP production in an independent manner without competition with each other.

Superoxide radicals in the metabolism of the red cell.

The formation of O2- radicals as primary products of O2 reduction by human erythrocytes is demonstrated. O2- radicals react directly with GSH and OxyHb as 1e-donors forming GSSG and MetHb. After exhaustion of the glutathione system MetHb increases greatly and secondary radicals formation ensues with breakdown of Hb. The toxicity of O2- radicals is caused mainly by secondary radicals.

Balance of ATP consumption of reticulocytes.

The sum-total of ATP produced amounts to 135 mmoles X 1 reticulocytes-1 X hour-1. About 70% of the ATP consumption has been accounted for by specific processes. The main contributing processes are: the globin synthesis with about 28%; the Na+/K+-ATPase with 23%, and the ATP-dependent share of proteolysis with more than 15%. 30% of ATP consumption has not been accounted for. Cycloheximide (20 X 10(-6) M) leads to a dissociation between synthesis and degradation of proteins, which argues against any obligatory connection between these processes. About 90% of the lysine liberated from mitochondria by proteolysis were reutilized for the globin synthesis demonstrating the high nitrogen economy of reticulocytes. Each of the ATP consuming processes studied appears to control ATP production in an independent manner without competition with each other.

Quantification of pathways of glucose utilization and balance of energy metabolism of rabbit reticulocytes.

In this work it is demonstrated that glucose constitutes the main substrate of energy metabolism of rabbit reticulocytes under aerobic conditions in the presence of 5 mM glucose. Amino acids and fatty acids are minor sources of energy. The shares of processes utilizing glucose in reticulocytes were estimated from tracer experiments. A new mathematical technique used permits the derivation of closed terms for the specific radioactivity of single positions of C atoms of the metabolites of the citrate cycle. By means of regression analysis, the undetermined flux rates in the citrate cycle were calculated. On the basis of the data an overall balance sheet of glucose utilization and of ATP generation is given. About 45% of the glucose of reticulocytes is catabolized via the citrate cycle, about the same percentage yields lactate. Only 2% of the glucose was oxidized in the oxidative pentose pathway whereas the remainder is used for the formation of serine and glycine required for hemoglobin synthesis. These results are related to knowledge about the main processes utilizing ATP in reticulocytes, i.e. the synthesis of hemoglobin and the energy-dependent proteolysis. Our approach to the investigation of metabolic relations in the reticulocytes can be applied to other tissues in which equilibria between large metabolite pools play a role.

Balance of glucose utilization in rabbit reticulocytes.

The shares of glucose utilizing processes in red blood cells under aerobic conditions were estimated in tracer experiments and with mathematical methods. A complex method of these pathways in reticulocytes is given. Our approach to the solution of the problem in reticulocytes can be transferred to other tissues. The new mathematical technique used is suitable for derivation of closed terms for the specific radioactivity of single positions of C-atoms of the metabolites of citrate cycle. Therefore, by means of regressions analysis, the calculation of unknown concentrations and flux rates is possible.

Nitrogen economy and the metabolism of serine and glycine in reticulocytes of rabbits.

In this work it is demonstrated the NH3 potentially available from the oxidation of amino acids by the reticulocyte is utilized for the new-synthesis of serine via transamination reactions with hydroxypyruvate and phosphohydroxypyruvate. These compounds are derived from glucose, which furnishes in this manner the carbon skeleton of serine. It was shown that serine is mainly degraded via glycine, while glycine is in the main formed from serine. Furthermore it was found that serine synthesis is localized in the cytosol, while the reversible transformation of serine to glycine takes place in the mitochondria. A transfer of methylenetetrahydrofolate occurs in both directions across the mitochondrial membrane. A concentrative uptake of serine by the mitochondria was found, while glycine is transported slowly in both directions. On the basis of the data an overall balance is given of the quantitative relations between protein breakdown and hemoglobin synthesis as well as for the new-formation and utilization of serine and glycine. The new-formation of serine may amount to about one tenth of the glucose utilized by the reticulocyte and furnishes about one-half of the serine and glycine moieties required for the synthesis of heme and globin. The remainder is provided by the energy-dependent protein breakdown.

NADPH production in the oxidative pentose phosphate pathway as source of reducing equivalents in glycolysis of human red cells in vitro.

Studies have been carried out on human erythrocytes in vitro to clarify the deficit of pyruvate formation under conditions when 2,3 DPG is degraded. The results lead to the conclusion that there exist a cross connection between the glycolytic and the oxidative pentose phosphate pathway which is mediated by the NADP/NADPH couple. NADPH serves as additional reducing equivalent in the reaction of the LDH. In the absence of glucose the pool of the metabolites of the pentose phosphate pathway is able to supply glucose-6-phosphate for the production of NADPH by recombination. The reaction of NADPH at the LDH is probably of significance under in vivo conditions.

[Nitrogen-economy and synthesis of serine and glycine in reticulocytes]. / Stickstoff-Okonomie und Synthese von Serin und Glyzin in Retikulozyten.

Amino acids constitute the main substrate of the reticulocyte. The amino acid pool of reticulocytes represents a characteristic selection. The composition of the amino acid pool is dependent on maturation. From the preferential oxidation of short-chain amino acids one would expect a ratio about of 5:1 between the oxygen consumption and ammonia formation. If one corrects for NH3-formation by the deamination of nucleotides the ratio between the oxygen consumption and NH3-formation is about an order of magnitude higher than the theoretical ratio. The small liberation of NH3 in energy production from amino acids results from the re-utilization of their alpha-NH2-group for the synthesis of serine and glycine, while the C-skeleton stems from glucose. Serine is formed via OH-pyruvate and OH-pyruvate. Serine and glycine serve preferentially for synthesis of hemoglobin. In reticulocytes there exists a compartmentation of glycine which accounts for differences between serine and glycine in isotopic experiments. From the time dependent change of the specific activities of pulse-labelled serine and glycine one may calculate that the serine synthesis amounts to 15--30% of the glucose utilization.

[Studies of antimycin-A-resistant respiration of erythroid cells]. / Untersuchungen zur Antimyzin a-resistenten Atmung erythroider Zellen.

In bone marrow of rabbits and in erythroid cells of other species there exists an Antimycin A-resistant respiration. Its mechanism corresponds to that postulated for the Antimycin A-resistant respiration of rabbit reticulocytes.

[Chacterization of human reticulocytes: respiration, Pasteur effect, and electron microscopic findings on mitochondria]. / Charakterisierung von Retikulozyten des Menschen: Atmung, Pasteur-Effekt und elektronenmikroskopische Befunde an Mitochondrien

On 5 blood samples of newborns, whose reticulocytes had been enriched by density gradient centrifugation, and on 25 blood samples of different reticulocytoses of man were determined: the extent of intra- and extramitochondrial respiration, coupling of the electron transfer with the oxidative phosphorylation and the electronmicroscopic appearance, and the number of mitochondria. The reticulocytes occurring in the flowing human blood are in general relatively stiff and are characterized by the following properties:--low respiration--low capacity of the respiratory chain enzymes--weakened Pasteur effect --varying proportion of intramitochondrial respiration and total respiration--decoupling of a major part of the intramitochondrial respiration--low number of mitochondria--qualitative changes of mitochondria. However, there are situations of erythropoiesis where immature reticulocytes are discharged in man (similar to the socalled "stress reticulocytes" of rabbits). On the other hand, it could be shown that the reticulocytes of rabbits are mature in the normal state.