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1.
The cytosolic precursor for the mitochondrial glutamate dehydrogenase of rat liver was synthesized in a cell-free reticulocyte lysate using messenger RNA from rat liver. To check whether this precursor had enzymatic activity, a highly sensitive fluorimetric method, which can measure picogram quantities of enzyme, was used together with competitive dissociation of the precursor from an immunoprecipitate with inactive glutamate dehydrogenase. Glutamate dehydrogenase activity, corresponding to that estimated from incorporation of [35S]-methionine, was detected in the precursor. The significance of this finding is discussed.  相似文献   

2.
Antibodies to purified glycerol-3-phosphate dehydrogenase were raised in rabbits and purified from serum by affinity chromatography on enzyme-bound Sepharose columns. RNA from membrane-free polyribosomes, or poly(A)+ RNA (total cellular RNA) of rat liver, was translated in a rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, and the glycerol-3-phosphate dehydrogenase synthesized was isolated by immunoprecipitation using the antibody. The in vitro product moved on sodium dodecyl sulfate-polyacrylamide gels as a polypeptide that was about 5,000 daltons larger than the subunit of the mature enzyme (74,000 daltons). Digestion of both the mature and the in vitro newly synthesized forms of the enzyme yielded respective sets of peptide fragments which had similar patterns upon sodium dodecyl sulfate-gel electrophoresis. When the presumptive precursor that had been synthesized in vitro was incubated with isolated intact rat liver mitochondria, it was converted to "mature" subunits that were no longer susceptible to externally added proteases. Import of the presumptive precursor is dependent upon an electrochemical potential across the inner mitochondrial membranes. The mature form of the protein is assembled in its native location (the outer surface of the inner mitochondrial membrane).  相似文献   

3.
An in vitro expression plasmid (pGRAP) that contained the cDNA coding for the rat mitochondrial aldehyde dehydrogenase precursor was constructed, mRNA was synthesized then translated, and the in vitro synthesized precursor of aldehyde dehydrogenase was used in an in vitro import assay. As expected the 19 amino acid signal peptide of the precursor allowed import of the precursor into rat liver mitochondria. This in vitro system was used to examine the effect of alcohols on import. It was found that the alcohols (ethyl, butyl, hexyl, and octyl) tested inhibited the import of the aldehyde dehydrogenase precursor. Pretreatment of the mitochondria with alcohol was responsible for the inhibition. The inhibition appeared to be relatively specific for pre-aldehyde dehydrogenase as the precursor of ornithine transcarbamylase was still imported in the presence of alcohols. Of potential physiological significance was finding that ethanol inhibited import in a dose-response fashion; 50% inhibition occurred at 75 mM, a concentration achievable during the ingestion of alcohol. In addition, the concentrations of alcohols required to produce an inhibitory effect on import decreased as the hydrocarbon chain length of alcohols increased. The inhibitory effect of alcohols appeared to be specific as other solvents examined did not inhibit import. We postulate that alcohols may perturb the mitochondrial membrane and affect the receptor-translocator necessary for the import of the aldehyde dehydrogenase precursor.  相似文献   

4.
Messenger RNA template activity for glutamate dehydrogenase was detected in poly(A)-rich RNA extracted from rat liver polysomes. Enzyme synthesized in cell-free reticulocyte system was detected by measuring enzyme activity in the translation incubation mixture using dual wavelength spectrophotometric technique. The translation product was also identified by a partial purification of the labeled synthesized enzyme and by coelectrophoresis with the carrier enzyme preparation from mitochondrial matrix.  相似文献   

5.
We have previously reported that the precursor of rat liver mitochondrial malate dehydrogenase, synthesized in vitro, is about 1,500 to 2,000 Mr larger than the mature enzyme and can be processed to the mature size by isolated mitochondria from Chinese hamster ovary cells (Chien, S.-M. and Freeman, K. B. (1984) J. Biol. Chem. 259, 3337-3342). Furthermore, binding, but not processing, was observed in the presence of an uncoupler. Binding was insensitive to temperature and was completed within 2.5 min at 0 degrees C. The role of binding in the overall process of import of the precursor is now further characterized. The precursor form, bound either in the presence of an uncoupler or at 0 degrees C, was sensitive to trypsin suggesting that binding occurs on the mitochondrial outer membrane. Saturation of binding was observed with a limited amount of mitochondria and an excess of in vitro translated rat liver proteins indicating that there is a finite number of binding sites. Furthermore, when the precursor was prebound to mitochondria at 0 degrees C for 5 min, the precursor was processed to the mature size and the rate of processing was independent of the volume of reaction mixture. In contrast, the rate of processing of unbound precursor was dependent on reaction volume. These results strongly suggest that binding of the precursor of malate dehydrogenase to the mitochondrial outer membrane is an intermediate step in its import.  相似文献   

6.
Changes in the activity of three mitochondrial enzymes in rat liver after in vitro ischemia have been determined by enzyme histochemical methods. The changes were correlated with the appearance in the electron microscope of flocculent densities in the mitochondria indicative of irreversible cell injury. The flocculent densities were observed in rat liver after about 2 h of ischemia in vitro at 37 degrees C. At the same time the activity of glutamate dehydrogenase, localized in the mitochondrial matrix, started to decrease. However, the activities of succinate dehydrogenase localized in the inner membrane of mitochondria, as well as monoamine oxidase of the mitochondrial outer membrane did not change at that stage. It is concluded from the results of this study and those of others that flocculent densities are formed by denaturation of proteins of the mitochondrial matrix in which glutamate dehydrogenase takes part. It should be considered more as a sign than as the cause of cell death.  相似文献   

7.
The precursor polypeptides of a large subunit of succinate dehydrogenase and ornithine aminotransferase (the enzymes which are located in the mitochondrial inner membrane and matrix respectively) were synthesized as a larger molecular mass than their mature subunits, when rat liver RNA was translated in vitro. These precursor polypeptides were also detected in vivo in ascites hepatoma cells (AH-130 cells). When the 35S-labeled precursor polypeptides were incubated with isolated rat liver mitochondria at 30 degrees C in the presence of an energy-generating system, these two precursors were converted to their mature size and the 35S-labeled mature-size polypeptides associated with mitochondria. Furthermore, these mature-size polypeptides were recovered from their own locations, the inner mitochondrial membrane and the matrix. The precursor of ornithine aminotransferase incubated with rat liver mitochondria at 0 degree C was specifically and tightly bound to the surface of the mitochondria even in the presence of an uncoupler of oxidative phosphorylation. This precursor, bound to the mitochondria, was imported into the matrix when the mitochondria were reisolated and incubated at 30 degrees C in the presence of an energy-generating system, suggesting that a specific receptor may be involved in the binding of the precursor. The processing enzyme for both precursor polypeptides seemed to be located in the mitochondrial matrix and was partially purified from the mitochondria. A metal-chelating agent strongly inhibited the processing enzyme and the inhibition was recovered by the addition of Mn2+ or Co2+.  相似文献   

8.
The very high affinity for GTP of glutamate dehydrogenase was used to purify this enzyme by affinity chromatography. After periodic acid oxidation, GTP was covalently bound to an activated Sepharose. When crude mitochondrial extracts were applied on a column of this GTP-Sepharose, glutamate dehydrogenase was retained with very few other proteins. Glutamate dehydrogenase from rat liver was eluted with a KCl gradient with only one contaminating protein. From a pig heart mitochondrial extract the enzyme was purified 300-fold in one step. A chromatography on hydroxyapatite was sufficient to achieve the purification. This very simple technique avoids the long and troublesome crystallization steps generally involved in glutamate dehydrogenase purification.  相似文献   

9.
Outer mitochondrial membrane was purified from rat liver. Its constituent proteins were analyzed by SDS-polyacrylamide gel electrophoresis and by electrophoretic immunoblotting employing antibodies raised against total outer mitochondrial membrane. Anti-outer mitochondrial membrane antiserum reacted with only one polypeptide (15 kDa) in rough microsomes, whereas no immunological cross-reactivity was observed with other mitochondrial compartments (intermembrane space, inner membrane, or matrix) or with lysosomes or total cytosol. The antiserum was employed to characterize precursors of outer mitochondrial membrane proteins synthesized in vitro in a rabbit reticulocyte cell-free system. One product (a 68 kDa polypeptide designated OMM-68) bound efficiently to mitochondria in vitro but did not interact with either dog pancreas or rat liver microsomes, either co-translationally or post-translationally. OMM-68 was synthesized exclusively by the membrane-free class of polyribosomes. Attachment of precursor OMM-68 to mitochondria was not accompanied by processing of the polypeptide to a different size.  相似文献   

10.
11.
In vitro synthesis of general acyl CoA dehydrogenase [EC 1.3.99.3], one of the mitochondrial flavoenzymes, was carried out to elucidate its biosynthetic mechanism. Poly(A)+ RNA isolated from pig kidney was translated in vitro using wheat germ lysate system and the synthesized enzyme was immunoprecipitated by the antibody against purified pig kidney general acyl CoA dehydrogenase. The apparent molecular weight of the synthesized protein was estimated to be approximately 1,000 daltons larger than that of the mature enzyme, indicating that general acyl CoA dehydrogenase in pig kidney is synthesized as a precursor with a larger molecular weight.  相似文献   

12.
The precursor of the rat mitochondrial flavoenzyme dimethylglycine dehydrogenase (Me(2)GlyDH) has been produced in Escherichia coli as a C-terminally 6-His-tagged fusion protein, purified by one-step affinity chromatography and identified by ESI-MS/MS. It was correctly processed into its mature form upon incubation with solubilized rat liver mitoplasts. The purified precursor was mainly in its apo-form as demonstrated by immunological and fluorimetric detection of covalently bound flavin adenine dinucleotide (FAD). Results described here definitively demonstrate that: (i) covalent attachment of FAD to Me(2)GlyDH apoenzyme can proceed in vitro autocatalytically, without third reactants; (ii) the removal of mitochondrial presequence by mitochondrial processing peptidase is not required for covalent autoflavinylation.  相似文献   

13.
1. Glutamate oxidation in brain and liver mitochondrial systems proceeds mainly through transamination with oxaloacetate followed by oxidation of the α-oxoglutarate formed. Both in the presence and absence of dinitrophenol in liver mitochondria this pathway accounted for almost 80% of the uptake of glutamate. In brain preparations the transamination pathway accounted for about 90% of the glutamate uptake. 2. The oxidation of [1-14C]- and [5-14C]-glutamate in brain preparations is compatible with utilization through the tricarboxylic acid cycle, either after the formation of α-oxoglutarate or after decarboxylation to form γ-aminobutyrate. There is no indication of γ-decarboxylation of glutamate. 3. The high respiratory control ratio obtained with glutamate as substrate in brain mitochondrial preparations is due to the low respiration rate in the absence of ADP: this results from the low rate of formation of oxaloacetate under these conditions. When oxaloacetate is made available by the addition of malate or of NAD+, the respiration rate is increased to the level obtained with other substrates. 4. When the transamination pathway of glutamate oxidation was blocked with malonate, the uptake of glutamate was inhibited in the presence of ADP or ADP plus dinitrophenol by about 70 and 80% respectively in brain mitochondrial systems, whereas the inhibition was only about 50% in dinitrophenol-stimulated liver preparations. In unstimulated liver mitochondria in the presence of malonate there was a sixfold increase in the oxidation of glutamate by the glutamate-dehydrogenase pathway. Thus the operating activity of glutamate dehydrogenase is much less than the `free' (non-latent) activity. 5. The following explanation is put forward for the control of glutamate metabolism in liver and brain mitochondrial preparations. The oxidation of glutamate by either pathway yields α-oxoglutarate, which is further metabolized. Since aspartate aminotransferase is present in great excess compared with the respiration rate, the oxaloacetate formed is continuously removed by the transamination reaction. Thus α-oxoglutarate is formed independently of glutamate dehydrogenation, and the question is how the dehydrogenation of glutamate is influenced by the continuous formation of α-oxoglutarate. The results indicate that a competition takes place between the α-oxoglutarate-dehydrogenase complex and glutamate dehydrogenase, probably for NAD+, resulting in preferential oxidation of α-oxoglutarate.  相似文献   

14.
The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [3H]dTTP or [3H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.  相似文献   

15.
Carbamyl phosphate synthase-I and glutamate dehydrogenase both form a complex with mitochondrial aspartate aminotransferase. Instead of these two enzymes competing for the aminotransferase, carbamyl phosphate synthase-I enhances glutamate dehydrogenase-aminotransferase interaction. This suggests that a complex can be formed between all three enzymes. Since this complex is stable in the presence of substrates and modifiers of the three enzymes, it could conceivably convert NH4+ produced from aspartate into carbamyl phosphate. Furthermore, since carbamyl phosphate synthase-I is the predominant protein in liver mitochondria, it could play a major role in placing the aminotransferase and glutamate dehydrogenase in close proximity. Malate removes glutamate dehydrogenase from the tri-enzyme complex and thus could play a role in determining whether glutamate dehydrogenase interacts with carbamyl phosphate synthase-I or is available to participate in reactions with the Krebs cycle. Palmitoyl-CoA has a high affinity for both carbamyl phosphate synthase-I and glutamate dehydrogenase. ATP and malate which, respectively, decrease and enhance binding of palmitoyl-CoA to glutamate dehydrogenase, respectively decrease and enhance the ability of this enzyme to compete with carbamyl phosphate synthase-I for palmitoyl-CoA. Since carbamyl phosphate synthase-I is present in high levels in liver mitochondria and has a high affinity for palmitoyl-CoA, it could play a major role as a reservoir for palmitoyl-CoA.  相似文献   

16.
Threonine is a precursor of glycine in the rat, but the metabolic pathway involved is unclear. To elucidate this pathway, the biosynthesis of glycine, and of aminoacetone, from l-threonine were studied in rat liver mitochondrial preparations of differing integrities. In the absence of added cofactors, intact mitochondria formed glycine and aminoacetone in approximately equal amounts from 20 mM l-threonine, but exogenous NAD+ decreased and CoA increased the ratio of glycine to aminoacetone formed. In intact and freeze-thawed mitochondria, the ratio of glycine to aminoacetone formed was markedly sensitive to the concentration of l-threonine, glycine being the major product at low l-threonine concentrations. Disruption of mitochondrial integrity by sonication (1 min) decreased the ratio of glycine to aminoacetone formed, and in 20 000 × g supernatant fractions from sonicated (3 min) mitochondria, aminoacetone was the major product. The main non-nitogenous tow-carbon compound detected when intact mitochondria catabolized l-threonine to glycine was acetate, which was probably derived from deacylation of acetyl-CoA. These results suggest that glycine formation from l-threonine in rat liver mitochondria occured primarily by the coupled activities of threonine dehydrogenase and 2-amino-3-oxobutyrate CoA-ligase, the extent of coupling between the enzymes being dependent upon a close physical relationship and upon the flux through the dehydrogenase reaction. In vivo glycine synthesis would predominate, and aminoacetone would be a minor product.  相似文献   

17.
1. l-Leucine strongly activated intramitochondrial glutamate dehydrogenase in the direction of glutamate synthesis. 2. In the deamination direction, the enzyme was not stimulated by leucine. This was probably due to a rate-limiting transport of glutamate across the mitochondrial membrane. 3. The effect of leucine on the kinetic constants of glutamate dehydrogenase in a mitochondrial sonicate was studied. 4. In isolated mitochondria, leucine did not stimulate the synthesis of citrulline with glutamate as the source of NH(3). 5. Leucine very markedly stimulated the synthesis of glutamate from added 2-oxoglutarate+NH(4)Cl. 6. Under conditions where glutamate and citrulline could be synthesized simultaneously from added NH(4)Cl, leucine greatly increased glutamate synthesis at the expense of citrulline synthesis. 7. It is suggested that the intramitochondrial leucine concentration may be a factor influencing the nitrogen metabolism of the liver cell.  相似文献   

18.
Serine: pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide. In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, STPp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.  相似文献   

19.
Calcium-activated proteolytic activity in rat liver mitochondria   总被引:1,自引:0,他引:1  
Soluble extracts from sonicated rat liver mitochondria and rat liver cytosol were each chromatographed on DEAE-cellulose columns, and the fractions assayed for Ca2+-activated proteolytic activity using 14C-casein as a substrate. The mitochondrial preparations were shown to be free of cytosolic and microsomal contamination by the lack of alcohol dehydrogenase activity, a cytosolic marker enzyme, and by a lack of cytochrome P-450 activity, a microsomal marker enzyme. Two peaks of Ca2+-activated neutral endoprotease activity were resolved from the mitochondrial fractions. One protease was half-maximally activated with 25 μM Ca2+, and the other by 750 μM Ca2+. Rat liver cytosol contained only a high Ca2+-requiring protease peak. This is the first demonstration of Ca2+-activated proteases in mitochondria.  相似文献   

20.
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