Cell-free synthesis of a putative precursor to the rat liver mitochondrial glycerol-3-phosphate dehydrogenase

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).     

Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc₁ complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.     

In vitro synthesis of superoxide dismutases of rat liver

The syntheses of copper, zinc-superoxide dismutase (Cu,Zn-SOD) and manganese-superoxide dismutase (Mn-SOD) in vitro were studied. Both Cu,Zn-SOD and Mn-SOD were preferentially synthesized by free polysomes. Mn-SOD was synthesized as a large precursor (26,000 daltons), which was processed to the mature size (22,500 daltons) by in vitro incubation with a rat liver mitochondrial fraction. On the other hand, Cu,Zn-SOD was synthesized as the mature size product. It was shown that Cu,Zn-SOD and Mn-SOD synthesized in vitro represented 0.018% and 0.016% of the total translation products of free polysomes, respectively.     

Biosynthesis of rat liver transhydrogenase in vivo and in vitro

The biosynthesis of pyridine dinucleotide transhydrogenase, a homodimeric inner mitochondrial membrane redox-linked proton pump, has been studied in isolated rat hepatocytes. Newly synthesized transhydrogenase, having an apparent molecular weight identical to the enzyme of isolated liver mitochondria, was selectively immunoprecipitated from detergent extracts of isolated hepatocytes which were labeled with [35S]methionine. That the enzyme is a nuclear gene product is indicated since 1) synthesis was inhibited by cycloheximide, but not by chloramphenicol and 2) no synthesis could be demonstrated in hepatocyte ghosts which are competent only in mitochondrial translation. In addition to the mature form of the enzyme, a species about 2000 daltons larger was also immunoprecipitated from pulse-labeled cells. The half-life of the larger form during a subsequent chase at 37 degrees C was about 2 min, whereas the mature form was not degraded. The relationship between the two forms of the enzyme was established by in vitro studies. A protein approximately 2000 daltons larger than mature transhydrogenase was immunoisolated from a rabbit reticulocyte lysate system programmed with sucrose gradient fractionated rat liver mRNA. This protein was converted to a species having the same size as mature enzyme after incubation with either intact rat liver mitochondria or a soluble matrix fraction derived from mitoplasts. These studies indicate that transhydrogenase is synthesized in the cytoplasm as a higher molecular weight precursor which is post-translationally processed to the mature protein by a soluble matrix protease during or after membrane insertion.     

Isozymes of S-adenosylmethionine synthetase from rat liver: isolation and characterization

S-Adenosylmethionine synthetase exists in at least two distinct forms, alpha- and beta-forms, in adult liver. The beta-form was purified to homogeneity from the soluble fraction of rat liver with a yield of about 10%. An antiserum directed against the purified beta-form from rat liver was prepared by injecting the purified enzyme into a rabbit. Ouchterlony double diffusion analysis and immunochemical titrations revealed that the isozymes, alpha- and beta-forms, are identical. Thus, the alpha-form was isolated from rat liver as a single protein using immunoaffinity chromatography against the beta-form. The molecular weights of the beta- and alpha-forms were determined to be 48,000 each by sodium dodecyl sulfate disc gel electrophoresis, and about 100,000, and 200,000, respectively, by Sephacryl S-200 gel filtration. These results indicate that the beta-form consisted of two subunits of 48,000 daltons and the alpha-form of four subunits of 48,000 daltons. The sedimentation coefficient was calculated to be 5.5S for the beta-form and 8.0S for the alpha-form.     

Identity of long-chain acyl-coenzyme A synthetase of microsomes, mitochondria, and peroxisomes in rat liver

The identity of long-chain acyl-CoA synthetase in microsomes, mitochondria, and peroxisomes of rat liver was examined by using the antibody raised against a purified preparation of the microsomal enzyme. The enzyme activities of these three organelles and the purified microsomal enzyme were titrated by the antibody in a very similar fashion when the activity was measured in terms of palmitoyl-CoA synthetase activity. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitates and by Western blot analysis that the enzymes of all three organelles consisted of a polypeptide with the same molecular weight as that of the purified enzyme, and that the specific enzyme activity of the antigenic protein in all three subcellular compartments was nearly the same. The presence of other palmitoyl-CoA synthetase activity in these organelles could not be confirmed. Immunocytochemical study to locate the antigenic site with protein A-gold complex showed that the gold particles were closely associated with the membranes of these organelles. The cell-free translation product in a rabbit reticulocyte lysate protein-synthesizing system and the subunit of the mature enzyme labeled with [35S]methionine in the liver slices exhibited the same mobility as the subunit of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme in microsomes, mitochondria, and peroxisomes was labeled at nearly the same rate when the liver slices were incubated with [35S]methionine.     

Comparison of populations of mRNA coding fumarase in rat brain and liver

The populations of mRNA encoding mitochondrial and cytosolic fumarases in the poly A+ RNA fractions purified from polysomes of rat brain and liver were examined. When the in vitro translation products programmed by the poly A+ RNA fraction obtained from rat brain were purified by immunoprecipitation with anti-fumarase antibody and analyzed by SDS polyacrylamide gel electrophoresis and fluorography, only one polypeptide of 50 KD was detected as a precursor of fumarase. In contrast, by the same method, two polypeptides of 50 KD and 45 KD, which is the same size as mature fumarase, were detected as precursors of rat liver fumarase. These results suggest that rat brain polysomes contain only one population of mRNA coding a 50 KD precursor of mitochondrial fumarase with little or no mRNA of the cytosolic fumarase, whereas rat liver polysomes contain two types of mRNA for mitochondrial and cytosolic fumarases, respectively. These findings are consistent with the fact that the brain is the only organ in rats known not to contain cytosolic fumarase.     

delta-Aminolevulinate synthetases in the liver cytosol fraction and mitochondria of mice treated with allylisopropylacetamide and 3,5-dicarbethoxyl-1,4-dihydrocollidine.

Hepatic delta-aminolevulinate (ALA) synthetase was induced in mice by the administration of allylisopropylacetamide (AIA) and 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC). In both cases, a significant amount of ALA synthetase accumulated in the liver cytosol fraction as well as in the mitochondria. The apparent molecular weight of the cytosol ALA synthetase was estimated to be 320,000 by gel filtration, but when the cytosol ALA synthetase was subjected to sucrose density gradient centrifugation, it showed a molecular weight of 110,000. In the mitochondria, there were two different sizes of ALA synthetase with molecular weights of 150,000 and 110,000, respectively; the larger enzyme was predominant in DDC-treated mice, whereas in AIA-treated mice and normal mice the enzyme existed mostly in the smaller form. When hemin was injected into mice pretreated with DDC, the molecular size of the mitochondrial ALA synthetase changed from 150,000 to 110,000. The half-life of ALA synthetase in the liver cytosol fraction was about 30 min in both the AIA-treated and DDC-treated mice. The half-life of the mitochondrial ALA synthetase in AIA-treated mice and normal mice was about 60 min, but in DDC-treated mice the half-life was as long as 150 min. The data suggest that the cytosol ALA synthetase of mouse liver is a protein complex with properties very similar to those of the cytosol ALA synthetase of rat liver, which has been shown to be composed of the enzyme active protein and two catalytically inactive binding proteins, and that ALA synthetase may be transferred from the liver cytosol fraction to the mitochondria with a size of about 150,000 daltons, followed by its conversion to enzyme with a molecular weight of 110,000 within the mitochondria. The process of intramitochondrial enzyme degradation seems to be affected in DDC-treated animals.     

Hepatic mitochondrial cytochrome P-450 system. Identification and characterization of a precursor form of mitochondrial cytochrome P-450 induced by 3-methylcholanthrene

Hepatic mitoplasts from 3-methylcholanthrene-treated rats contain cytochrome P-450 which can metabolize polycyclic aromatic hydrocarbons like benzo(a)pyrene. Mitochondrial cytochrome P-450 was partially purified and reconstituted in vitro using adrenodoxin and the adrenodoxin reductase electron transfer system and [3H]benzo(a)pyrene as the substrate. A polyclonal antibody to purified microsomal P-450c (a major 3-methylcholanthrene-inducible form) inhibited the activity of mitochondrial enzyme in a concentration-dependent manner and also reacted with a 54-kDa protein on the immunoblots. A monoclonal antibody having exclusive specificity for P-450c, on the other hand, did not inhibit the aryl hydrocarbon hydroxylase activity of the mitochondrial enzyme and showed no detectable cross-reaction with the 54-kDa mitochondrial protein. Similarly, two-dimensional analysis and immunodetection using the polyclonal antibody showed distinct molecular properties of the mitochondrial enzyme different from the similarly induced microsomal P-450c with respect to the isoelectric pH. In vitro translation of free polysomes from 3-methylcholanthrene-induced liver, transport of precursor proteins by isolated mitochondria in vitro, and immunoprecipitation with the polyclonal antibody showed the presence of a 57-kDa putative precursor which is transported and processed into mature 54-kDa species. These results present evidence for the true intramitochondrial location of the P-450c-antibody reactive isoform detected in 3-methylcholanthrene-induced rat liver mitochondria.     

Immunochemical studies on induction of rat liver mitochondrial serine: pyruvate aminotransferase by glucagon.

The 7- to 10-fold increase in the rat liver serine:pyruvate aminotransferase activity after glucagon administration was shown to occur mainly in the mitochondrial matrix of parenchymal cells. The enzyme was purified from glucagon-treated rat liver mitochondria to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A specific rabbit antibody was prepared against the purified enzyme. Upon Ouchterlony double diffusion analysis, the mitochondrial extracts of glucagon-treated rat liver produced a single and fused precipitin line between the purified enzyme against the antibody. The supernatant fraction of glucagon-treated rat liver and the mitochondrial extracts of normal liver were also shown to make a single and fused precipitin line with the purified enzyme, when applied in large quantities. The quantitative immunotitration demonstrated that the glucagon-induced increase in the activity of liver serine:pyruvate aminotransferase were accompanied by the parallel increase in the amount of the enzyme antigen. Isotopic leucine incorporation studies showed that the relative rate of synthesis of the enzyme was increased approximately 10-fold by glucagon administration under the conditions employed. The rate of the degradation of the aminotransferase in the normal rat liver was a relatively slow process with a half-life of approximately 30 h. Thus the accumulation of serine:pyruvate aminotransferase in rat liver mitochondria by glucagon treatment can be ascribed mainly to the rise in the rate of enzyme synthesis.     

The interaction of rat liver carbamoyl phosphate synthetase and ornithine transcarbamoylase with inner mitochondrial membranes

The intramitochondrial localization of the urea cycle enzymes, carbamoyl phosphate synthetase and ornithine transcarbamoylase, has been examined by both in vitro and in situ studies. The following three lines of evidence are presented to establish that significant fractions of the rat liver enzymes are loosely associated with the inner mitochondrial membrane: 1) when the mitochondrion is fractionated, the enzymes partition between the matrix and membrane fractions in the absence of detergent and partition solely to the matrix in the presence of detergent; 2) the purified enzymes associate with purified inner membrane preparations; and, 3) protein A-gold electron microscopic immunocytochemical analysis of rat liver sections reveals a nonrandom arrangement of the enzyme, with the maximal enzyme density adjacent to the inner mitochondrial membrane. These findings serve as the basis for novel potential mechanisms for regulation of the activity of the enzymes and provide additional evidence for the extensive organization of the mitochondrial matrix. The membrane interaction might also serve as the organizing factor for a carbamoyl phosphate synthetase-ornithine transcarbamoylase or other multienzyme complex.     

Isolation and characterization of argininosuccinate synthetase from human liver

This communication describes the purification and characterization of argininosuccinate synthetase from human liver. By numerous criteria including electrophoresis in sodium dodecyl sulfate containing gels, electrophoresis in nondissociating gels, and analytical ultracentrifugation, the protein is homogeneous at a specific activity of 4.2 mumol/(min mg) assayed at 37 degrees C in the direction of argininosuccinate synthesis. The enzyme has a molecular weight of 183,000, as determined by gel filtration. Electrophoresis in the presence of sodium dodecyl sulfate yielded a single band migrating with an Rf corresponding to 43,000 daltons. Thus, the enzyme is considered to contain four subunits of identical molecular weight. The s20,w of the enzyme is 8.2 S. Antibodies were prepared in rabbits directed against the purified protein. These antibodies react specifically with argininosuccinate synthetase, as determined by electrophoretic analysis of the immunoadsorbed product from crude extracts of human liver. The human enzyme has very similar properties to those published for the beef and rat liver enzymes.     

Cloning and nucleotide sequence of cDNA encoding human liver serine-pyruvate aminotransferase

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.     

Transport of proteins into mitochondrial matrix. Evidence suggesting a common pathway for 3-ketoacyl-CoA thiolase and enzymes having presequences

Rat liver 3-ketoacyl-CoA thiolase, a mitochondrial matrix enzyme which catalyzes a step of fatty acid beta-oxidation, was synthesized in a rabbit reticulocyte lysate cell-free system. The in vitro product was apparently the same in molecular size and charge as the subunit of the mature enzyme. The enzyme synthesized in vitro was transported into isolated rat liver mitochondria in an energy-dependent manner. In pulse experiments with isolated rat hepatocytes at 37 degrees C, the radioactivity of the newly synthesized enzyme in the cytosolic fraction remained essentially unchanged during 5-20 min of incubation, whereas that of the enzyme in the particulate fraction increased with time during the incubation. The pulse-labeled enzyme disappeared with an apparent half-life of less than 3 min from the cytosolic fraction, in pulse-chase experiments. Purified 3-ketoacyl-CoA thiolase inhibited the mitochondrial uptake and processing of the precursors of the other matrix enzymes, ornithine carbamoyltransferase, medium-chain acyl-CoA dehydrogenase and acetoacetyl-CoA thiolase. These results indicate that 3-ketoacyl-CoA thiolase has an internal signal which is recognized by the mitochondria and suggest that this enzyme and the three others are transported into the mitochondria by a common pathway.     

A major polypeptide component of rat liver mitochondria: carbamyl phosphate synthetase.

One of the major components of rat liver mitochondria detected by gel electrophoresis in sodium dodecyl sulfate is a 165,000 molecular weight polypeptide that makes up 15 to 20% of the total mitochondrial protein. This component appears to be a single molecular species. Evidence is presented here for the identification of this protein with the polypeptide chain of a urea cycle enzyme, carbamoylphosphate synthetase I (EC 2.7.2.5). The 165,000 molecular weight polypeptide was solubilized from mitochondria with Triton X-100 and purified to 90% homogeneity by DEAE-cellulose chromatography. This component co-migrated with carbamyl phosphate synthetase activity when mitochondrial proteins were separated by gel filtration or sucrose gradient centifugation. The identification of the 165,000 molecular weight polypeptide with this activity was also supported by the presence or absence of this protein in a variety of rat tissue mitochondria, in liver and kidney mitochondria from various ureotelic and nonureotelic species, and in fetal rat liver mitochondria.     

In vitro synthesis of pig kidney general acyl CoA dehydrogenase

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.     

Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro

Total polysomal RNA of rat liver was translated in vitro in a rabbit reticulocyte lysate system. The translation products were mixed with a postnuclear supernatant fraction of rat liver and incubated post-translationally at 26 degrees C for 15-60 min. The import assay mixture was separated into a particulate fraction and supernatant by centrifugation, both of which were analyzed by immunoprecipitation with a goat antibody against rat liver peroxisomal proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. One peroxisomal translation product (Mr 72,000) appeared in the particulate fraction, was partly proteinase K-resistant, and addition of detergents prior to proteolysis abolished this resistance. In isopycnic centrifugation of the uptake assay mixture, the protease-resistant 35S-polypeptide of Mr 72,000 cosedimented with the peroxisomes. This translation product was identified immunochemically as fatty acyl-CoA oxidase; both before and after import it was indistinguishable in size from subunit A of the purified enzyme by prolonged sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the cell-free translation products were incubated with highly purified peroxisomes, 35S-catalase entered peroxisomes (by the criterion of protease resistance), and its entry was stimulated by the addition of a high speed supernatant (cytosolic) fraction of rat liver. These results demonstrate the post-translational import into peroxisomes in vitro of at least two cell-free translation products.     

Translocation of proteins into rat liver mitochondria. Existence of two different precursor polypeptides of liver fumarase and import of the precursor into mitochondria

Two different putative precursor polypeptides of rat liver fumarase were synthesized when RNA prepared from rat liver were translated in vitro using the rabbit reticulocyte lysate system. One of these putative precursor polypeptides (P1) was synthesized as a larger molecular mass than the mature subunit of fumarase (45,000 daltons) by about 5,000 daltons and the other (P2) had the same molecular mass as the mature enzyme. When the 35S-labeled cell-free translation products were incubated with rat liver mitochondria at 30 degrees C, P1 and the 35S-labeled mature size fumarase were associated with the mitochondria. Of these, the 35S-labeled mature size fumarase was resistant to externally added protease, but P1 was not, indicating that the 35S-labeled mature size fumarase was located in the mitochondrial matrix. The following observations strongly suggested that the 35S-labeled mature size fumarase in mitochondria was derived from P1, which was energy-dependently imported and concomitantly processed to the mature size. 1) The amount of the 35S-labeled mature size fumarase recovered from the mitochondria increased proportionally to the duration of incubation, while the amount of P1 recovered from the post-mitochondrial and mitochondrial fractions decreased with the duration of the incubation. 2) Only P1 could bind with the mitochondrial outer membrane at 0 degrees C even in the presence of an uncoupler of the oxidative phosphorylation but P2 did not. 3) P1 bound to the mitochondrial outer membrane was imported into the matrix, when the mitochondria binding only P1 at 0 degrees C was reisolated and incubated at 30 degrees C in the presence of an energy-generating system. The specific receptor was involved in the binding of P1 to mitochondria, since a high concentration of NaCl did not interfere with the binding of P1 to the membrane and did not discharge P1 bound onto the membrane. It was shown that P1 formed an aggregate composed of 6 to 8 molecules and P2 was a dimer in the cell-free translation mixture and that P1 and P2 were enzymatically inactive. These results suggest that the precursor for the mitochondrial enzyme has a larger molecular weight than that of the mature enzyme, whereas the precursor for the cytosolic enzyme has the same molecular weight as the mature enzyme.     

Levels of carbamoyl phosphate synthetase I in livers of young and old rats assessed by activity and immunoassays and by electron microscopic immunogold procedures

Carbamoyl phosphate synthetase I, the most abundant protein of rat liver mitochondria, plays a key role in synthesis of urea. Because aging affects some liver functions, and because there is no information on the levels of carbamoyl phosphate synthetase I during aging, we assayed the activity of this enzyme and determined immunologically the level of carbamoyl phosphate synthetase I in liver homogenates from young (4 months) and old (18 or 26 months) rats. In addition, we used electron microscopic immunogold procedures to locate and measure the amount of the enzyme in the mitochondrial matrix. There is no significant change in enzyme activity or enzyme protein content with age, although there is a higher concentration of the enzyme in the mitochondria (c. 1.5 times greater) from old rats, which is compensated by a decrease in the fractional volume of the mitochondrial compartment during aging.     

Partial purification of a metalloprotease catalyzing the processing of adrenodoxin precursor in bovine adrenal cortex mitochondria

In vitro translation of bovine adrenal cortex RNA in rabbit reticulocyte lysate cell-free system produced the precursor form of adrenodoxin having a molecular weight of approximately 22,000 daltons, which was about 10,000 daltons larger than mature adrenodoxin. The precursor of adrenodoxin was efficiently imported into adrenal cortex mitochondria in vitro. The precursor was also imported into rat liver mitochondria, suggesting the lack of tissue specificity and species specificity of the import process. The enzyme which processed the precursor of adrenodoxin to the mature form was in the matrix fraction from bovine adrenal cortex mitochondria, and the processing protease was partially purified from the matrix fraction. The apparent molecular weight of the processing protease was about 60,000 daltons as determined by Sephadex G-150 gel filtration, and its activity was optimal at pH 8.5. The processing protease was not inhibited by various bacterial protease inhibitors examined. Metal chelators (EGTA, GTP, 8-hydroxyquinoline, and Zincon) inhibited the processing, and EDTA and o-phenanthroline were more strongly inhibitory than other chelators. The processing protease was completely inactivated by incubation with 10 microM EDTA, and its activity was restored by addition of excess amounts of Mn2+, Fe2+, or Co2+. These results indicate that the maturation of the precursor of adrenodoxin is catalyzed by a soluble metalloprotease in the matrix.