Satoshi Miura

Cell-free synthesis of the enzymes of peroxisomal β-oxidation

Three enzymes of peroxisomal β-oxidation of rat liver were synthesized in a cell-free protein-synthesizing system derived from rabbit reticulocyte lysate. The invitro products of acyl-CoA oxidase and enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase multifunctional protein were similar in size to or slightly larger than the subunit of the respective mature enzymes. The invitro product of peroxisomal 3-ketoacyl-CoA thiolase was about 3,000 daltons larger than the mature subunit. The hepatic levels of translatable mRNAs coding for these three enzymes were about 10 times higher in rats fed a di(2-ethylhexyl)phthalate-containing diet than in control animals.

Ornithine transcarbamylase in liver mitochondria

SummaryOrnithine transcarbamylase (ornithine carbamoyltransferase, EC 2.1.3.3), the second enzyme of urea synthesis, is localized in the matrix of liver mitochondria of ureotelic animals. The enzyme is encoded by a nuclear gene, synthesized outside the mitochondria, and must then be transported into the organelle. The rat liver enzyme is initially synthesized on membrane-free polysomes in the form of a larger precursor with an amino-terminal extension of 3 400–4 000 daltons. In rat liver slices and isolated rat hepatocytes, the pulse-labeled precursor is first released into the cytosol and is then transported with a half life of 1 2 min into the mitochondria where it is proteolytically processed to the mature form of the enzyme. The precursor synthesized in vitro exists in a highly aggregated form and has a conformation different from that of the mature enzyme. The precursor has an isoelectric point (pI = 7.9) higher than that of the mature enzyme (pI = 7.2).The precursor synthesized in vitro can be taken up and processed to the mature enzyme by isolated rat liver mitochondria. The mitochondrial transport and processing system requires membrane potential and a high integrity of the mitochondria. The transport and processing activities are conserved between mammals and birds or amphibians and is presumably common to more than one precursor. Potassium ion, magnesium ion, and probably a cytosolic protein(s), in addition to the transcarbamylase precursor and the mitochondria, are required for the maximal transport and processing of the precursor.A mitochondrial matrix protease which converts the precursor to a product intermediate in size between the precursor and the mature subunit has been highly purified. The protease has an estimated molecular weight of 108 000 and an optimal pH of 7.5–8.0, and appears to be a metal protease. The protease does not cleave several of the protein and peptide substrates tested. The role of this protease in the precursor processing remains to be elucidated.Rats subjected to different levels of protein intake and to fasting show significant changes in the level of enzyme protein and activity of ornithine transcarbamylase. The dietary-dependent changes in the enzyme level are due mainly to an altered level of functional mRNA for the enzyme. In contrast, during fasting, the increase in the enzyme level is associated with a decreased level of translatable mRNA forthe enzyme.Pathological aspects of ornithine transcarbamylase including the enzyme deficiency and reduced activities of the enzyme in Reyes syndrome are also described. A possibility that impaired transport of the enzyme precursor into the mitochondria leads to a reduced enzyme activity, is proposed.

A metalloprotease involved in the processing of mitochondrial precursor proteins

A protease that cleaves the precursor of ornithine carbamoyltransferase (EC 2.1.3.3), a mitochondrial matrix enzyme, has been partially purified from the matrix fraction of rat liver mitochondria. The protease cleaved the precursors of several other matrix proteins at apparently correct sites. The protease was inhibited by 1,10-phenanthroline and EDTA, was reactivated by excess Mn2+ or Co2+, and did not cleave the alkali-denatured precursor proteins. These and other results indicate that this protease is responsible for the processing of at least several matrix protein precursors, and that the enzyme recognizes some three-dimensional conformation of the precursors as well as the amino acid sequences around the cleavage sites.

Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex

Pyruvate dehydrogenase complex was purified from rat heart. The complex showed four polypeptide bands on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, corresponding to lipoate acetyltransferase (mol.wt. 68 000), lipoamide dehydrogenase (mol.wt. 56 000), alpha-subunit (mol.wt. 41 000) and beta-subunit (mol.wt. 35 000) of pyruvate dehydrogenase. Rat heart pyruvate dehydrogenase complex was dissociated into three component enzymes and the antibodies against each component enzyme were prepared. Anti-pyruvate dehydrogenase and anti-lipoate acetyltransferase antibodies effectively precipitated pyruvate dehydrogenase complex, but an anti-lipoamide dehydrogenase antibody released lipoamide dehydrogenase from the complex and effectively precipitated lipoamide dehydrogenase. Lipoamide dehydrogenase was synthesized in a cell-free reticulocyte lysate system with total RNA from rat liver. Its translation product was detected as a putative precursor which is 3000 Da larger than the mature subunit. In cell-free translation programmed with free and membrane-bound polysomes, activity of mRNA coding for the precursor of the enzyme was much higher in free polysomes than in membrane-bound polysomes.

In vitro synthesis of a putative precursor of serine: pyruvate aminotransferase of rat liver mitochondria.

Abstract Serine:pyruvate aminotransferase [EC 2.6.1.51] of rat liver, an enzyme induced by glucagon in mitochondria, was synthesized in cell-free protein synthesizing systems derived from nuclease-treated rabbit reticulocyte lysate and wheat germ extract as a putative precursor which was approximately 2,000 daltons larger than the subunit of mature enzyme. The hepatic level of translatable messenger RNA coding for the putative precursor was approximately 40 times higher in rats received a glucagon administration 3.5 h before sacrifice than in control animals.

Level of translatable messenger RNA coding for argininosuccinate synthetase in the liver of the patients with quantitative-type citrullinemia

SummaryThe translation activity of mRNA coding for argininosuccinate synthetase in total RNA extracted from the liver of three patients with quantitative-type citrullinemia was determined using a cell-free translation system. In two patients, the hepatic content of the enzyme was about 20% of the control value, whereas translatable mRNA level for the enzyme was similar to or slightly lower than those of control livers. In the third patient, the enzyme content was about 50% of the control value, and mRNA activity for the enzyme was low normal. These results indicate that at least in the first two patients, the decrease in the enzyme protein is due either to increased degradation of the enzyme or to decreased translation in the patients liver.

Cell-free synthesis and transport of precursors of mutant ornithine carbamoyltransferases into mitochondria

Synthesis, mitochondrial transport and processing of ornithine carbamoyltransferase (EC 2.1.3.3) were studied in mutant mice strains (sparse-fur, spf, and sparse-fur with abnormal skin and hair, spf-ash) which exhibit a deficiency in this enzyme. Spf mice have an increased amount (about 150% of control) of the enzyme with abnormal kinetic properties, whereas spf-ash mice have a decreased amount (about 10% of control) of the enzyme with apparently normal kinetic properties. Precursors of the mutant enzymes were synthesized in a reticulocyte lysate cell-free system. The hepatic level of translatable mRNA coding for the enzyme and the rate of the enzyme synthesis in liver slices of spf mice were 58 and 60% of the controls, respectively. In the case of spf-ash mice the activity of translatable mRNA for the enzyme was 10% of the controls. These results indicate that the decreased amount of ornithine carbamoyltransferase protein in spf-ash mice is due mainly to a decreased level of translatable mRNA for the enzyme, whereas the increase in the enzyme amount in spf mice is presumably the result of a decreased rate of enzyme degradation. The subunit molecular weight of the spf enzyme precursor was practically the same as that of the normal enzyme precursor (Mr 40 000). Both precursors synthesized in vitro could be taken up and processed similarly to an apparently mature form (Mr 37 000). In the case of spf-ash enzyme, two discrete in vitro products were observed on sodium dodecyl sulfate polyacrylamide gel; one comigrated with the normal enzyme precursor and the other moved slightly slower. Both products appeared to be taken up and processed to the mature form of the enzyme.

Active forms of chymotrypsin C isolated from autolyzed porcine pancreas glands

Four active forms of chymotrypsin C (C1, C2A, C2B, and C3) were isolated from the autolyzed porcine pancreas glands. Their molecular weights were estimated by SDS-polyacrylamide gel electrophoresis to be 29 100 for C1, 26 300 for C2A and C3, and 25 500 for C2B. The kinetic analyses of esterase activity of the enzymes toward Ac-LLeu-OEt and Ac-LPhe-OEt showed that chymotrypsin C1 hydrolyzed the two substrates more efficiently than did chymotrypsin C3. Chymotrypsin C1 consisted of chain A (H-Cys-...-Asn-OH, Mr 886) and chain BC (H-Val-...-Lys-OH, Mr 28 200). Chymotrypsin C3 consisted of the two components of C3L and C3S that could be dissociated in the presence of 2.3% SDS. C3L consisted of the chain A and the chain C (H-Ser-...-Lys-OH, Mr 13 600). C3S was the chain B (H-Val-...-Lys-OH, Mr 11 800). These kinetic and chemical analyses show that chymotrypsins C1 and C3 correspond to chymotrypsin A delta and A alpha, respectively.

Transport of proteins into mitochondria: A high conservation of precursor uptake and processing system

Ornithine transcarbamylase (EC 2.1.3.3) of rat (Rattus norvegicus var. albus) liver, a urea cycle enzyme, is synthesized extramitochondrially as a larger precursor which is transported posttranslationally into mitochondria and processed to the mature enzyme. The precursor synthesized in vitro was taken up and processed to the mature enzyme by isolated pigeon (Columba livia var. domestica) liver and frog (Rana catesbeiana) liver mitochondria. Carp (Cyprinus carpio) liver mitochondria could also process the precursor. These results indicate that the mitochondrial transport and processing activities are conserved between mammalian and bird, amphibian or fish systems. However, attempts to demonstrate the precursor uptake and processing by Saccharomyces cerevisiae mitochondria were unsuccessful.

Synthesis and intracellular transport of mitochondrial carbamyl phosphate synthetase I and ornithine transcarbamylase.

Carbamyl phosphate synthetase I (CPS) and ornithine transcarbamylase (OTC), the first two enzymes of urea synthesis, are localized in the liver mitochondrial matrix of ureotelic animals.1 The two enzymes are coded by nuclear genes, synthesized on cytoplasmic 80 S ribosomes, and subsequently transported across the two mitochondrial membranes to the matrix space. Studies in our2,3 and other laboratories4,5 have shown that both enzymes are synthesized in larger precursor forms (pCPS and pOTC) in cell-free protein-synthesizing systems. These precursors form large aggregates and have conformations different from those of the mature enzymes.6 We further showed that pOTC was transported into isolated rat liver mitochondria in association with the processing of pOTC to the mature form of the enzyme.3,7,8 It has been shown that both pCPS9 and pOTC10 are synthesized on membrane-free polysomes, released into the cytosol and then transported rapidly into mitochondria. The present paper describes detailed kinetic studies of the synthesis, processing and intracellular transport of pCPS and pOTC in isolated rat hepatocytes. The paper also deals with the chemical nature of pOTC and its transport into isolated mitochondria in vi vitro.