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In quiescent Swiss 3T3 cells, metabolic flux through phosphoribosyl pyrophosphate into nucleotides increased within 1 h when various growth factors were added (S. Ishijima, K. Kita, N. Kinoshita, T. Ishizuka, N. Suzuki, and M. Tatibana. J. Biochem. 104: 570-575, 1988). The divalent cation ionophore A23187 mimicked the stimulatory effect of epidermal growth factor plus insulin, thereby suggesting involvement of divalent cation mobilization in signaling the stimulation by the growth factors. The stimulation induced by the growth factors was nil in medium devoid of added Mg2+ but was not affected by the omission of Ca2+. The dependency on external Mg2+ was also observed with the stimulations by bombesin plus insulin, fibroblast growth factor, and A23187. In contrast, the mitogenic stimulation of glycolysis was observed irrespective of the presence or absence of Mg2+, indicating that initial events in the signaling process, including mitogen binding to receptors, took place in the absence of exogenous Mg2+. These results suggest that a Mg2(+)-dependent process, subsequent to growth factor binding to receptors, plays an essential role in signaling the early mitogenic stimulation of nucleotide synthesis.

N-Acetyl-L-glutamate synthetase (EC 2.3.1.1) catalyses the synthesis of N-acetyl-L-glutamate, an allosteric activator of carbamoyl-phosphate synthetase I in the liver of ureotelic animals, and the first enzyme is activated specifically by arginine. We have proposed that arginine can stimulate acetylglutamine synthetase in vivo and thereby increase the mitochondrial content of acetylglutamate. The effects of arginine on acetylglutamate synthesis in isolated mitochondria were investigated in detail in the present work. When rat liver mitochondria were isolated and incubated with [14C]glutamate and unlabelled acetate as substrates, acetyl[14C]glutamate synthesis in the mitochondria was more extensive in the presence than in the absence of L-arginine. There was no significant difference between the specific radioactivities of intramitochondrial [14C]glutamate in the presence and absence of arginine. When rat liver mitochondria were incubated with [14C]acetate and unlabelled glutamate as substrates, arginine also stimulated acetyl[14C]glutamate synthesis in the isolated mitochondria. L-Lysine or L-homoarginine, which does not activate acetylglutamate synthetase, had no effect on acetylglutamate synthesis, in the isolated mitochondria. The arginine concentration giving half-maximal synthesis of acetylglutamate in isolated mitochondria was about 50 microM, which is in the range of physiological concentrations of arginine in the liver. As we previously reported [Kawamoto, Ishida, Mori & Tatibana (1982) Eur. J. Biochem. 123, 637-641], the sensitivity of acetylglutamate synthetase to arginine activation undergoes marked changes after food ingestion. The extent of arginine activation of acetylglutamate synthesis in isolated mitochondria correlated well with the sensitivity of acetylglutamate synthetase extracted from the mitochondria to arginine activation. These data lend further support to the idea that arginine itself activates the mitochondrial synthesis of acetylglutamate.

Three peroxisomal enzymes of beta-oxidation from rat liver were synthesized in a cell-free protein-synthesizing system derived from a lysate of rabbit reticulocytes. The in vitro products of acyl-CoA oxidase (EC 1.3.99.3) and a bifunctional protein containing enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities were apparently the same in size and charge as the subunit of the respective mature enzymes; that of 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was about 3,000 Da larger and more basic than its mature subunit. The free polysome fraction of rat liver was 3.1-5.7 times more active than the membrane-bound polysome fraction in the synthesis of the three peroxisomal enzymes; these values were similar to those for cytosolic enzymes and differed from that for serum albumin. In isolated rat hepatocytes, radiolabeled acyl-CoA oxidase and bifunctional protein increased with time with no appreciable change in the subunit size. On the other hand, the labeled putative precursor of 3-ketoacyl-CoA thiolase, as well as the mature form of the enzyme, was detected in the hepatocytes. The radioactivity of the putative precursor reached a plateau in 30 min; that of the mature subunit appeared after a lag time of about 5 min and increased with time up to 90 min. In pulse-chase experiments, the putative precursor disappeared with an apparent half-life of several minutes. When the hepatocytes were fractionated into the cytosolic and the particulate fractions, one half of labeled acyl-CoA oxidase and 60% of the bifunctional protein were recovered in the cytosolic fraction after 10 min of labeling, whereas 70-80% of the labeled enzymes were recovered in the particulate fraction after 40-60 min of labeling. These results indicate that the three enzymes of peroxisomal beta-oxidation are synthesized on free polysomes, released into the cytosol, and then transported into peroxisomes. Our findings also indicate that 3-ketoacyl-CoA thiolase undergoes proteolytic processing during maturation. The temporal sequence of the proteolytic cleavage and intracellular transport of the thiolase remains to be determined.

Messenger RNA of rat ornithine carbamoyltransferase (EC 2.1.3.3), a mitochondrial matrix enzyme, was enriched by immunoprecipitation of rat liver free polysomes, and recombinant plasmids were prepared from the enriched mRNA by a vector-primer method. The cDNA clones for ornithine carbamoyltransferase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pOTC-1, contained a 1.6-kilobase insert and hybridized to a mRNA of approximately equal to 1.8 kilobases in rat liver. The cDNA clone was subjected to nucleotide sequence analysis. The deduced amino acid sequence indicates that the ornithine carbamoyltransferase precursor consists of the mature enzyme of 322 amino acid residues and an NH2-terminal peptide extension (presequence) of 32 amino acid residues. The presequence contains 8 basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The amino acid sequence of the rat ornithine carbamoyltransferase was compared with the recently reported sequence of the human enzyme [Horwich, A. L., Fenton, W. A., Williams, K. R., Kalousek, F., Kraus, J. P., Doolittle, R. F., Konigsberg, W. & Rosenberg, L. E. (1984) Science 224, 1068-1074]. The sequences of the mature enzyme portion are 93% identical, whereas those of the presequences are 69% identical. There are two highly conserved segments in the presequences of the rat and human enzymes. One of the two conserved segments is significantly similar to a segment of the presequence of yeast mitochondrial elongation factor EF-Tu. These results suggest that the homologous segments are important for the proteins that are synthesized in the cytosol to be transported into the mitochondrial matrix.

Carbamoyl-phosphate synthetase (ammonia) (EC 6.3.4.16) and ornithine carbamoyltransferase (EC 2.1.3.3) are matrix enzymes synthesized outside the mitochondria in the form of larger precursors and are transported rapidly into mitochondria, in association with post-translational proteolytic processing to the mature enzymes. Treatment of isolated rat hepatocytes with 40 micrograms/ml of rhodamine 123, a laser dye which specifically stains mitochondria, resulted both in a strong inhibition of the processing of the enzyme precursors and in accumulation. Rhodamine 123 did not specifically inhibit the synthesis of the synthetase and the transcarbamylase precursors in a reticulocyte lysate cell-free system programmed with rat liver free polysomes. The dye strongly inhibited the uptake and processing of the ornithine transcarbamylase precursor by isolated rat liver mitochondria. When the mitochondria and the medium were separated by centrifugation, the unprocessed precursor was recovered almost exclusively in the medium. These results indicate that rhodamine 123 inhibits either the binding of the enzyme precursors to the mitochondria or their transport into the organelle.

N-Acetylglutamate synthetase was purified 33,000-fold to apparent homogeneity from the matrix fraction of rat liver mitochondria. The purification procedure involved treatment of mitochondria with digitonin, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, Affi-Gel blue chromatography, acetylglutaminyl Bio-Gel chromatography, sucrose density gradient centrifugation, and isoelectric focusing. As the enzyme lost activity in contact with a glass surface, glassware coated with silicone was used throughout the purification. Triton X-100 stabilized the enzyme and was included in most buffers at a concentration of 0.1% (w/v). The purified acetylglutamate synthetase has a specific activity of 92.4 mumol min-1 (mg of protein)-1, in the presence of 1 mM L-arginine. The enzyme had a sedimentation coefficient (S20,w) of 8.1 S and its molecular weight was estimated to be about 160,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 57,000, indicating a composition of three subunits of similar size. The purified acetylglutamate synthetase showed a high substrate specificity for L-glutamate and acetyl-CoA, confirming the previous results with a less purified preparation (Shigesada, K., and Tatibana, M. (1978) Eur. J. Biochem. 84, 285-291). Among other 14 acyl-CoAs tested, only propionyl-CoA could substitute for acetyl-CoA, as an acyl donor, with a reaction rate 4.3% of that with acetyl-CoA at 0.5 mM. Among amino acids other than L-glutamate and derivatives or analogues of glutamate tested, the following served as an acyl acceptor: glycine at a rate 2.7% of that with L-glutamate at 1.0 mM, L-glutamine (5.0%), L-glutamate gamma-hydroxamate (15.5%), DL-alpha-aminoadipate (5.2%), and DL-alpha-aminopimelate (4.0%). The purified enzyme is markedly stimulated by L-arginine. The specificity of L-arginine as a low molecular weight activator was strict; only L-argininic acid could activate the enzyme to a lesser extent. Cationic polypeptides, such as protamine, polyarginine and polylysine, also activated the synthetase. The effect was additive to that of arginine.

The synthesis and intracellular transport of the mitochondrial matrix enzymes ornithine transcarbamylase (carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3.) and carbamoyl-phosphate synthetase (ammonia) I [carbon-dioxide:ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] were studied in isolated rat hepatocytes. In pulse experiments at 37 degrees C, the larger precursors of the two enzymes appeared in the cytosol of the liver cells, where radioactivity levels of the precursors reached a plateau in 10-20 min after the pulse. The pulse-labeled mature enzymes appeared in the particulate fraction (containing mitochondria) after a time lag and increased almost linearly with time up to 40 min. The specific radioactivities of the precursors in the cytosol were much higher than those of the mature enzymes in the particulate fraction. In pulse--chase experiments, the labeled precursors disappeared from the cytosol with estimated half-lives of about 1-2 min. These results indicate that ornithine transcarbamylase and carbamoyl-phosphate synthetase I are initially synthesized as larger precursors and exist in a cytosolic pool from which they are transported into mitochondria and processed there to the mature enzymes concomitantly with or immediately after transport. Although the rates of synthesis, transport, and processing were decreased about 3-fold at 25 degrees C (as compared to incubation at 37 degrees C), the pool size of the precursors in the cytosol were somewhat larger at this temperature.

The precursor of rat liver ornithine transcarbamylase (ornithine carbamoyltransferase; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) (pre-ornithine transcarbamylase), which was synthesized in a reticulocyte lysate cell-free system, was converted to an apparently mature form of the enzyme by isolated rat liver mitochondria. The proteolytic processing involved two steps: (i) conversion of pre-ornithine transcarbamylase (39,400 daltons) to a product of about 37,000 daltons and (ii) further conversion to the apparently mature form of the enzyme (36,00 daltons). When mitochondria were subfractionated by digitonin treatment followed by sonication of a mitoplast fraction, the proteolytic activity catalyzing the first step was recovered mainly in a matrix fraction. Some activity was found in an intermembrane space fraction. The enzyme activity in the matrix fraction has an optimal pH at about 7.5. The activity was inhibited almost completely by 2 mM leupeptin and partly by 2 mM antipain but not significantly by other microbial protease inhibitors or serine protease inhibitors. It was inhibited strongly by 2 mM EDTA, 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate, 2 mM p-chloromercuriphenylsulfonate, and 2 mM Hg(CH3COO)2 but not by N-ethylmaleimide or iodoacetamide. These results suggest that pre-ornithine transcarbamylase is first transported into the mitochondrial matrix and converted there to the mature form of the enzyme by a novel neutral protease(s).

The rat Yoshida sarcoma (YS) cDNA library was screened using oligonucleotide probes designed from peptide sequences of rat phosphoribosylpyrophosphate synthetase, and two distinct clones were obtained. Nucleotide sequencing revealed that both clones encode 317 amino acids containing the peptide sequences. The deduced amino acid sequences of the two differ only by 13 residues (96%) identity), whereas the nucleotide sequences are relatively divergent (81% identity in the coding regions). These results, together with N-terminal amino acid sequencing data, suggest the existence of two different subunits of this enzyme, designated as PRS I and II (their genes as PRPS1 and PRPS2). Transcripts of 2.3 and 3.7 kilobases were detected in YS cells and rat liver by Northern blot analysis, using PRS I and II cDNAs respectively, as probes. In the liver, the expression of both genes increased after partial hepatectomy.

A potent nucleoside triphosphate hydrolase (EC 3.6.1.3) with a number of unusual properties has been found in the parasitic protozoan (Toxoplasma gondii) and has been purified to homogeneity. The enzyme is localized in the cytosol and constitutes 3-4% of the total cytosolic protein. It has a molecular weight of 240,000-260,000 and contains four equivalent subunits of Mr = 63,000. Dithiol compounds such as dithiothreitol, dithioerythritol, or dimercaptopropanol were essential activators of the enzyme. Monothiol compounds had no effect. The specific activity of the purified enzyme was 2,500 mumol/min/mg at 37 degrees C under optimal conditions. Magnesium was the most effective activating metal ion, although manganese and calcium were also active. A higher excess of magnesium over total ATP was essential for maximal activity. Anions were found to inhibit the enzyme activity in an almost chaotropic order. The enzyme demonstrated a wide substrate specificity for both ribo- and deoxyribonucleoside triphosphate and hydrolyzed these nucleotides at almost the same rate. ADP was also a substrate and was hydrolyzed at a rate of 18% of that for ATP. Slight activity was seen with inorganic tri- and tetrapolyphosphates but not with monophosphate compounds. Km values for MgATP2- and MgADP- were 0.12 +/- 0.01 mM and 0.70 +/- 0.06 mM, respectively.

The precursor of rat liver ornithine carbamoyltransferase (EC 2.1.3.3) synthesized in vitro was taken up and processed to the mature enzyme by isolated rat liver mitochondria. Potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s), in addition to the precursor and the mitochondria, were required for maximal transport and processing of the precursor. The concentrations of potassium and magnesium ions required for maximal transport and processing were about 120 and 0.8-1.6 mM, respectively. Dialyzed postribosomal supernatant of rabbit reticulocyte lysate (36 mg of protein/ml), in combination with potassium and magnesium ions, stimulated the transport and processing severalfold. The stimulatory activity of the dialyzed lysate was inactivated by trypsin treatment or heating at 100 degrees C for 2 min. No significant amount of the precursor was associated with the mitochondria when incubation was performed in the absence of these components. These results suggest that potassium ion, magnesium ion, and a reticulocyte cytosolic protein(s) stimulate the binding and transport of the ornithine carbamoyltransferase precursor to the mitochondria. Dialyzed supernatant of rabbit erythrocyte lysate was equally effective in stimulating the precursor transport and processing, and a dialyzed cytosol fraction of Ehrlich ascites cells was partly stimulatory. On the other hand, dialyzed cytosol fractions of rat liver and rat kidney, and dialyzed supernatant of wheat germ extracts did not stimulate the precursor transport and processing but rather inhibited it.