Cloning and expression in Escherichia coli of cDNA for serine: pyruvate aminotransferase of rat liver

Cloned cDNAs for rat liver serine: pyruvate aminotransferase were obtained by screening of a cDNA expression bank of rat liver with an antibody against the enzyme. Nineteen clones were isolated from 33 000 transformants and most of them had common fragments of cDNA on analysis by digestion with some restriction enzymes. These clones were identified as those containing cDNA for serine:pyruvate aminotransferase by the following criteria. (a) At the nucleic acid level, a 500-base-pair fragment of cDNA prepared by digestion of cDNAs with EcoRI and PstI hybridized with the mRNA coding for serine:pyruvate aminotransferase as judged by hybrid-selected and hybrid-arrested translations. (b) Specific proteins were detected in nine bacterial clones, a 40-kDa protein in one clone and a 39-kDa protein in eight clones. Among them only the 40-kDa protein was found to be solubilized from the cell by sonication, and this protein was immunoprecipitated with an antibody against serine:pyruvate aminotransferase of rat liver. (c) High activity of serine:pyruvate aminotransferase was expressed both in whole cell suspension and sonicated extract prepared from the transformant producing the 40-kDa protein, and 99% of the activity was immunoreactive with the antibody. Two types of mRNA for serine:pyruvate aminotransferase were detected on the RNA blot analysis by using cloned cDNA fragment as a probe. The larger mRNA (approximately 1600 nucleotides) was glucagon-inducible while the smaller one (approximately 1500 nucleotides) was not affected by the hormone.     

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.     

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

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.     

Induction of mitochondrial serine:pyruvate aminotransferase of rat liver by glucagon and insulin through different mechanisms

Studies were performed in the rat liver to examine whether or not insulin as well as glucagon causes the induction of mitochondrial serine:pyruvate aminotransferase (SPTm) [EC 2.6.1.51] and if so, whether the mechanisms of induction are similar or different for the two hormones. Not only glucagon but also insulin induced SPTm. Cell-free translation assaying and RNA blot analysis showed that both hormones cause an increase in the hepatic level of mRNA for the precursor of SPTm. Their effects were virtually additive, and the time course of the increase in the mRNA level differed between the hormones. The maximal increase induced by glucagon was observed 3.5 h after the hormone injection while that by insulin was found after 6 h. The increase in the mRNA due to insulin was completely inhibited by the co-administration of cycloheximide, while that due to glucagon was not. The finding suggests that a newly synthesized, insulin-dependent protein(s) is involved in the regulation of the mRNA level by insulin. On the other hand, hydrocortisone treatment selectively suppressed the increase in the mRNA due to glucagon. These data indicate that the synthesis of the mRNA for SPTm is regulated by glucagon and insulin through different mechanisms. The size of the hormone-induced mRNA for SPTm gradually decreased with time, but the cell-free translation products did not exhibit size alteration. RNase H digestion to remove the poly(A) tail of the mRNA indicated that shortening of the poly(A) sequence might be responsible for the time-dependent size alteration of the mRNA.     

Uptake and processing of serine: pyruvate aminotransferase precursor by rat liver mitochondria in vitro and in vivo

Processing and uptake of the precursor of serine: pyruvate aminotransferase [EC 2.6.1.51] by mitochondria were studied in vitro and in vivo. Serine: pyruvate aminotransferase was synthesized mainly on free ribosomes as judged by immunoprecipitation of puromycin-labeled nascent peptides prepared from free and bound ribosomes. The precursor of rat liver serine:pyruvate aminotransferase (pSPT) synthesized in vitro was post-translationally processed to an apparently mature form by isolated rat liver mitochondria. Available evidence indicated that the processed product was localized in the matrix of mitochondria. Mature serine:pyruvate aminotransferase did not inhibit the in vitro processing, suggesting that the extra peptide was necessary for the mitochondrial uptake of the precursor. In the livers of rats fed a vitamin B6-deficient high-protein diet, the induction by glucagon of serine:pyruvate aminotransferase occurred and most of the induced enzyme existed in mitochondria as the apo-form, suggesting that pSPT was taken up by mitochondria and processed in the apo-form under the conditions employed. In the in vitro system, on the other hand, the processing of pSPT proceeded both in the absence and presence of pyridoxal 5'-phosphate. Should the precursor also bind the prosthetic molecule, therefore, it would be transported into mitochondria in both the apo- and holo-forms. When isolated rat hepatocytes were labeled with [35S]methionine, labeled pSPT appeared in the cytosolic fraction and was transported rapidly into mitochondria in association with the processing. This uptake and processing were inhibited by a fluorescent laser dye, rhodamine 123, and the precursor accumulated in the cytosol in the presence of the dye.     

Nucleotide sequence of rat liver tyrosine aminotransferase gene fragment

The sequence of a 3677 nucleotide EcoRI fragment was determined that codes for part of the rat liver tyrosine aminotransferase gene. The sequence was compared with the previously determined cDNA sequence and the intron and exon boundaries were deduced.     

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.     

Nucleotide sequence of a human heart cDNA encoding the mitochondrial phosphate carrier.

We have isolated and characterized a full length cDNA clone encoding the precursor of the human heart mitochondrial phosphate carrier protein. The entire clone is 1330 bp in length with 5'- and 3'-untranslated regions of 48 and 184 bp, respectively. The open reading frame encodes the mature protein consisting of 312 amino acids, preceded by a presequence of 49 amino acids. The amino acid sequence of the mature human phosphate carrier is 93.6, 94.2 and 33.6% identical to that of the phosphate carrier from beef, rat and yeast, respectively. Like other mitochondrial transport proteins, the human phosphate carrier has a tripartite structure. Each of the three repeats contains two hydrophobic regions which presumably span the membrane in the form of alpha-helices.     

Nucleotide sequence of a cDNA coding for mitochondrial fumarase from human liver

The nucleotide sequence of a 1.46 kb cDNA, selected from a human liver library by the expression of fumarase antigenic determinants, was determined using the dideoxy chain termination method. The cDNA contained an open reading frame extending from the extreme 5-base and coding for a protein with 468 amino acids. This protein, with the exception of an N-terminal methionine, was identified as mitochondrial fumarase. The protein showed a high degree of identity of structure with the fumarase fromBacillus subtilis (56.6 %) and a fumarase fromEscherichia coli (product of thefumC gene, 59.3 %), and a lower degree of identity with the aspartase ofE. coli (37.2 %).     

Purification and characterization of rat liver mitochondrial phenylalanine pyruvate aminotransferase.

Phenylalanine pyruvate aminotransferase in rat liver was found in both the mitochondrial and supernatant fractions. Phenylalanine pyruvate aminotransferase was purified from rat liver mitochondria. The purified enzyme was specific for pyruvate, exhibiting no activity with 2-oxoglutarate as aminoacceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order of activity: L-phenylalanine > L-tyrosine > L-histidine > 3,4-dihydroxy-DL-phenylalanine. Very little activity was observed with L-tryptophan and 5-hydroxy-L-tryptophan. The apparent Km values for L-phenylalanine and L-histidine were 2.6 mM and 2.7 mM, respectively. The Km values for pyruvate were 5.0 mM and 1.5 mM with phenylalanine and histidine as amino donors, respectively. The pH optimum was near 9.0. Sucrose density gradient centrifugation gave a molecular weight of approximately 68,000. On the basis of subcellular distributions, substrate specificities, substrate inhibition, pH optima, polyacrylamide gel electrophoresis and some other properties, it was suggested that mitochondrial phenylalanine pyruvate aminotransferase was identical with mitochondrial histidine pyruvate aminotransferase.     

Nucleotide sequence of cDNA encoding the mitochondrial precursor protein of the ATPase inhibitor from the giant panda (Ailuropoda melanoleuca)

Mitochondrial ATP synthase (F1Fo-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, using RT-PCR combined with in silico cloning, we isolated and sequenced the cDNA encoding the inhibitor protein of the giant panda (Ailuropoda melanoleuca). The deduced protein sequence showed that the protein is composed of 106 amino acids and the estimated molecular weight of the ATPIF(1) protein is 12.32 kDa with an isoelectric point (pI) of 10.17. Alignment analysis revealed that the deduced protein sequence shares 66%, 78.3%, 66%, 72.6%, 77.4%, and 78.3% homology with that of Mus musculus, Pan troglodytes, Rattus norvegicus, Bos taurus, Macaca mulatta, and Homo sapiens, respectively. Topology prediction showed that there are three protein kinase C phosphorylation sites, one amidation site, three N-myristoylation sites, one casein kinase II phosphorylation site, and one tyrosine kinase phosphorylation site in the ATPase inhibitor. In particular, amino acids in the region between 39 and 72, which is the minimum sequence showing ATPase inhibitory activity, were highly conserved in the protein.