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.     

Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat liver medium chain acyl coenzyme A dehydrogenase

cDNA encoding the precursor of rat liver medium chain acyl-CoA dehydrogenase (EC 1.3.99.3) was cloned and sequenced. The longest cDNA insert isolated was 1866 bases in length. This cDNA encodes the entire protein of 421-amino acids including a 25-amino acid leader peptide and a 396-amino acid mature polypeptide. The identity of the medium chain acyl-CoA dehydrogenase clone was confirmed by matching the amino acid sequence predicted from the cDNA to the NH2-terminal and nine internal tryptic peptide sequences derived from pure rat liver medium chain acyl-CoA dehydrogenase. The calculated molecular masses of the precursor medium chain acyl-CoA dehydrogenase, the mature medium chain acyl-CoA dehydrogenase, and the leader peptide are 46,600, 43,700, and 2,900 daltons, respectively. The leader peptide contains five basic amino acids and only one acidic amino acid; thus, it is positively charged, overall. Cysteine residues are unevenly distributed in the mature portion of the protein; five of six are found within the NH2-terminal half of the polypeptide. Comparison of medium chain acyl-CoA dehydrogenase sequence to other flavoproteins and enzymes which act on coenzyme A ester substrates did not lead to unambiguous identification of a possible FAD-binding site nor a coenzyme A-binding domain. The sequencing of other homologous acyl-CoA dehydrogenases will be informative in this regard.     

Molecular cloning of cDNA for rat liver catalase

For the studies on the induction of peroxisomal enzymes by hypolipidemic agents, we have tried to isolate a cDNA clone for rat liver catalase. A recombinant clone, pMJ501, was isolated, of which cDNA insert specifically hybridized to catalase mRNA in hybridization-selected translation. On RNA blot hybridization, it hybridized to 2.4-kilobases RNA which was increased about 1.5-fold by the administration of di-(2-ethylhexyl)phthalate to the rats. The nucleotide sequence of the cDNA contains a reading frame for 109 amino acid residues which match the reported amino acid sequence of bovine liver catalase at the carboxyl end with 82% homology. It is concluded that pMJ501 contains a cDNA sequence for rat liver catalase.     

Molecular cloning of cDNA for rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase

Messenger RNA for 3-hydroxyacyl-CoA dehydrogenase, a mitochondrial matrix enzyme of fatty acid beta-oxidation, was purified from livers of di(2-ethylhexyl)phthalate-treated rats by immunoadsorption of hepatic free polysomes to fixed cells of Staphylococcus aureus and enrichment for poly(A)-rich RNA by oligo(dT)-cellulose chromatography. Plasmid cDNA was constructed from this poly(A)-rich RNA by a modification of the method of Okayama and Berg and was transformed into the Escherichia coli DH1 strain. Plasmids containing cDNA sequences coding for 3-hydroxyacyl-CoA dehydrogenase were screened by differential colony hybridization, and were identified by hybrid-arrested translation and hybrid-selected translation. Plasmid pHADH-1, which contains a 1400-base-pair insert, hybridized to rat 3-hydroxyacyl-CoA dehydrogenase mRNA with a length of 1700 bases. Determination of the dehydrogenase mRNA by in vitro translation and dot-blot analysis with the cDNA probe showed that the induction of the enzyme in rat liver by di(2-ethylhexyl)phthalate could be attributed to an increase in the mRNA concentration.     

Molecular cloning of cDNA for rat liver lipoate acetyltransferase a component of pyruvate dehydrogenase complex

One cDNA clone for lipoate acetyltransferase, a component enzyme of pyruvate dehydrogenase complex, was isolated from a rat liver cDNA library prepared in the phage expression vector λgt11 using immunological screening with affinity purified anti-lipoate acetyltransferase antibody. It was identified that cDNA insert in this clone codes for lipoate acetyltransferase by immunoblotting of lysogen carrying the isolated clone. Lipoate acetyltransferase antigenic polypeptide in fusion protein was about 11,000 daltons, agreeing with the size of cDNA insert to be 300 base pairs.     

Molecular cloning, sequencing, and expression of cDNA for rat liver microsomal aldehyde dehydrogenase.

The cDNA clone for rat liver microsomal aldehyde dehydrogenase (msALDH) was isolated and sequenced. The deduced amino acid sequence consisting of 484 amino acid residues revealed that the carboxyl-terminal region of msALDH has a hydrophobic segment, which is probably important for the insertion of this enzyme into the endoplasmic reticulum membrane. COS-1 cells transfected with the expression vector pcD containing the full-length cDNA showed that the active enzyme was expressed and localized mainly on the cytoplasmic surface of the endoplasmic reticulum membranes. It has been proposed that ALDH isozymes form a superfamily consisting of class 1, 2, and 3 ALDHs (Hempel, J., Harper, K., and Lindahl, R., (1989) Biochemistry 28, 1160-1167). Comparison of the amino acid sequence of rat liver msALDH with those of rat other class ALDHs showed that msALDH was 24.2, 24.0, and 65.5% identical to phenobarbital-inducible ALDH (variant class 1), mitochondrial ALDH (class 2), and tumor-associated ALDH (class 3), respectively. Several amino acid residues common to the other known ALDHs, however, were found to be conserved in msALDH. Based on these results, we proposed to classify msALDH as a new type, class 4 ALDH.     

Molecular cloning and expression of rat liver bile acid CoA ligase

Bile acid CoA ligase (BAL) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5'-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa protein with a molecular mass of 75,960 Da, and a 138 nucleotide 3'-nontranslated region followed by a poly(A) tail. Identity of the cDNA was established by: 1) the rBAL-1 open reading frame encoded peptides obtained by chemical sequencing of the purified rBAL protein; 2) expressed rBAL-1 protein comigrated with purified rBAL during SDS-polyacrylamide gel electrophoresis; and 3) rBAL-1 expressed in insect Sf9 cells had enzymatic properties that were comparable to the enzyme isolated from rat liver. Evidence for a relationship between fatty acid and bile acid metabolism is suggested by specific inhibition of rBAL-1 by cis-unsaturated fatty acids and its high homology to a human very long chain fatty acid CoA ligase. In summary, these results indicate that the cDNA for rat liver BAL has been isolated and expression of the rBAL cDNA in insect Sf9 cells results in a catalytically active enzyme capable of utilizing several different bile acids as substrates.     

Molecular cloning of cDNA for argininosuccinate lyase of rat liver

A cDNA expression library constructed from poly(A)+ RNA of rat liver was screened immunologically using an antibody against argininosuccinate lyase (EC 4.3.2.1), a urea cycle enzyme, of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.5 kilobase pairs in length. In the bacterial clone, a specific protein of Mr = about 25,000 was expressed. The argininosuccinate lyase mRNA of about 2.1 kilobases long was detected in the liver and in a lesser amount in the kidney and spleen, but not in the small intestine and heart of the rats.     

Molecular cloning and nucleotide sequence of the cDNA for human liver glutamate dehydrogenase precursor

Two cDNA clones (lambda GDHh1 and lambda GDHn61) for glutamate dehydrogenase (GDH) were isolated from a human liver cDNA library in lambda gt11. The clone, lambda GDHh1, was isolated from the library using a synthetic 45mer oligodeoxy-ribonucleotide, the sequence of which was derived from the known amino acid sequence near the NH2-terminus of human liver GDH. Subsequently, lambda GDHn61 was isolated from the same library using lambda GDHh1 as a probe. The inserts of both clones contained an overlapping cDNA sequence for human liver GDH, consisting of a 5'-untranslated region of 70 bp, an open reading frame of 1677 bp, a 3'-untranslated region of 1262 bp and a 15 base poly(A) tract. The predicted amino acid sequence revealed that the human liver GDH precursor consisted of a total of 558 amino acid residues including the NH2-terminal presequence of 53 amino acids. The sequence deduced for the mature enzyme showed 94% homology to the previously reported amino acid sequence of human liver GDH.     

Molecular cloning of cDNA for rat liver gap junction protein

An affinity-purified antibody directed against the 27-kD protein associated with isolated rat liver gap junctions was produced. Light and electron microscopic immunocytochemistry showed that this antigen was localized specifically to the cytoplasmic surfaces of gap junctions. The antibody was used to select cDNA from a rat liver library in the expression vector lambda gt11. The largest cDNA selected contained 1,494 bp and coded for a protein with a calculated molecular mass of 32,007 daltons. Northern blot analysis indicated that brain, kidney, and stomach express an mRNA with similar size and homology to that expressed in liver, but that heart and lens express differently sized, less homologous mRNA.     

Molecular cloning of a cDNA for the E1 alpha subunit of rat liver branched chain alpha-ketoacid dehydrogenase

We have isolated a cDNA encoding the branched chain alpha-ketoacid dehydrogenase E1 alpha subunit. A rat liver lambda gt11 expression library was screened with antibody reactive with the 2-oxoisovalerate dehydrogenase (lipoamide) component. A positive clone, lambda BZ304, contains a 1.7-kilobase pair cDNA insert with a 1323-base pair open reading frame. Translation of the open reading frame predicts the 24 residues of the previously reported phosphorylation sites 1 and 2 for the bovine kidney and rabbit heart enzymes. The N-terminal sequence of purified E1 alpha was determined, and this sequence was found 40 residues from the beginning of the deduced peptide sequence. Northern blots of rat liver and muscle RNA demonstrate a single mRNA species of approximately 1.8 kilobase pairs in each tissue, suggesting that this cDNA is nearly full length.     

Molecular cloning of a cDNA for rat liver monoamine oxidase B

The cDNA for rat monoamine oxidase B mRNA was isolated from liver cDNA library in lambda gt11 using specific antibody and oligonucleotide probes derived from FAD-containing peptide of the enzyme. The primary structure of the protein, deduced from the nucleotide sequence, consisted of 520 amino acid residues and its molecular weight was calculated to be 58.4 kD which is in good agreement with that of the in vitro-synthesized peptide. FAD-binding site is located in the carboxy-terminal region. There is no typical structural feature common to the targeting signals for mitochondria, the periodic distribution of basic amino acids spaced by several uncharged residues, at its amino-terminal region. This region has an uninterrupted stretch of 14 hydrophobic residues.     

Molecular cloning and expression of rat liver 3 alpha-hydroxysteroid dehydrogenase

Complementary DNA clones encoding 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. The sizes of the cDNA inserts ranged from 1.3-2.3 kilobases. Sequence analysis indicated that variation in the DNA size was due to heterogeneity in the length of 3' noncoding sequences. A full-length cDNA clone of 1286 basepairs contained an open reading frame encoding a protein of 322 amino acids with an estimated mol wt of 37 kDa. When expressed in E. coli, the encoded protein migrated to the same position on sodium dodecyl sulfate-polyacrylamide gels as the enzyme purified from rat liver cytosols. The protein expressed in bacteria was highly active in androsterone reduction in the presence of NAD as cofactor, and this activity was inhibited by indomethacin, a potent inhibitor of 3 alpha HSD. The predicted amino acid sequence of 3 alpha HSD was related to sequences of several other enzymes, including bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase, and frog lens epsilon-crystalline, suggesting that these proteins belong to the same gene family.     

Molecular cloning and characterization of the cDNA encoding the rat liver gamma-butyrobetaine hydroxylase

Carnitine biosynthesis from lysine and methionine involves five enzymatic reactions. γ-butyrobetaine hydroxylase (BBH; EC 1.14.11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of γ-butyrobetaine to carnitine. The cDNA encoding this enzyme has been isolated and characterized. The cDNA contained an open reading frame of 1161 bp encoding a protein of 387 amino acids with a deduced molecular weight of 44.5 kDa. The sequence of the cDNA showed an important homology with the human cDNA recently isolated. Northern analysis showed γ-butyrobetaine hydroxylase expression in the liver and in some extend in the testis and the epididymis. During this study, it also appeared that BBH mRNA expression was undetectable by Northern analysis during the perinatal period. During the development of the rat, the amount of BBH mRNA appeared after the weaning of the young rat and reached a maximal expression at the adult stage.     

The subcellular distribution of acyl CoA: Dihydroxyacetone phosphate acyl transferase in guinea pig liver

Upon differential centrifugation, the enzyme acyl CoA: dihydroxyacetone phosphate acyl transferase (EC 2.3.1.42) in guinea pig liver is shown to sediment in a lysosomal-peroxisomal fraction. Comparison of the distribution of the marker enzymes and of DHAP acyl transferase indicates that the acyl transferase is localized in peroxisomes (microbodies).     

Molecular cloning and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine synthetase.

We previously reported the isolation of a cDNA encoding the liver-specific isozyme of rat S-adenosylmethionine synthetase from a lambda gt11 rat liver cDNA library. Using this cDNA as a probe, we have isolated and sequenced cDNA clones for the rat kidney S-adenosylmethionine synthetase (extrahepatic isoenzyme) from a lambda gt11 rat kidney cDNA library. The complete coding sequence of this enzyme mRNA was obtained from two overlapping cDNA clones. The amino acid sequence deduced from the cDNAs indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,715 Da. The predicted amino acid sequence of this protein shares 85% similarity with that of rat liver S-adenosylmethionine synthetase. This result suggests that kidney and liver isoenzymes may have originated from a common ancestral gene. In addition, comparison of known S-adenosylmethionine synthetase sequences among different species also shows that these proteins have a high degree of similarity. The distribution of kidney- and liver-type S-adenosylmethionine synthetase mRNAs in kidney, liver, brain, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.4-kilobase (kb) mRNA species hybridizable with a probe for kidney S-adenosylmethionine synthetase was found in all tissues examined except for liver, while a 3.4-kb mRNA species hybridizable with a probe for liver S-adenosylmethionine synthetase was only present in the liver. The 3.4-kb kidney-type isozyme mRNA showed the same molecular size as the liver-type isozyme mRNA. Thus, kidney- and liver-type S-adenosylmethionine synthetase isozyme mRNAs were expressed in various tissues with different tissue specificities.     

Molecular cloning of a cDNA for alpha-subunit of rat liver electron transfer flavoprotein

Two cDNA clones for the alpha-subunit of rat liver electron transfer flavoprotein were isolated and their nucleotide sequences were determined. The longer cDNA contained a protein-coding region of 900 nucleotides and 3'-noncoding region of 335 nucleotides. The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the sequence of one of the lysyl endopeptidase-digested peptides from the purified alpha-subunit. The molecular weight of the protein calculated from the protein-coding nucleotides was approx. 3,000 daltons smaller than that of the precursor, suggesting that the cDNA was not of full length. The derived amino acid composition fairly agreed with the chemically determined amino acid composition of the purified alpha-subunit, indicating that the protein-coding region contains most of the mature alpha-subunit.     

Molecular cloning of cDNA for rat acyl-CoA oxidase

Poly(A+) RNA was prepared from hepatic free polysomes of rats which had been fed di(2-ethylhexyl) phthalate for the induction of peroxisomal beta-oxidation enzymes. This preparation was enriched for the mRNAs of these enzymes by sucrose density gradient centrifugation, and used for the synthesis of double-stranded cDNA. Recombinant plasmids were constructed from the cDNA and pBR322 by dG X dC-tailing method and used for the transformation of an Escherichia coli strain, chi 1776. By differential colony hybridization using [32P]cDNA of partially purified liver poly(A+) RNA from induced and noninduced rats as probes, and then by hybridization-selected translation, we obtained two clones with cDNA inserts which specifically selected acyl-CoA oxidase mRNA. On Northern blotting, both cDNA inserts hybridized to 3.8-kilobase RNA which was increased about 10-fold by di(2-ethylhexyl) phthalate treatment of the rats. The cleavage maps of the cDNA inserts showed they overlap with each other. We conclude that the above two recombinant plasmid clones contain cDNA sequences for rat acyl-CoA oxidase.     

Molecular cloning and characterization of cDNA for androgen-repressible rat liver protein, SMP-2

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.     

Molecular cloning of cDNA for rat glycine methyltransferase

Using a highly purified preparation of glycine methyltransferase mRNA, double-stranded cDNA was synthesized and inserted into the PstI site of pBR322. The resulting recombinant DNA was used to transform E. coli X 1776 by conventional methods. Among tetracycline-resistant transformants, a number of colonies were found to contain cDNA sequence for glycine methyltransferase as examined by hybrid-selected translation. A restriction endonuclease cleavage map was constructed covering about 720 base pairs. With the cDNA as the probe, the content of the glycine methyltransferase mRNA was quantitated in various rat tissues and was found to be proportional to the specific enzyme activity.