Cloning and sequence analysis of the human liver rhodanese: comparison with the bovine and chicken enzymes

The cDNA for the human rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), a nuclearly encoded protein of the mitochondrial matrix, was isolated from a human fetal liver cDNA library. Nucleotide sequence revealed an open reading frame coding for a polypeptide of 295 amino acids, which presented a 57% and 58% identity with the bovine and avian rhodanese, respectively. The analysis of the 5'-ends of the coding region gave no evidence for the presence of a cleavable signal sequence as found in other mitochondrial proteins. A comparison with two available amino acid sequences (cow and chicken) showed that sequence similarity is not restricted to the alpha-helices and beta-structures motifs which are remarkably superimposable in the two halves of bovine rhodanese, but extends to adjacent regions.     

A cDNA clone for the precursor of rat mitochondrial ornithine transcarbamylase: comparison of rat and human leader sequences and conservation of catalytic sites.

We have cloned a DNA complementary to the messenger RNA encoding the precursor of ornithine transcarbamylase from rat liver. This complementary DNA contains the entire protein coding region of 1062 nucleotides and 86 nucleotides of 5'- and 298 nucleotides of 3'-untranslated sequences. The predicted amino acid sequence has been confirmed by extensive protein sequence data. The mature rat enzyme contains the same number of amino acid residues (322) as the human enzyme and their amino acid sequences are 93% homologous. The rat and human amino-terminal leader sequences of 32 amino acids, on the other hand, are only 69% homologous. The rat leader contains no acidic and seven basic residues compared to four basic residues found in the human leader. There is complete sequence homology (residues 58-62) among the ornithine and aspartate transcarbamylases from E. coli and the rat and human ornithine transcarbamylases at the carbamyl phosphate binding site. Finally, a cysteine containing hexapeptide (residues 268-273), the putative ornithine binding site in Streptococcus faecalis, Streptococcus faecium, and bovine transcarbamylases, is completely conserved among the two E. coli and the two mammalian transcarbamylases.     

Rhodanese as a thioredoxin oxidase

A major catalytic difference between the two most common isoforms of bovine liver mitochondrial rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been observed. Both isoforms were shown to be capable of using reduced thioredoxin as a sulfur-acceptor substrate. However, only the less negative form in common with the recombinant mammalian rhodanese expressed in E. coli, can also catalyze the direct oxidation of reduced thioredoxin evidently by reactive oxygen species. These activities are understood in terms of the established persulfide structure (R-S-SH) of the covalently substituted rhodanese in the sulfurtransferase reaction and an analogous sulfenic acid structure (R-S-OH) when the enzyme acts as a thioredoxin oxidase. The observations suggest a role for one rhodanese isoform in the detoxication of intramitochondrial oxygen free radicals.     

On the size and chemical nature of the polypeptide chain of bovine liver rhodanese.

Evidence from molecular weight studies and sequence analysis of bovine liver rhodanese indicates that the enzyme is a single polypeptide of molecular weight 35,200, and not a dimer of identical subunits half this size. The rhodanese molecule contains 317 amino acids including 5 methionines, 4 cysteines, and 5 tryptophans. As expected, six fragments were produced by cleavage with cyanogen bromide and these have been aligned in the enzyme with the aid of overlapping tryptic peptides isolated from a [¹⁴C] carboxymethylmethionyl rhodanese derivative. The cyanogen bromide fragments account for all of the amino acid residues of the parent rhodanese molecule. Methionine residues are located at positions 72, 112, 214, 217, and 235 in the polypeptide chain and the active site cysteine is at position 251, in the carboxyl-terminal segment of the molecule.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase.

The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.     

Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli 1,4-alpha-D-glucan:1,4-alpha-D-glucan 6-alpha-D-(1, 4-alpha-D-glucano)-transferase as deduced from the nucleotide sequence of the glg B gene

The nucleotide sequence of the glg B gene, coding for branching enzyme (EC 2.4.1.18), was elucidated. It consists of 2181 base pairs specifying a protein of 727 amino acids. The deduced amino acid sequence was consistent with the amino acid analysis that was obtained with the pure protein as well as with the molecular weight determined from sodium dodecyl sulfate-gel electrophoresis. The deduced amino acid sequence was also consistent with the amino-terminal amino acid sequence and the amino acid sequence analysis of various peptides obtained from CNBr degradation of purified branching enzyme.     

The amino acid sequence of goat alpha-lactalbumin

The amino acid sequence of goat α-lactalbumin has been established from the structures of peptides isolated from trypsin and thermolysin digests of the reduced aminoethylated protein and from a chymotrypsin digest of the reduced carboxamidomethylated protein. The amino-terminal sequence was confirmed by automatic sequencer analysis. Of the previously sequenced species variants of α-lactalbumin, the bovine protein is most similar to the goat, differing in only 12 amino acid substitutions. One difference between these proteins corresponds to a substitution found in the bovine A genetic variant (Arg¹⁰ → Gln). The relevance of the structure to the evolutionary relationships in the α-lactalbumin-lysozyme family of proteins is discussed.     

Immunological evidence for a conformational difference between recombinant bovine rhodanese and rhodanese purified from bovine liver

Rhodanese has been utilized as a model enzyme for the study of protein structure-function relationships. The enzyme has recently been cloned and the recombinant enzyme is now available for investigation. However, prior to use in structure-function studies, the recombinant enzyme must be shown to have the same structure and activity as the bovine liver enzyme used in the previous studies. An immunological study of the conformations of these enzyme conformers is described. Three antibodies (two monoclonal and one polyclonal, site-directed antibody) were shown to detect distinct and nonoverlapping epitopes. The epitopes of the monoclonal antirhodanese antibodies (R207 and MAB11) were mapped to the same CNBr digest fragment of the amino terminal domain of rhodanese, and the epitope of the site-directed antibody prepared against the interdomain tether sequence of rhodanese (PAT-T1) was mapped to that region of rhodanese (residues 142–156). The rhodanese conformers were studied by monitoring the accessibility of the epitopes recognized by each antibody in each conformer using an indirect ELISA. None of the antibodies could detect its epitope on the purified liver enzyme. Two of the antibodies (R207 and PAT-T1) could also not detect their epitopes on the recombinant enzyme. However, MAB11 did detect a conformational difference between the natural and recombinant rhodanese conformers, indicating the conformational difference is localized in the first 73 amino acids of rhodanese. This difference presumably reflects the difference in the histories of the two enzymes and may be due to differences in enzyme folding, differences in the purification procedures, and differences in storage conditions—all of which could influence the final conformation of the enzyme.     

Amino-terminal amino acid sequence of the nonspecific phospholipid exchange protein from bovine liver

The amino-terminal amino acid sequence of the nonspecific phospholipid exchange protein from bovine liver has been determined. The first 52 amino-terminal residues in the sequence were identified. The sequence determined failed to show statistically significant homology to any previously published protein sequence. However, a stretch of 12 amino acids at the end of the sequence displays homology to the phosphatidylcholine-specific phospholipid exchange protein.     

Molecular cloning of a ubiquitously distributed microtubule-associated protein with Mr 190,000

A heat-stable microtubule-associated protein (MAP) with apparent molecular weight of 190,000 is a major non-neural MAP which distributes ubiquitously among bovine tissues (termed here MAP-U). Previously we reported that microtubule-binding chymotryptic fragments of MAP-U and tau contain a common assembly-promoting (AP) sequence of 22 amino acid residues (Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1989) J. Biol. Chem. 264, 5885-5890). We isolated cDNA clones for MAP-U containing the whole coding sequence. Northern blot analysis revealed that a major species of MAP-U mRNA is 5 kilobases in length and is expressed ubiquitously among bovine tissues. Nucleotide sequence analysis revealed the complete amino acid sequence of MAP-U which consists of 1,072 amino acid residues. Analysis of the deduced amino acid sequence of MAP-U indicated that this molecule is clearly divided into two domains in terms of electrostatic charge distribution: an amino-terminal acidic domain (residues 1-640) and a carboxyl-terminal basic domain (residues 641-1072). The amino-terminal domain of MAP-U shows no significant sequence homology with other known protein sequences including neural MAPs, tau, and MAP-2. The amino-terminal domain of MAP-U contains unique 18 1/2 repeats of 14-amino acid motif which have not been observed in other MAPs. The carboxyl-terminal domain of MAP-U is further divided into three regions: a Pro-rich region (residues 641-880), an AP sequence region (residues 881-1003), and a short hydrophobic tail (residues 1004-1072). The Pro-rich region is mainly composed of five species of amino acid residues, Pro, Ala, Lys, Ser, and Thr. The AP sequence region contains four tandem repeats of AP sequences, and thus, this region is considered to play a leading role in the interaction of MAP-U with microtubules.     

Proteolytic interconversion of electrophoretic variants of the enzyme rhodanese

It has been confirmed that the enzyme rhodanese, although a homogeneous single polypeptide chain protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is separable by electrophoresis under nondenaturing conditions into four species which differ in net surface charge (I-IV in the order of increasing positive charge). Limited proteolysis can interconvert these species. Chymotrypsin converts IV and III to II and forms a small amount of I. Carboxypeptidase B converts IV to III. The total protein among the species remains constant, and two-dimensional gels show that the change induced is below the resolution of the sodium dodecyl sulfate-polyacrylamide gel system. The suggestion that the products can be produced in the order IV, III, and II is supported by the results of sequential treatment of rhodanese first with carboxypeptidase B and then with chymotrypsin. It is concluded that there are covalent differences among the rhodanese species identified to date and an interconversion of forms can be triggered by proteolysis at the COOH-terminal end of the Mr = 33,000 single polypeptide chain which comprises the enzyme. This conclusion is strengthened by the close similarity between the amino acid composition of the peptide released by chymotrypsin and the composition expected on the basis of the known sequence. Furthermore, it appears that form IV is the primary in vivo product and the other species arise from it.     

Recombinant bovine dihydrofolate reductase produced by mutagenesis and nested PCR of murine dihydrofolate reductase cDNA

Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quantities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.     

Isolation of bovine liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase cDNA: bovine liver and heart forms of the enzyme are separate gene products.

In order to ascertain whether the heart and liver forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were products of two different genes or arose via alternative splicing of a single gene, the bovine liver cDNA of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a lambda gt10 phage library and its sequence compared with that of bovine heart cDNA. The deduced amino acid sequence of the bovine liver cDNA was also compared with the amino acid sequence of the human and rat liver phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme. The bovine liver cDNA codes for a protein that has 81.6% amino acid identity with the bovine heart form and 97.0 and 98.3% identity with the rat and human liver forms of the enzyme, respectively. Comparison of the nucleotide sequences of the two bovine cDNAs and their deduced amino acid sequences demonstrates that while there is conservation of the active sites of liver/muscle and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases they are encoded by different genes.     

Rat gastric prepepsinogen: in vitro synthesis and partial amino-terminal signal sequence

The major translation product of rat gastric mucosa RNA in a wheat germ cell-free system was identified as prepepsinogen by electrophoretic analysis of its immunoprecipitate on sodium dodecyl sulfate (SDS)-polyacrylamide gels and amino-terminal sequence determination. The translation product containing radioactive amino acids, purified by SDS-polyacrylamide gel electrophoresis, was shown to have an amino-terminal extension peptide comprising 16 amino acid residues. A partial amino acid sequence of this extension peptide is as follows: Met-X-X-Met-Val-Val-X-Leu-Leu-X-Leu-X-Leu-Leu-X-X-pepsinogen.     

Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis

The 5' regulatory region and the portion of the structural gene coding for the amino-terminal sequence of alkaline phosphatase I (APase I) were isolated from Bacillus licheniformis MC14 using a synthetic oligodeoxynucleotide deduced from the amino acid sequence of the enzyme. The DNA sequence analysis of this region revealed an open reading frame of 129 amino acids containing the amino-terminal sequence of the mature APase protein. The protein sequence was preceded by a putative signal sequence of 32 amino acid residues. The predicted amino acid sequence of the partial APase clone as well as the experimentally determined amino acid sequence of the enzyme indicated that B. licheniformis APase retains the important features conserved among other APases of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and various human tissues. Heterologous expression studies of the promoter using a fusion with the lacZ gene indicated that it functions as a very strong inducible promoter in B. subtilis that is tightly regulated by phosphate concentration.     

Isolation and characterization of cDNA clones encoding the murine NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase

Forty cDNA clones corresponding to the bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase enzyme were isolated from a mouse lambda gt11 library. Two classes of cDNA clones were shown by Northern analysis to correspond to the two mRNA species of 1.7 and 2.0 kilobases present in transformed cells but not in normal tissues and that apparently are derived from alternate polyadenylation signals. The 1050-base pair coding region encodes a protein of 350 amino acids which contains a putative mitochondrial-targeting signal peptide of 34 amino acids following the initiator methionine. The 20 amino acids immediately following the signal peptide correspond exactly to those determined by sequence analysis of the amino terminus of the purified protein. The derived amino acid sequence of the NAD-dependent dehydrogenase-cyclohydrolase shows extensive homology with the corresponding amino-terminal sequence of the trifunctional NADP-dependent dehydrogenase-cyclohydrolase-synthetase enzyme from human cells (approximately 40%), yeast cytosol (approximately 36%), and yeast mitochondria (approximately 45%).     

Cloning and characterization of the yeast nuclear gene for subunit 5 of cytochrome oxidase

The nuclear gene COX5 coding for subunit 5 of cytochrome oxidase has been cloned by transformation of the cox5-1 mutant aE4-238/AL1 with a library of yeast genomic DNA. The recombinant plasmid pG46/ST2 bearing a nuclear DNA insert of 1.17 kilobase pairs restores the ability of cox5 mutants to respire and to synthesize a wild type subunit 5. The COX5 gene has been sequenced and determined to code for a 153-amino acid long protein with a molecular weight of 17,121. The amino-terminal 20 residues comprise the signal peptide. The sequence starting from residue 21 matches the partial sequence reported for the mature subunit 5. The sequence of the subunit 5 gene indicates that the mature protein has a molecular weight of 14,858 which agrees with previous size estimates based on electrophoretic migration. The primary sequence and polarity profile of yeast subunit 5 establishes that it is homologous to subunit 4 of bovine cytochrome oxidase.