Protein structure of pig liver 4-aminobutyrate aminotransferase and comparison with a cDNA-deduced sequence.

The amino acid sequence of pig liver 4-aminobutyrate aminotransferase has been determined by gas-phase sequencing of proteolytically derived peptide fragments. The sequence differs substantially from that predicted for the same enzyme on the basis of the sequence of cDNA derived from pig brain in recently published work [Kwon, O., Park, J. & Churchich, J. E. (1992) J. Biol. Chem. 267, 7215-7216]. Apart from a few minor differences, the two sequences are completely different in the segment of protein comprising the 36 residues at positions 107-142. Insertion of a cytosine between bases 402 and 403 in the cDNA sequence, together with deletion of the guanine at position 510, results in a DNA sequence which predicts exactly the amino acid sequence determined by peptide analysis in the present work. The mammalian enzyme has approximately 44% sequence identity with the same enzyme from two unicellular eukaryotes (Saccharomyces cerevisiae, Aspergillus nidulans) and 22% identity with that from Escherichia coli.     

Isolation and sequence of a cDNA encoding mouse IMP dehydrogenase.

Inosinic acid (IMP) dehydrogenase (IMPD) catalyzes the conversion of IMP to XMP as the first committed step in GMP biosynthesis de novo. We have isolated a cDNA containing the complete coding region of mouse IMPD by its ability to complement a bacterial mutant lacking IMPD activity. Two independent cDNA clones were isolated by complementation, of which the longest was 1.7 kb in length. Northern analyses, using the IMPD cDNA as a probe, indicated that mature IMPD mRNA was a single species approx. 2.0 kb in size. Mouse IMPD is almost identical to Chinese hamster and human IMPDs and is highly conserved between Escherichia coli and mouse, with a direct amino acid (aa) identity of 39%, which increases to 60% if conserved aa are considered. The leader region of our longest cDNA clone is G + C-rich and contains two tandem copies of a G + C-rich direct repeat.     

Isolation and sequence analysis of a cDNA encoding rat liver alpha-L-fucosidase.

cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5' untranslated sequence, 78 nucleotide residues of 3' untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].     

Rotational dynamics of 4-aminobutyrate aminotransferase

The fluorescence dye 1-anilinonaphthalene-8-sulfonate (ANS) was used as a probe of non-polar binding sites in 4-aminobutyrate aminotransferase. ANS binds to a single binding site of the dimeric protein with a K_d of 6 μM. Nanosecond emission anisotropy measurements were performed on the ANS-enzyme in an effort to detect independent rotation of the subunits in the native enzyme. The observed rotational correlation time (φ = 65 ns) corresponds to the rotation of a rather rigid dimeric structure. The microenvironment surrounding the natural probe pyridoxal-5-P covalently bound to the dimeric structure was explored using ³¹P-NMR at 72.86 MHz. In the native enzyme, the pyridoxal-5-P ³¹P-chemical shift is pH-independent, indicating that the phosphate group is well protected from the solvent. The correlation time determined from the ³¹P-spectrum of the aminotransferase exceeds the value calculated for the hydrated spherical model (φ = 40 ns). It is concluded that the phosphate of the pyridoxal-5-P molecule is rigidly bound to the active site of 4-aminobutyrate aminotransferase.     

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.     

Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliaceum, an NAD-malic-enzyme-type C4 plant and screened the library for AspAT isozymes. A full-length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondrial AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAT are synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28-amino-acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P. miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.     

Isolation and characterization of a soybean cDNA clone encoding the plastid form of aspartate aminotransferase

Five aspartate aminotransferase (EC 2.6.1.1; AAT) isozymes were identified in soybean seedling extracts and designated AAT1 to AAT5 based on their rate of migration on non-denaturing electrophoretic gels. AAT1 was detected only in extracts of cotyledons from dark-grown seedlings. AAT3 and AAT4 were detected in crude extracts of leaves and in cotyledons of seedlings grown in the light. AAT2 and AAT5 were detected in all tissues examined. A soybean leaf cDNA clone, pSAT17, was identified by hybridization to a carrot AAT cDNA clone at low stringency. pSAT17 had an open reading frame which could encode a 50 581 Da protein. Alignment of the deduced amino acid sequence from the pSAT17 open reading frame with mature AAT protein sequences from rat disclosed a 60 amino acid N-terminal extension in the pSAT17 protein. This extension had characteristics of a plastid transit peptide.A plasmid, pEXAT17, was constructed which encoded the mature protein lacking the putative chloroplast transit polypeptide. Transformed Escherichia coli expressed a functional soybean AAT isozyme, which comigrated with the soybean AAT5 isozyme during agarose gel electrophoresis. Differential sucrose gradient sedimentation of soybean extracts indicated that AAT5 specifically cofractionated with chloroplasts. Antibodies raised against the pEXAT17-encoded AAT protein specifically reacted with the AAT5 isozyme of soybean and not with any of the other isozymes, indicating that the soybean cDNA clone, pSAT17, encodes the chloroplast isozyme, AAT5.     

Isolation and nucleotide sequence of a cDNA clone encoding rat mitochondrial malate dehydrogenase.

We have determined the complete sequence of the rat mitochondrial malate dehydrogenase (mMDH) precursor derived from nucleotide sequence of the cDNA. A single synthetic oligodeoxynucleotide probe was used to screen a rat atrial cDNA library constructed in lambda gt10. A 1.2 kb full-length cDNA clone provided the first complete amino acid sequence of pre-mMDH. The 1014 nucleotide-long open reading frame encodes the 314 residue long mature mMDH protein and a 24 amino acid NH2-terminal extension which directs mitochondrial import and is cleaved from the precursor after import to generate mature mMDH. The amino acid composition of the transit peptide is polar and basic. The pre-mMDH transit peptide shows marked homology with those of two other enzymes targeted to the rat mitochondrial matrix.     

Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human alpha 2-macroglobulin has indicated regions of pronounced sequence similarity. Examination of cytoplasmic RNA prepared from human liver and the human hepatoma cell line Hep G2 by Northern transfer has indicated a C5 mRNA species of about 5.2 kilobase pairs.     

Cloning and sequence of a functionally active cDNA encoding the mouse ubiquitin-activating enzyme E1.

A cDNA encoding the ubiquitin-activating enzyme, E1, was isolated from the mouse mammary carcinoma cell line, FM3A, and shown to complement mutant mouse cells deficient in the enzyme. The 3495-bp cDNA encodes 1058 amino acids (aa), and shares extensive homology with the human E1 enzyme at both the nucleotide and aa sequence levels.     

The reaction of ethanolamine O-sulphate with 4-aminobutyrate aminotransferase.

Homogeneous rat alpha-lactalbumin was prepared from whey by chromatography on DEAE-Sephadex A-50 and Ultrogel AcA 44. Two biologically active forms of alpha-lactalbumin were apparent after ion-exchange chromatography, but on gel filtration the combined forms were eluted as a single peak with a molecular weight of approx. 33000. The molecular weight when determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was 15100. Antiserum to alpha-lactalbumin was prepared from rabbits, and single radial immunodiffusion was used to measure the concentration of alpha-lactalbumin in milk expressed from rats during lactation and for 2 days after the cessation of lactation. A significant positive correlation (r = + 0.89) between the concentrations of alpha-lactalbumin and lactose was obtained for the first 20 days of lactation. This is consistent with the suggestion that alpha-lactalbumin may control the concentration of lactose in milk. However, a significant negative correlation (r = -0.91) between the concentration of alpha-lactalbumin and lactose was obtained for 2 days after the cessation of lactation on day 20.     

Kinetic and spectral properties of rabbit brain 4-aminobutyrate aminotransferase.

Purification and 4-aminobutyrate-2-oxoglutarate aminotransferase (EC 2.6.1.19) from rabbit brain is described. The method was used as a routine to give between 5 and 10mg of pure enzyme from 750 g of rabbit brain. The enzyme is a dimer made up of subunits each with a mol. wt. of 58000. An absorption spectrum of the freshly prepared enzyme shows peaks at 415 and 330 nm. Treatment of the enzyme with the substrate 4-amino-butyrate or glutamate produces a decrease in the 415 nm and an increase in the 330 nm peak. This conversion, which is attributed to an aldimine into ketimine step in the reaction, is sufficiently slow when 4-aminobutyrate is the substrate to allow it to be followed by stopped-flow spectrophotometry. A first-order rate constant was determined for this step (12s-1) and compared with the turnover number for the enzyme derived by steady-state methods (9.5S-1). The first-order rate constant when glutamate was used as substrate was estimated to be approx. 30s-1.     

Isolation and sequence of a cDNA encoding the major structural protein of peripheral myelin

The myelin sheath is a multilayered membrane, unique to the nervous system, which functions as an insulator to increase greatly the velocity of axonal impulse conduction. We have used the techniques of differential screening and hybrid selection to identify a cDNA clone encoding the Schwann cell glycoprotein P0, the major structural protein of the peripheral myelin sheath. The sequence of this protein, deduced from the nucleotide sequence of the cloned cDNA, indicates that P0 is an integral membrane protein containing a single membrane-spanning region, a large hydrophobic extracellular domain, and a smaller basic intracellular domain. The structure of the protein suggests that each of these domains plays an essential role in generating the highly ordered structure of the myelin sheath. Furthermore, we find that the induction of P0 mRNA coincides with the initiation of myelin formation, and we propose a model in which the glycoprotein serves as a molecular guidepost for this process.     

Kinetics of inactivation of 4-aminobutyrate aminotransferase by 3-bromopyruvate.

3-Bromopyruvate inhibited 4-aminobutyrate aminotransferase (EC 2.6.1.19) from Pseudomonas fluorescens, apparently irreversibly. Kinetics of this inactivation were studied by continuously monitoring the enzyme reaction at 30 degrees C in the presence of inhibitor. Irrespective of how high an inhibitor concentration was present, a maximum rate of inactivation was eventually achieved (5.9 x 10(-3) s-1), indicating the formation of a reversible inhibitor-enzyme complex before the final inactivation step. The dissociation constant of this complex was found to be 6.5 microM. This affinity labelling by 3-bromopyruvate suggests the presence of essential sulphydryl groups on the enzyme, since this compound is known to preferentially alkylate cysteinyl residues.     

Catalytic and structural properties of trypsin-treated 4-aminobutyrate aminotransferase

4-Aminobutyrate aminotransferase (EC 2.6.1.19, 4 aminobutyrate:2-oxoglutarate aminotransferase) is cleaved by trypsin, yielding an enzymatically active species which can be separated from the split peptides by gel filtration. The shortened enzyme derivative gives one band (Mr = 95,000) on polyacrylamide gradient gel electrophoresis. Changes in protein conformation induced by tryptic digestion were studied by fluorescence spectroscopy. The native enzyme tagged with the chromophore fluorescein yields a rotational relaxation time of 106 ns, whereas the trypsin-digested enzyme gives a rotational relaxation time of 33 ns. The decrease in rotational relaxation time is attributed to flexibility of the polypeptide chain with enhanced rotational freedom of the probe covalently linked to one thiol group. The reactivity of sulfhydryl groups toward 5,5'-dithiobis(2-nitrobenzoic acid) is also affected by trypsin cleavage. More--SH groups (2.6/dimer) become reactive toward 5,5'-dithiobis(2-nitrobenzoic acid) as a result of trypsin digestion. Local conformational fluctuations are induced as a result of tryptic cleavage, but the catalytic sites remain intact. The peptides released from 4-aminobutyrate aminotransferase were characterized by fingerprint analysis and their amino acid composition determined.