Use of immuno-blot techniques to discriminate between the glutathione S-transferase Yf, Yk, Ya, Yn/Yb and Yc subunits and to study their distribution in extrahepatic tissues. Evidence for three immunochemically distinct groups of transferase in the rat.

The glutathione S-transferases are dimeric enzymes whose subunits can be defined by their mobility during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis as Yf (Mr 24,500), Yk (Mr 25,000), Ya (Mr 25,500), Yn (Mr 26,500), Yb1 (Mr 27,000), Yb2 (Mr 27,000) and Yc (Mr 28,500) [Hayes (1986) Biochem. J. 233, 789-798]. Antisera were raised against each of these subunits and their specificities assessed by immuno-blotting. The transferases in extrahepatic tissues were purified by using, sequentially, S-hexylglutathione and glutathione affinity chromatography. Immune-blotting was employed to identify individual transferase polypeptides in the enzyme pools from various organs. The immuno-blots showed marked tissue-specific expression of transferase subunits. In contrast with other subunits, the Yk subunit showed poor affinity for S-hexylglutathione-Sepharose 6B in all tissues examined, and subsequent use of glutathione and glutathione affinity chromatography. Immuno-blotting was employed to identify a new cytosolic polypeptide, or polypeptides, immunochemically related to the Yk subunit but with an electrophoretic mobility similar to that of the Yc subunit; high concentrations of the new polypeptide(s) are present in colon, an organ that lacks Yc.     

Amino acid sequence of chicken liver cathepsin L

The complete amino acid sequences of the heavy and light chains of chicken liver cathepsin L have been determined by automated gas-phase Edman degradation. The heavy and light chains contained 176 and 42 amino acid residues respectively. A glucosamine-based oligosaccharide group was attached to Asn-109 of the heavy chain. Chicken liver cathepsin L had high sequence homology with rat cathepsin H, but exhibited less similarity with rat cathepsin B. Comparisons of cathepsin L with plant cysteine proteinases, such as papain, actinidin and aleurain, reveal high degree of homology.     

Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

A 4175-bp EcoRI fragment of DNA that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1 alpha subunit (368 amino acids, Mr 41,312, without the initiating methionine residue) and E1 beta subunit (324 amino acids, Mr 35,306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1 beta gene begins just 3 bp downstream from the E1 alpha stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1 alpha and E1 beta subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1 alpha and E1 beta subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1 alpha chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.     

Amino acid sequence of rat liver cathepsin L

The complete amino acid sequences of the heavy and light chains of rat liver cathepsin L (EC 3.4.22.15) were determined at the protein level. The heavy and light chains consisted of 175 and 44 amino acid residues, respectively, and their Mr values without glycosyl groups calculated from these sequences were 18941 and 5056, respectively. The amino acid sequence was also determined from the N-terminal sequences of the heavy and light chains, and the sequences of cleavage fragments of the heavy chain with lysylendopeptidase and cyanogen bromide. The fragments were aligned by comparison with the amino acid sequence deduced from the sequence of cDNA of rat preprocathepsin L. The sequence of rat liver cathepsin L determined at the protein level was identical with that deduced from the cDNA sequence except that in the heavy chain, residues 176-177 (Asp-Ser) were not present at the C-terminus and alanine was replaced by proline at residue 125. Asn-108 in the heavy chain is modified with carbohydrate.     

鹅白细胞介素 2基因的克隆与分子模型

对鸡、鸭、火鸡IL-2的核苷酸序列进行比较,在其保守区设计引物,通过RT-PCR方法扩增和克隆了鹅白介素2 (goIL-2) 的核苷酸序列。该序列由768 nt组成,编码一条由141个氨基酸组成的前体蛋白。goIL-2核苷酸序列和氨基酸序列与鸭IL-2(duIL-2)核苷酸序列和氨基酸序列的同源性为90.1%和83.6%,与鸡、火鸡和鹌鹑IL-2的同源性为69.7%-75%和61.0%-63.1%,与哺乳动物IL-2的同源性为25%-30%和14%-17%。氨基酸序列分析表明,N端存在一长21个氨基酸的信号肽,含有形成2个链内二硫键的4个半胱氨酸。goIL-2 mRNA的体外表达动力学分析表明,脾脏T淋巴细胞经Con A诱导2 h至24 h均可检测到goIL-2 mRNA的表达。三维结构预测表明,goIL-2蛋白由A、B、C、D 4个α-螺旋和2个?-折叠构成。遗传进化分析表明,goIL-2和duIL-2的亲缘关系最近。     

The chemical characterization of favin, a lectin isolated from Vicia faba.

We have determined the subunit structure of the glucose- and mannose-binding lectin favin, from Vicia faba. The molecule is composed of two nonidentical polypeptide chains held together by noncovalent interactions. We have determined the complete amino acid sequence of the smaller alpha chain (Mr = 5,571) and shown that it is homologous to the alpha chain of the lectins from lentil and pea and to residues 72 to 120 of concanavalin A (Con A). The larger beta chain (Mr = 20,000) contains carbohydrate and is homologous to the beta chain of lentil, pea, soybean, peanut, and red kidney bean lectins and is homologous to a portion of the Con A molecule beginning at residue 122. Favin also contains a minor component, beta' (Mr = 18,700), that closely resembles the beta chain but lacks carbohydrate and may, on the basis of apparent molecular weight, lack some part of the COOH-terminal region of the polypeptide chain. Although favin is similar to Con A, it, like the lentil and pea lectins, appears to lack residues corresponding to positions 1 to 71 of Con A. Because these residues contribute significantly to the carbohydrate binding site of Con A, the lack of this region in the otherwise homologous lectin favin suggests that the carbohydrate binding site of favin differs from that of Con A or that the region represented by residues 1 to 71 of Con A is located in a different portion (i.e. in the beta chain) of the favin molecule.     

Nucleoside diphosphate kinase from human erythrocytes. Structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme.

Human erythrocyte nucleoside-diphosphate kinase (NDP kinase) is a hexameric enzyme consisting of two kinds of polypeptide chains, A and B. By random association (A6, A5B...AB5, B6) these polypeptides form isoenzymes differing in their isoelectric point. Chains A and B of NDP kinase were purified by ion-exchange chromatography under denaturing conditions. Upon mixing and renaturation, the isozymic pattern of NDP kinase obtained by conventional methods was restored. Antibodies raised against purified chains showed significant cross-reactivity, both in immunoblot experiments and activity inhibition studies. Sequence determination showed that both chains consisted of 152 amino acid residues corresponding to Mr or 17,143 (chain A) and 17,294 (chain B), respectively. There was high homology between the two sequences (88% identity). The phosphorylation site on the enzyme is located at His-118. Chain A was identical with human Nm23 protein, which has been reported as a potential suppressor protein in tumor metastasis and chain B was identical with Nm23-H2 protein.     

The primary structure of human gamma-glutamyl transpeptidase

A cDNA hybridizable to that of rat gamma-glutamyl transpeptidase (GGT) was cloned from a cDNA library of human fetal liver. The insert of the cDNA clone contained 1866 bp consisting of an open reading frame (ORF) of 1709 bp (569 amino acids (aa), N-terminal portion truncated) and a 135-bp 3'-untranslated region followed by a polyadenylated tail. In parallel, amino acid sequences of N-terminal portions of heavy and light chains of a purified human GGT were determined. Two stretches of amino acid sequences identical to the N-terminal sequences of heavy and light chains were found in the ORF. We therefore concluded that the clone is a cDNA for human GGT. From the amino acid sequence deduced from cDNA, the heavy and the light chains of the purified enzyme are estimated to be composed of 351 aa (Mr 38,336) and of 189 aa (Mr 20,000), respectively. The heavy chain is preceded by a signal peptide of at least 29 aa presumed to be cleaved by bromelain treatment. Six putative N-glycosylation sites are present in the heavy subunit region and one in the light subunit region. Primary structure and hydrophobicity profile are closely similar to those of rat GGT.     

Mouse liver cytochrome P-450 (P-450IIIAM1): its cDNA cloning and inducibility by dexamethasone.

A full-length cDNA complementary to mouse liver mRNA coding for one of the cytochromes P-450 (P-450) in the P-450IIIA family, namely P-450IIIM1, was isolated and completely sequenced. The sequence of this cDNA clone, pMDex13, revealed that it encoded a polypeptide of 504 deduced amino acid residues (Mr = 57,853). The deduced amino acid sequence showed 87.3 and 84.9% identity with rat P-450IIIA1 and P-450IIIA2, respectively. The NH2-terminal 24 amino acid sequences of P-450IIIAM1 were completely identical with purified mouse P-450UT protein. RNA blot analysis showed that mRNA content of hepatic P-450IIIAM1 was remarkably increased by treatment of mice with dexamethasone.     

Separation, purification, partial characterization and comparison of the heavy and light chains of botulinum neurotoxin types A, B, and E

Clostridium botulinum produces botulinum neurotoxin (NT) in antigenically distinct forms. When isolated from bacterial cultures type E is a single chain, type B is a mixture of single and two-chain molecules, and type A is essentially a two-chain molecule (Mr approximately 150,000). Protease(s) in the cultures or trypsin nick single-chain NT to the two-chain form. The heavy (Mr approximately 100,000) and light (Mr approximately 50,000) chains of the two-chain molecule remain held together by -S-S-bond(s). The two chains are presumed to have different functions. NT binds to nerve cells via the heavy chain and then light chain enters the cell and blocks release of acetylcholine (Simpson, L. L. (1981) Pharmacol. Rev. 33, 155-188). We nicked single-chain NT to form the two-chain form with trypsin, minimizing secondary cleavages, then separated and purified the heavy and light chains using ion-exchange chromatography. The technique, with minor modifications, is a generalized method for types A, B, and E. These subunit chains (each a single band in sodium dodecyl sulfatepolyacrylamide gel electrophoresis) were analyzed for their complete amino acid compositions. The amino acid contents of the heavy and light chains agreed well with the parent two-chain molecule. This affirms that NT is composed of two chains. The two subunit chains are now usable for amino acid sequence and other studies. Comparison of the amino acid contents indicates more similarity among the light chains than the heavy chains of the three NT types, a similarity that agrees with our published partial amino acid sequences (first 13-18 residues) of these chains. Several (up to 9) different amino acid residues of the heavy chain (which is twice the size of the light chain) are present in double the number of corresponding residues in the light chain.     

Botulinum neurotoxin type B (strain 657): partial sequence and similarity with tetanus toxin

The type B neurotoxin (NT) isolated from Clostridium botulinum (strain 657) behaved as a mixture of single (unnicked) and dichain (nicked) proteins, both of Mr approximately 150 kDa. When the dichain NT was reduced by mercaptoethanol, the two chains migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as separate polypeptides of Mr approximately 100 and 50 kDa that appeared similar to the heavy and light chains of other serotypes of botulinum NT. The N-terminal amino acid sequences of the two chains were determined. They were as follows: light chain: Pro-Val-Thr-Ile-Asn-Asn-Phe-Asn-Tyr-Asn-Asp-Pro-Ile-Asp-Asn-Asn-Asn-Ile- Ile-Met - Met-Glu-Pro-Pro-Phe-Ala-Arg-Gly-Met-Gly-Arg-Tyr-Tyr-Lys-Ala-Phe-Lys-Ile- Thr-Asp - Arg-Ile-Trp-Ile-; and heavy chain: Ala-Pro-Gly-Ile-X-Ile-Asp-Val-Asp-Asn-Glu-Asp-Leu-Phe-Phe-Ile-Ala-Asp-Ly s-Asn- Ser-Phe-Arg-Asp-Asp-Leu-. These two sequences matched exactly with those of the light and heavy chains of type B NT (strain Okra) of which only 16 and 18 residues were known (J. Biol. Chem. (1985) 260, 10461). The above sequences were different from those of type A NT. Immunoprecipitation reactions of type B NT isolated from strains 657 and Okra were indistinguishable against polyclonal anti-type B NT serum. These two preparations did not produce precipitin reactions with polyclonal anti-type A NT serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of the biotin-binding site of chicken liver acetyl-CoA carboxylase

Limited proteolysis of chicken liver acetyl-CoA carboxylase by staphylococcal serine proteinase yielded a fragment of 31 kDa which contained the biotinyl active site. This polypeptide was purified by preparative polyacrylamide gel electrophoresis and characterized. The complete amino acid sequence of this polypeptide has been deduced from the nucleotide sequence of cloned DNA complementary to the chicken liver acetyl-CoA carboxylase mRNA. A highly conserved sequence of Met-Lys-Met was found in the biotin-binding site. Appreciable homology was observed among the sequences in close vicinity of the biotin sites of chicken liver acetyl-CoA carboxylase and other biotin-dependent carboxylases including biotin carboxyl carrier protein of Escherichia coli acetyl-CoA carboxylase.     

Cloning of cDNA encoding rat TCP-1.

We have isolated and sequenced a cDNA encoding a rat homolog of the mouse t-complex polypeptide 1 (TCP-1). Its deduced gene product is a polypeptide of 556 amino acids, with a predicted Mr of 60,341. The similarity between mouse Tcp-1 and the rat homolog is about 94.0% at the nucleotide level and 97.1% at the amino acid level showing the evolutionary conservation of this protein. The similarity of the amino acid sequence of the rat TCP-1 is not significantly biased to any of those from wild (TCP-1B) or from t-haplotype mice (TCP-1A). From a comparison of deduced amino acid sequences of eukaryotic TCP-1 proteins, we found highly conserved domains. Southern blot analysis revealed that there are at least two similar sequences to Tcp-1 in the rat, one is a structural gene and the other seems to be a processed pseudogene.     

Chicken IgA H chains. Implications concerning the evolution of H chain genes.

A cDNA clone (A1) encoding for a novel chicken Ig H chain isotype was isolated. In sequence comparison to mammalian H chains, A1 C region was most closely related to the alpha isotype. For example, identities of 35%, 32%, and 31% at the amino acid level to rabbit C alpha, C mu, and C gamma were observed, respectively. Distribution of the glucosamine acceptor sites (Asn-X-Ser/Thr) in A1 C region was typical of alpha H chains. Moreover, A1 C region probe hybridized to a 2.2-kb RNA species expressed in the epithelial lymphoid tissues. Thus, A1 was identified as the avian homologue for mammalian alpha H chains. Interestingly, the chicken C alpha was structurally consistent with four complete CH domains, whereas only three domains are present in the mammalian C alpha genes. In addition, interdomain sequence alignments suggested that the homologue for the chicken C alpha 2 domain is missing from the mammalian alpha H chains. Thus, the present data suggest evolution of the IgA isotype before the segregation of avian and mammalian species. Also, the first C alpha gene may have consisted of four CH domains, whereas reorganization of the C alpha 2 region led to the generation of hinge region in the mammalian alpha H chains.     

Isolation and characterization of the third complement component of axolotl (Ambystoma mexicanum)

1. Using a monoclonal anti-human C3 antibody and a polyclonal anti-cobra venom factor antibody as probes, a protein homologous to the mammalian third complement component (C3) was purified from axolotl plasma and found to be axolotl C3. 2. Axolotl C3 consists of two polypeptide chains (Mr = 110,000 and 73,000) linked by disulfide bonds. An internal thiolester bond in the alpha chain was identified by the incorporation of [14C]methylamine and NH2-terminal sequence from the C3d fragment of C3. 3. Digestion of C3 by trypsin resulted in the cleavage of both the alpha and beta chains, generating fragments with a cleavage pattern similar to that of human C3. 4. The amino acid composition of axolotl C3 and the amino acid sequences of the thiolester site (and the surrounding amino acids), the cleavage site for the C3-convertase, and one of the factor I cleavage sites are similar to C3 from other vertebrates. 5. In contrast to human C3, which has concanavalin A binding carbohydrates on both the alpha and beta chains, only the beta chain of axolotl C3 contains such carbohydrates.     

Chicken chromosomal protein HMG-14 and HMG-17 cDNA clones: isolation, characterization and sequence comparison

A cDNA clone coding for the chicken high-mobility group 14 (HMG-14) mRNA has been isolated from a chicken-liver cDNA library by screening with two synthetic oligodeoxynucleotide pools whose sequences were derived from the partial amino acid sequence of the HMG-14 protein. A chicken HMG-17 cDNA clone was also isolated in a similar fashion. Comparison of the two chicken HMG cDNA clones to the corresponding human cDNA sequences shows that chicken and human HMG-14 mRNAs and polypeptides are considerably less similar than are the corresponding HMG-17 sequences. In fact, the chicken HMG-14 is almost as similar to the chicken HMG-17 in amino acid sequence as it is to mammalian HMG-14 polypeptides. HMG-14 and HMG-17 mRNAs seem to contain a conserved sequence element in their 3'-untranslated regions whose function is at present unknown. The chicken HMG-14 and HMG-17 genes, in contrast to their mammalian counterparts, appear to exist as single-copy sequences in the chicken genome, although there appear to exist one or more additional sequences which partially hybridize to HMG-14 cDNA. Chicken HMG-14 mRNA, about 950 nucleotides in length, was detected in chicken liver RNA but was below our detection limits in reticulocyte RNA.     

Comparative studies of vertebrate aldehyde dehydrogenase 3: sequences, structures, phylogeny and evolution. Evidence for a mammalian origin for the ALDH3A1 gene

Mammalian ALDH3 genes (ALDH3A1, ALDH3A2, ALDH3B1 and ALDH3B2) encode enzymes of peroxidic and fatty aldehyde metabolism. ALDH3A1 also plays a major role in anterior eye tissue UV-filtration. BLAT and BLAST analyses were undertaken of several vertebrate genomes using rat, chicken and zebrafish ALDH3-like amino acid sequences. Predicted vertebrate ALDH3 sequences and structures were highly conserved, including residues involved in catalysis, coenzyme binding and enzyme structure as reported by Liu et al. [27] for rat ALDH3A1. Phylogeny studies of human, rat, opossum, platypus, chicken, xenopus and zebrafish ALDH3-like sequences supported three hypotheses: (1) the mammalian ALDH3A1 gene was generated by a tandem duplication event of an ancestral vertebrate ALDH3A2 gene; (2) multiple mammalian and chicken ALDH3B-like genes were generated by tandem duplication events within genomes of related species; and (3) vertebrate ALDH3A and ALDH3B genes were generated prior to the appearance of bony fish more than 500 million years ago.     

Localization of the charge differences in the actins of rabbit skeletal muscle and chicken gizzard by two-dimensional gel electrophoretic analysis of tryptic fragments.

Partial tryptic cleavage products of pure actin from rabbit skeletal muscle and chicken gizzard are compared by two-dimensional electrophoresis in polyacrylamide gels with respect to isoelectric point and molecular weight. While the intact polypeptides (Mr 42,000) have different isoelectric points, two large cleavage products (Mr 35,000) generated from both both actin species have identical isoelectric points and identical molecular weights. These relatively trypsin-resistant cleavage products are presumably identical to the known "core actin" fragments which lack the aminoterminal region of the polypeptide chain. Therefore the differences that are responsible for the different isoelectric points of rabbit skeletal muscle actin and chicken gizzard actin seem to be restricted to the aminoterminal part of the actin polypeptide chains as was proposed on the basis of partial amino acid sequence data.