Primary structure of a hemorrhagic metalloproteinase, HT-2, isolated from the venom of Crotalus ruber ruber

Crotalidae and Viperidae snake venoms contains several kinds of metalloproteinases which cause localized hemorrhage by direct action on blood vessel walls. We report here the entire amino acid sequence and the disulfide bridge locations of HT-2, one of the hemorrhagic toxins isolated from the venom of Crotalus ruber ruber (red rattlesnake). The non-reduced protein was first cleaved at methionine residues to provide a set of 8 fragments, which covered the entire sequence of HT-2. The disulfide bridge locations of HT-2 were also determined by using these primary fragments. The unambiguous sequence for the whole protein was then established by conventional methods using lysyl endopeptidase and thermolysin digests. HT-2 consisted of 202 amino acid residues with two disulfide bridges, which were assigned to Cys-117-Cys-197 and Cys-157-Cys-164. HT-2 had a typical zinc-chelating sequence His-Glu-X-X-His (residues 142-146) found in thermolysin, and its overall sequence showed, respectively, 50, 52, and 53% identities to those of HR2a, H2-proteinase, and the metalloproteinase domain of HR1B. However, the disulfide bridge locations of HT-2 were different from those in the other metalloproteinases. The primary structure of HT-2 was more closely related to that of Ht-d from Crotalus atrox recently determined (81% identity). From the structural comparison with five metalloproteinases so far elucidated, six conservative amino acid residues, which may possibly be related to the induction of the hemorrhagic activity, were suggested to be present in these toxins.     

Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom.

The complete amino acid sequence of fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom, has been determined. This is the first report of the sequence of a direct-acting, nonhemorrhagic fibrinolytic enzyme found in snake venom. The majority of the sequence was established by automated Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. The amino-terminus is blocked by a cyclized glutamine (pyroglutamic acid) residue, and the sequence of this region of the molecule was determined by mass spectrometry. Fibrolase is composed of 203 residues in a single polypeptide chain with a molecular weight of 22,891, as determined by the sequence. Its sequence is homologous to the sequence of the hemorrhagic toxin Ht-d of Crotalus atrox venom and with the sequences of two metalloproteinases from Trimeresurus flavoviridis venom. Microheterogeneity in the sequence was found at both the amino-terminus and at residues 189 and 192. All six cysteine residues in fibrolase are involved in disulfide bonds. A disulfide bond between cysteine-118 and cysteine-198 has been established and bonds between cysteines-158/165 and between cysteines-160/192 are inferred from the homology to Ht-d. Secondary structure prediction reveals a very low percentage of alpha-helix (4%), but much greater beta-structure (39.5%). Analysis of the sequence reveals the absence of asparagine-linked glycosylation sites defined by the consensus sequence: asparagine-X-serine/threonine.     

Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)     

MALDI-TOF mass spectrometry analysis of substrate specificity of lebetase, a direct-acting fibrinolytic metalloproteinase from Vipera lebetina snake venom

Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins which include both hemorrhagic and non-hemorrhagic proteinases. Despite apparent structural similarities, fibrinolytic and hemorrhagic proteinases differ significantly in substrate specificity. In this study, we have examined the activity of lebetase I against biologically active peptides (bradykinin, kallidin, substance P) and 6-10 amino acid residues containing peptides synthesized according to cleavage regions of alpha(2)-macroglobulin, pregnancy zone protein (PZP) and fibrinogen. Lebetase was found to have no activity against studied hexapeptides. Surprisingly, the best substrates for lebetase were substance P, and peptide fragment of PZP, both were cleaved at position Pro-Gln. Identification of the hydrolysis products of 15 peptides by MALDI-TOF mass spectrometry analysis indicates that lebetase possesses broad substrate specificity. The MALDI-TOF MS technique was proven to be highly efficient for the recovery and identification of the peptides released by lebetase hydrolysis.     

The complete amino acid sequence of the high molecular mass hemorrhagic protein HR1B isolated from the venom of Trimeresurus flavoviridis

Hemorrhage is a common occurrence in a victim bitten by crotalid and viperid snakes, and hemorrhagic components in these various venoms have been isolated and characterized. Previously, we have shown that a low molecular weight hemorrhagic protein (HR2a, 202 amino acid residues) isolated from the venom of Trimeresurus flavoviridis is a member of a new subfamily of metalloproteinases. We now report the complete amino acid sequence of a high molecular mass hemorrhagic protein isolated from the same venom. This protein, HR1B, is a mosaic protein composed of 416 residues containing four asparagine-linked oligosaccharide chains. The amino-terminal half (residues 1-203) of HR1B contains a metalloproteinase domain, the sequence of which is 62% identical to that of HR2a and 52% identical to that of hemorrhagic toxin d isolated from Crotalus atrox venom. The most interesting finding is that the middle region (residues 204-300) of HR1B shows a striking similarity to disintegrins, Arg-Gly-Asp-containing platelet aggregation inhibitors, recently found in several viper venoms. Interestingly, however, this region of HR1B does not contain the Arg-Gly-Asp sequence which is known to be a putative binding site in the disintegrins for the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. We also found that the carboxyl-terminal region (residues 213-336) of the middle part of HR1B shows 30% identity to residues 1543-1656 of von Willebrand factor and that the remaining region at the carboxyl-terminal end is unique and has a cysteine-rich sequence. These results suggest that the middle portion of HR1B, which shows structural similarities to the disintegrins and von Willebrand factor, may be important in synergistically stimulating hemorrhagic activity in the NH2-terminal metalloproteinase domain.     

五步蛇蛇毒金属蛋白酶cDNA的克隆和序列分析

抽提五步蛇毒腺总RNA,通过反转录PCR(RT-PCR)扩增出五步蛇毒腺中一种低分子量金属蛋白酶(aculysinl)的cDNA,克隆到pGMT-vector并测定了全序列.推导其编码的蛋白质序列,发现aculysinl是以酶原形式合成的分泌蛋白,酶原包括信号肽、前肽、金属蛋白酶成熟肽和间隔肽4个部分.金属蛋白酶成熟肽与其它蛇毒金属蛋白酶相比,蛋白质一级结构具有一定的同源性,有一个保守的Zn2+结合位点:HEXXHXXGXXH.Aculysinl含有6个半胱氨酸,推测形成3对链内二硫键.五步蛇低分子量金属蛋白酶cDNA的克隆,为研究蛇毒金属蛋白酶结构与功能的关系,以及开发治疗血栓药物打下了良好的基础     

The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing

The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora mitochondria was determined by cDNA and genomic DNA sequencing. A first cDNA was identified from a cDNA bank cloned in Escherichia coli by hybridization selection of mRNA, cell-free protein synthesis and immunoadsorption. Further cDNA and geonomic DNA were identified by colony filter hybridization. The N-terminal sequence of the mature protein was determined by automated Edman degradation. From the sequence a molecular mass of 24749 Da results for the precursor protein and of 21556 Da for the mature protein. The presequence consists of 32 amino acids with four arginines as the only charged residues. The mature protein consists of 199 amino acids. It is characterized by a small N-terminal hydrophilic part of 29 residues, a hydrophobic stretch of 25 residues and a large C-terminal hydrophilic domain of 145 residues. The only four cysteines of the protein, which are assumed to bind the 2 Fe-2S cluster, are located in a moderate hydrophobic region of this large domain. Cysteines 3 and 4 are unusually arranged in that they are separated by only one proline. From sequence data the arrangement of the subunit in the membrane is deduced.     

Characterization of the cDNA encoding bullfrog, Rana catesbeiana, osteocalcin and two forms of the protein isolated from bone

A full-length cDNA clone encoding osteocalcin from the bullfrog, Rana catesbeiana (bone Gla-protein, BGP) has been isolated, and the complete coding sequence for the 100-amino-acid pre-pro-osteocalcin protein was determined. The amino acid sequence of Rana catesbeiana osteocalcin, especially the mature 49-amino acid sequence, is closer to the mammalian than to the fish, Sparus osteocalcin. Rana mature osteocalcin has a similarity of 67% with human or 59% with rat osteocalcin, and only 42% with fish mature osteocalcin. The 51-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Arg-Arg sequence preceding the NH2-terminal Ser of the mature 49-amino-acid Rana osteocalcin. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, which targets vitamin K-dependent gamma-carboxylation of three specific Glu residues at positions 17, 21, and 24 in the mature protein. At the native protein expression levels, extraction from Rana cortical bone in the presence of protease inhibitor cocktail resulted in the isolation of two distinct forms of osteocalcin, P-1 and P-2, with a 3:2 distribution. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequence analysis of the N-terminal domain, we confirmed that P-1 is the intact 49-residue osteocalcin with N-terminal SNLRNAVFG., and that P-2 lacks four amino acids from the N-terminus, (NAVFG.). These results demonstrate the existence of a form of osteocalcin lacking four N-terminal amino acids in Rana bone, and that mature Rana osteocalcins remained highly conserved in their molecular evolution, especially with respect to the conservation of the C-terminal domain (residues 14-49).     

P-III hemorrhagic metalloproteinases from Russell's viper venom: cloning, characterization, phylogenetic and functional site analyses

Two homologous P-III hemorrhagic metalloproteinases were purified from Russell's viper venoms from Myanmar and Kolkata (eastern India), and designated as daborhagin-M and daborhagin-K, respectively. They induced severe dermal hemorrhage in mice at a minimum hemorrhagic dose of 0.8-0.9 microg. Daborhagin-M specifically hydrolyzed an Aalpha-chain of fibrinogen, fibronectin, and type IV collagen in vitro. Analyses of its cleavage sites on insulin chain B and kinetic specificities toward oligopeptides suggested that daborhagin-M prefers hydrophobic residues at the P(1), P(1)', and P(2)' positions on the substrates. Of the eight Daboia geographic venom samples analyzed by Western blotting, only those from Myanmar and eastern India showed a strong positive band at 65kDa, which correlated with the high risk of systemic hemorrhagic symptoms elicited by Daboia envenoming in both regions. The full sequence of daborhagin-K was determined by cDNA cloning and sequencing, and then confirmed by peptide mass fingerprinting. Furthermore, molecular phylogenetic analyses based on 27 P-IIIs revealed the co-evolution of two major P-III classes with distinct hemorrhagic potencies, and daborhagin-K belongs to the most hemorrhagic subclass. By comparing the absolute complexity profiles between these two classes, we identified four structural motifs probably responsible for the phylogenetic subtyping and hemorrhagic potencies of P-III SVMPs.     

Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326.

The gene encoding a novel milk protein-hydrolyzing proteinase was cloned on a 6.56-kb SstI fragment from Streptomyces sp. strain C5 genomic DNA into Streptomyces lividans 1326 by using the plasmid vector pIJ702. The gene encoding the small neutral proteinase (snpA) was located within a 2.6-kb BamHI-SstI restriction fragment that was partially sequenced. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 15,740, which corresponds very closely with the relative molecular mass of the purified protein (15,500) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified neutral proteinase was determined, and the DNA encoding this sequence was found to be located within the sequenced DNA. The deduced amino acid sequence contains a conserved zinc binding site, although secondary ligand binding and active sites typical of thermolysinlike metalloproteinases are absent. The combination of its small size, deduced amino acid sequence, and substrate and inhibition profile indicate that snpA encodes a novel neutral proteinase.     

Primary structure of H2-proteinase, a non-hemorrhagic metalloproteinase, isolated from the venom of the habu snake, Trimeresurus flavoviridis

The complete amino acid sequence of and the locations of disulfide bridges in H2-proteinase, a major non-hemorrhagic proteinase isolated from the venom of the habu Trimeresurus flavoviridis, have been determined and compared with those of HR2a, one of the hemorrhagic metalloproteinases in this venom. The strategy involved consisted of structural analysis of peptides in digests with cyanogen bromide, lysyl endopeptidase, trypsin, Staphylococcus aureus V8 protease and thermolysin. Peptides were purified by gel filtration followed by reversed-phase HPLC. H2-proteinase is a non-glycosylated single chain polypeptide consisting of 201 amino acids with an amino-terminal pyroglutamic acid, a calculated molecular weight of 22,991 and a net charge of +14 at neutral pH. There was no evidence of heterogeneity of the sequence. H2-proteinase has a typical zinc-chelating sequence and its overall sequence identity with HR2a is 73.6%. The 3 disulfide bridges in H2-proteinase link Cys-117 to Cys-196, Cys-158 to Cys-180, and Cys-160 to Cys-163, in the same manner as in the case of HR2a. In striking contrast to HR2a, it contains en extra free cysteine residue at position 94 which becomes reactive to a sulfhydryl reagent in the presence of a denaturant.     

Amino acid and cDNA sequences of a methionine-rich 2S protein from sunflower seed (Helianthus annuus L.)

The amino acid sequence of a methionine-rich 2S seed protein from sunflower (Helianthus annuus L.) and the sequence of a cDNA clone which codes for the entire primary translation product have been determined. The mature protein consists of a single polypeptide chain of 103 amino acids (molecular mass 12133 Da) which contains 16 residues of methionine and 8 residues of cysteine. The cDNA sequence established that the protein is synthesized as a precursor of 141 residues with a typical hydrophobic signal sequence of 25 residues followed by a further 13-residue hydrophobic pro-sequence which is presumably removed by post-translational cleavage. The sequence of the mature protein and that deduced from the cDNA were identical with no evidence of processing at the C-terminus. Comparison of the sunflower methionine-rich protein sequence with sequences of other seed 2S proteins from dicotyledons and monocotyledons showed limited but distinct sequence similarities; in particular the arrangement of the cysteine residues was conserved. The sunflower protein shows 34% identity with the methionine-rich Brazil nut 2S protein and the prepro regions of the precursors of these two proteins show about 50% identity. This similarity indicates that these methionine-rich 2S proteins have diverged as a subclass of the 2S superfamily of proteins which contain only 2-3% methionine. While the related 2S proteins from other dicotyledons are processed to a small and large subunit, the sunflower protein is not cleaved in this way.     

Purification and Characterization of a New Weak Hemorrhagic Metalloproteinase BmHF-1 from <Emphasis Type="Italic">Bothrops marajoensis</Emphasis> Snake Venom

BmHF-1, from the venom of Bothrops marajoensis, was purified by Sephadex G-75 and HPLC-RP on μ-Bondapak C-18 column chromatography. It presented a molecular mass of 27162.36 Da determined by MALDI-TOF MS. BmHF-1 had a sequence of 238 residues of amino acids. The multiple alignment of its amino acid sequence and those of other snake venom metalloproteinases showed high structural similarity, mainly among P–I class. The enzyme initially cleaves the Aα-chain of fibrinogen, followed by the Bβ-chain, and shows no effects on the γ-chain. BmHF-1 had, caseinolytic and weakly hemorrhagic activities, which were inhibited by EDTA. In contrast, PMSF did not affect these activities. The caseinolytic activity of BmHF-1 had a pH optimum of 8.0 and was stable in solution up to 40 °C; activity was completely lost at ≥70 °C. The proteolytic activity was also inhibited by sDa (opossum sera) and Da2-1, Da2-II, antihemorrhagic factors isolated from the opossum sera of Didelphis albiventris. BmHF-1 presents weak hemorrhagic activity, with a MHD of 41.14 μg and it induces dose-dependent edema. We could concluded that, despite its weak hemorrhagic activity, BmHF-1 contributes to local tissue damage by inducing edema, releasing pharmacologically active mediators from protein precursors due to its enzymatic action.     

cDNA sequence and predicted primary structure of the gamma subunit from the ATP synthase from Chlamydomonas reinhardtii

The 1701-base nucleotide sequence (not including the poly(A) tail) of a cDNA for the gamma subunit of the ATP synthase from Chlamydomonas reinhardtii was determined. A start translation sequence, 23 bases in from the 5' end, initiates an 1074-base-long open reading frame. The sequence of the first 21 amino acids at the amino-terminal end of the mature gamma subunit from C. reinhardtii was determined and compared to the deduced amino acid sequence of the open reading frame. From this it was determined that the mature protein contains 323 amino acids, with the first 35 amino acids probably being part of the transit peptide. The length of the mature protein is the same as that for the mature gamma subunit from spinach, for which only a few of the amino acids of the transit peptide are known. The similarity of the two mature proteins at the nucleotide level is 56% while at the amino acid level it is 77%. In addition, the 3 cysteines, which in spinach are involved in the energy-linked catalytic functions of the ATP synthase, are conserved in the predicted amino acid sequence for the gamma subunit from C. reinhardtii. In contrast, the mature C. reinhardtii gamma subunit contains 3 additional cysteine residues not found in the spinach gamma subunit.     

Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C(164)I(165)M(166), which characterize the "metzincin" superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four alpha-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single alpha-helix and several loops. The catalytic zinc ion is coordinated by the N(epsilon 2) nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.     

Phaiodactylipin, a glycosylated heterodimeric phospholipase A from the venom of the scorpion Anuroctonus phaiodactylus.

Phaiodactylipin was purified from the venom of the scorpion Anuroctonus phaiodactylus. It is the first protein to be purified from a scorpion of the family Iuridae and has a molecular mass of 19 172 atomic mass units. The mature protein is composed of two subunits, the large one consisting of 108 amino acid residues, whereas the small subunit has only 18 residues, and the structure is stabilized by five disulfide bridges. The heterodimer is expressed from a single message containing 769 base pairs and a signal peptide with 16 and/or 25 amino acid residues. During maturation an internal hexapeptide is excised. There are three putative sites of N-glycosylation, one of which is situated in the small subunit region. The carbohydrate composition of this site was determined by mass spectrometry analysis and was found to contain three hexoses, two N-acetyl-hexoses and two deoxyhexoses. The protein has a calcium dependent phospholipase A(2) type of activity. It is lethal to arthropods (insects and isopods), but not toxic to mammals, using doses up to 20 microg per 20 g mouse body weight. For crickets, a dose of 5 microg per animal is lethal; however, when injected into mice it is capable of causing only muscular inflammation, without rupture of the basal membrane of cells. It has a direct hemolytic effect in human erythrocytes and retards the coagulation time of blood. It is an unusual phospholipase A(2), with only 36% and 50% amino acid sequence identities to the closest known phospholipases, imperatoxin I and phospholipin, respectively. Identities with bee and Heloderma venom phospholipase are only in the order of 28%.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone

The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase.     

The amino acid sequence of porcine intestinal calcium-binding protein.

The complete amino acid sequence of the calcium-binding protein (CaBP) from pig intestinal mucosa has been determined: Ac-Ser-Ala-Gln-Lys-Ser-Pro-Ala-Glu-Leu-Lys-Ser-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Gln-Leu-Ile-Gln-Ala-Glu-Phe-Pro-Ser-Leu-Leu-Lys-Gly-Pro-Arg-Thr-Leu-Asp-Asp-Leu-Phe-Gln-Glu-Leu-Asp-Lys-Asn-Gly-Asn-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. The N-terminal octapeptide sequence was determined by mass spectrometric analysis by Morris and Dell. The first 45 residues of bovine CaBP differ only in six positions from the corresponding sequence of the porcine protein, except that the sequence starts in position two of the porcine sequence. The mammalian intestinal CaBP's belong to the troponin-C superfamily on the basis of an analysis by Barker and Dayhoff.