COOH-terminal Amino Acid Sequence Research Articles

Identification of a Novel Recognition Sequence for Fibronectin within the NH2-terminal β-Sandwich Domain of Tissue Transglutaminase

Tissue transglutaminase belongs to the multigene transglutaminase family of Ca2+-dependent protein cross-linking enzymes. Unlike other transglutaminases, it is involved in cell-matrix interactions and serves as an adhesion co-receptor for fibronectin. Previous work established that the fibronectin-binding motif(s) is located within the NH2-terminal proteolytic fragment of the protein consisting of residues 1-272. Here we identify a novel fibronectin recognition site within this sequence of tissue transglutaminase. Substitution of individual domains of tissue transglutaminase with those from homologous factor XIIIA showed that the major fibronectin-binding site is present within the first beta-sandwich domain of the protein. Experiments with deletion mutants of the first domain revealed that amino acids 81-140 of tissue transglutaminase are involved in fibronectin binding. Using synthetic peptides encompassing this region, we found that the peptide 88WTATVVDQQDCTLSLQLTT106 inhibited the interaction of tissue transglutaminase with fibronectin and decreased transglutaminase-dependent cell adhesion and spreading. In the three-dimensional structure of the first domain, amino acids 88-106 comprise an extended hairpin formed by antiparallel beta strands 5 and 6. Mutations of Asp94 and Asp97 within the beta5/beta6 hairpin to Ala significantly reduced the affinity of tissue transglutaminase for fibronectin, indicating that these residues are critical for fibronectin binding. Identification of the fibronectin-binding site on tissue transglutaminase will help to dissect the role of this protein in cell-matrix interactions.

Assessment of splice variant-specific functions of desmocollin 1 in the skin.

Desmocollin 1 (Dsc1) is part of a desmosomal cell adhesion receptor formed in terminally differentiating keratinocytes of stratified epithelia. The dsc1 gene encodes two proteins (Dsc1a and Dsc1b) that differ only with respect to their COOH-terminal cytoplasmic amino acid sequences. On the basis of in vitro experiments, it is thought that the Dsc1a variant is essential for assembly of the desmosomal plaque, a structure that connects desmosomes to the intermediate filament cytoskeleton of epithelial cells. We have generated mice that synthesize a truncated Dsc1 receptor that lacks both the Dsc1a- and Dsc1b-specific COOH-terminal domains. This mutant transmembrane receptor, which does not bind the common desmosomal plaque proteins plakoglobin and plakophilin 1, is integrated into functional desmosomes. Interestingly, our mutant mice did not show the epidermal fragility previously observed in dsc1-null mice. This suggests that neither the Dsc1a- nor the Dsc1b-specific COOH-terminal cytoplasmic domain is required for establishing and maintaining desmosomal adhesion. However, a comparison of our mutants with dsc1-null mice suggests that the Dsc1 extracellular domain is necessary to maintain structural integrity of the skin.

Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alternative splicing.

Three human glutaminase (hGA) isoforms were identified, two of which represent isoforms previously unidentified in any species. One isoform contains an open reading frame with high homology with the rat kidney-type glutaminase, suggesting that this isoform represents the human kidney-type glutaminase, hKGA. A second isoform, termed hGAC, contains an open reading frame that matches hKGA except for a unique COOH-terminal amino acid sequence. In addition, a third human glutaminase isoform was identified from a computer search and on further analysis was found to represent an additional unique isoform, hGAM. hKGA is expressed predominantly in brain and kidney but not in liver, hGAC is expressed principally in cardiac muscle and pancreas but not in liver or brain, and hGAM is expressed solely in cardiac and skeletal muscle. hGAC is the predominant isoform expressed by a human breast cancer cell line that exhibits a high rate of glutamine utilization and glutaminase activity. Genomic Southern analysis as well as isolation and analysis of five glutaminase genomic clones suggested that all three hGA isoforms originate from the same locus and therefore represent mRNA species that are produced by tissue-specific alternative splicing of a single pre-mRNA. Furthermore, an RT-PCR assay was developed that can be used to easily differentiate between hKGA and hGAC mRNA species.

Dominant Negative Isoform of Rat Norepinephrine Transporter Produced by Alternative RNA Splicing

We have cloned from rat brain a family of alternatively spliced cDNAs from a single gene, which encodes a norepinephrine transporter (NET) having variations at the 3'-region including both coding and noncoding regions. This produces two transporter isoforms, rNETa and rNETb, which differ at their COOH termini. The rNETa isoform reveals a COOH terminus homologous to human NET and transports norepinephrine. In contrast, rNETb revealed no detectable transport function but reduced functional expression of rNETa when both isoforms were expressed in the same cell. Thus, rNETb potentially functions as a dominant negative inhibitor of rNETa activity. Co-expression of rNETb with a gamma-aminobutyric acid transporter (rGAT1), a serotonin transporter (rSERT), and a dopamine transporter (rDAT) reduced their transport activity. No reduction was found with the glutamate/aspartate transporter (rGLAST). Alternative RNA splicing of NET suggests a novel mechanism for the regulation of synaptic transmission.

Maturation of Secreted Meprin α during Biosynthesis: Role of the Furin Site and Identification of the COOH-Terminal Amino Acids of the Mouse Kidney Metalloprotease Subunit

The α subunit of meprin A is synthesized as a type I integral membrane protein; however, the mature form contains no membrane-spanning region due to COOH-terminal proteolytic cleavage during biosynthesis. Previous studies with transfected mouse meprin α subunit cDNA, showed that the inserted (I) domain in the COOH-terminus was essential for proteolytic processing and transport of the subunit out of the endoplasmic reticulum. A furin site in the I domain was implicated as the site of cleavage in the rat meprin α subunit, however, this site was shown not to be required in the homologous mouse subunit. The present studies were designed to determine whether there is a species difference in the use of the furin site for processing of the subunit, and whether the different mutations used in the previous studies could account for the different conclusions regarding the importance of the furin site. When the furin sites in mouse and rat cDNAs were mutated, using similar amino acid substitutions, and expressed in human 293 cells, all mutants were secreted, and had comparable activities compared to the wild-types against a protein (azocasein) and peptide (bradykinin analog) substrate. These data revealed no difference between processing of the rat and mouse subunits, and indicated that the furin site is not essential for COOH-terminal processing in either species. Additional transfection investigations with brefeldin A or low temperature confirmed that COOH-terminal processing occurs in the endoplasmic reticulum, further supporting the contention that furin-type enzymes localized to the Golgi apparatus are not responsible for processing this subunit. COOH-terminal amino acid sequence analysis of the mature detergent-purified membrane form of meprin α from ICR mouse kidney indicated that the subunit ends at Arg615, which is NH2-terminal to the I domain in the AfterMATH domain. Mutation of Arg615 to an Ala did not affect secretion of the protein from 293 cells. The results indicate that the I domain enables or directs the final COOH-terminal processing of meprin α to a region NH2-terminal to the I domain, and that there are no essential dibasic, furin, or single base processing sites.

Processing of Mammalian and PlantS-Adenosylmethionine Decarboxylase Proenzymes

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl enzyme, and the pyruvate is formed in an intramolecular reaction that cleaves a proenzyme precursor and converts a serine residue into pyruvate. The wild type potato AdoMetDC proenzyme processed much faster than the human proenzyme and did not require putrescine for an optimal rate of processing despite the presence of three acidic residues (equivalent to Glu11, Glu178, and Glu256) that were demonstrated in previous studies to be required for the putrescine activation of human AdoMetDC proenzyme processing (Stanley, B. A., Shantz, L. M., and Pegg, A. E. (1994) J. Biol. Chem. 269, 7901-7907). A fourth residue that is also needed for the putrescine stimulation of human AdoMetDC proenzyme processing was identified in the present studies, and this residue (Asp174) is not present in the potato sequence. The site of potato AdoMetDC proenzyme processing was found to be Ser73 in the conserved sequence, YVLSESS, which is the equivalent of Ser68 in the human sequence. Replacement of the serine precursor with threonine or cysteine by site-directed mutagenesis in either the potato or the human AdoMetDC proenzyme did not prevent processing but caused a significant reduction in the rate. Although the COOH-terminal regions of the known eukaryotic AdoMetDCs are not conserved, only relatively small truncations of 8 residues from the human protein and 25 residues from the potato proenzyme were compatible with processing. The maximally truncated proteins show no similarity in COOH-terminal amino acid sequence but each contained 46 amino acid residues after the last conserved sequence, suggesting that the length of this section of the protein is essential for maintaining the proenzyme conformation needed for autocatalytic processing.

The efficient production of human epidermal growth factor by Bacillus brevis.

A system has been developed for the efficient production of heterologous proteins using Bacillus brevis as a host that secretes large amounts of cell wall protein into the medium. The promoter region and signal peptide-encoding region of the cell wall protein gene were used to construct an expression-secretion vector. Bacterial proteins such as amylases can be produced in large amounts by this system (1 g/l or more), but mammalian proteins such as human alpha-amylase are produced at a low level (one or two orders of magnitude less than for bacterial proteins). The highly efficient secretion of human epidermal growth factor (h-EGF, more than 1 g/l) was obtained with B. brevis HPD31 as the host and plasmid pHY481, derived from B. brevis 481, as the vector. Recombinant hEGF was purified easily from the culture supernatant by two steps. Purified hEGF had the identical NH2-terminal amino acid sequence and COOH-terminal amino acid sequence with those of the authentic hEGF, and it was fully active in biological assays. This recombinant hEGF has been shown to be successful for biological wool harvesting (CSIRO, Australia). These results, in combination with previous results, indicate that foreign proteins of diverse origins can be produced efficiently as functional proteins in B. brevis.

Molecular cloning and identification of a rat serum carboxylesterase expressed in the liver.

We have cloned and sequenced a carboxylesterase from rat liver and purified the corresponding protein from rat blood. The cDNA encodes the entire mature serum esterase protein. It is apparently identical to cDNAs cloned from rat liver by several groups (Long, R. M., Satoh, H., Martin, B. M., Kimura, S., Gonzales, F. J., and Pohl, L. R. (1988) Biochem. Biophys. Res. Commun. 156, 866-873; Takagi, Y., Morohashi, K.-i., Kawabata, S.-i., Go, M., and Omura, T. (1988) J. Biochem. (Tokyo) 104, 801-806; and Robbi, M., and Beaufay, H. (1992) Biochem. Biophys. Res. Commun. 183, 836-841). However, the identification of the protein encoded by this cDNA has not been previously reported. The COOH-terminal -TEHT sequence found in the rat serum carboxylesterase does not possess retention properties and is therefore responsible for its secretion and presence in the circulation. The rat serum carboxylesterase was purified to apparent homogeneity by affinity chromatography on immobilized antibody to rat liver microsomal acyl-CoA thioesterase followed by ion exchange chromatography. The purified protein, with a M(r) of approximately 70,000, was cleaved in situ in a polyacrylamide gel with trypsin, and two peptides were isolated and sequenced. Sequence analysis showed that both peptides were identical only to the corresponding deduced amino acid sequence of the cloned cDNA. Antibodies raised to the COOH-terminal amino acid sequence deduced from the cDNA cross-reacted with the purified rat serum carboxylesterase. Changes in serum esterase activity levels followed changes in protein mass in rat serum and changes in liver mRNA levels in response to various nutritional conditions while total liver esterase activity was essentially unchanged. The above experiments confirm the identity of the protein isolated from rat sera with the cDNA cloned from rat liver and suggest a function for the serum esterase in lipid metabolism.

Specific phosphorylation of a COOH-terminal site on the full-length form of the alpha 1 subunit of the skeletal muscle calcium channel by cAMP-dependent protein kinase.

The primary (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive calcium channels is present in full-length (212 kDa) and truncated (190 kDa) forms which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. In the present study, phosphorylation of the purified calcium channel by cA-PK followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed differential phosphorylation of the related 190- and 212-kDa forms. The 190-kDa form of the alpha 1 subunit was phosphorylated on three major and three minor tryptic phosphopeptides; the 212-kDa form was phosphorylated on all six of these phosphopeptides plus two that were unique. Time course experiments showed that a single site on the COOH-terminal portion of the full-length form of the alpha 1 subunit is most intensely and rapidly (within 10 s) phosphorylated. Phosphorylation occurs almost exclusively on this COOH-terminal site unless harsh conditions such as treatment with denaturing detergents are employed to expose phosphorylation sites within the 190-kDa segment of the molecule. Elution of phosphopeptides from the second dimension chromatograph followed by immunoprecipitation with an anti-peptide antibody (anti-CP1) directed against the COOH-terminal amino acid sequence enabled us to identify this major phosphorylation site as serine 1854. The nearby consensus sites for cA-PK phosphorylation at serines 1757 and 1772 were phosphorylated only after denaturation or proteolytic cleavage. Phosphorylation of serine 1854 may play a pivotal role in the regulation of calcium channel function by cA-PK.

Soluble forms of tumor necrosis factor receptors (TNF-Rs). The cDNA for the type I TNF-R, cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor.

Two proteins which specifically bind tumor necrosis factor (TNF) have recently been isolated from human urine in our laboratory. The two proteins cross-react immunologically with two species of cell surface TNF receptors (TNF-R). Antibodies against one of the two TNF binding proteins (TBPI) were found to have effects characteristic of TNF, including stimulating phosphorylation of specific cellular proteins. Oligonucleotide probes designed on the basis of the NH2-terminal amino acid sequence of TBPI were used to clone the cDNA for the structurally related cell surface type 1 TNF-R. It is notable that although this receptor can signal the phosphorylation of cellular proteins, it appears from its amino acid sequence to be devoid of intrinsic protein kinase activity. The extracellular domain of the receptor is composed of four internal cysteine-rich repeats, homologous to structures repeated four times in the extracellular domains of the nerve growth factor receptor and the B lymphocytes surface antigen CDw40. The amino acid composition and size of the extracellular domain of the type I TNF-R closely resemble those of TBPI. The COOH-terminal amino acid sequence of the four cysteine rich repeats within the extracellular domain of the type I TNF-R matches the COOH-terminal sequence of TBPI. Amino acid sequences in the extracellular domain also fully match other sequences found in TBPI. On the other hand, amino acid sequences in the soluble form of the type II TNF-R (TBPII), while indicating a marked homology of structure, did not suggest any identity between this protein and the extracellular domain of the type I TNF-R. CHO cells transfected with type I TNF-R cDNA produced both cell surface and soluble forms of the receptor. The receptor produced by CHO cells was recognized by several monoclonal antibodies against TBPI, reacting with several distinct epitopes in this molecule. These data suggest that the soluble forms of the TNF-Rs are structurally identical to the extracellular cytokine binding domains of these receptors and are consistent with the notion that the soluble forms are, at least partly, derived from the same transcripts that encode the cell surface receptors.

Isoprenylation of the low molecular mass GTP-binding proteins rac 1 and rac 2: Possible role in membrane localization

Ras proteins can be modified at their COOH-terminal cysteine in the motif Cys-Ali-Ali-Xaa by a farnesyl isoprenoid. This modification is essential for membrane association and biological activity of ras proteins. A similar COOH-terminal amino acid sequence, Cys-Xaa-Ali-Xaa, exists in the ras-related GTP-binding proteins rac 1 and rac 2. To determine whether these proteins were similarly modified, COS cells were transfected with rac 1 and rac 2 cDNA and expressed proteins were labeled with [ 3H]mevalonic acid. We report here that both rac 1 and rac 2 are post-translationally modified by addition of an isoprenoid group, the likely site of which is the COOH-terminal cysteine. Isoprenylation was found only in racs associated with particulate cell fractions, suggesting that this modification may be associated with membrane localization of the proteins. These data specifically identify mammalian low molecular mass GTP-binding proteins other than ras that undergo post-translational modification and further define the COOH-terminal consensus sequence, Cys-Ali-Ali-Xaa, as an isoprenylation signal. This sequence may identify a larger family of low molecular mass GTP-binding proteins which are isoprenylated.

Molecular cloning of cDNA encoding a 55-kDa multifunctional thyroid hormone binding protein of skeletal muscle sarcoplasmic reticulum.

A cDNA clone encoding 55-kDa multifunctional, thyroid hormone binding protein of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 509 amino acids, and a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that an 18-residue NH2-terminal signal sequence was removed during synthesis. The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma beta-subunit of prolyl 4-hydroxylase and hen oviduct glycosylation site binding protein. The protein contains two repeated sequences Trp-Cys-Gly-His-Cys-Lys proposed to be in the active sites of protein disulfide isomerase. Northern blot analysis showed that the mRNA encoding rabbit skeletal muscle form of the protein is present in liver, kidney, brain, fast- and slow-twitch skeletal muscle, and in the myocardium. In all tissues the cDNA reacts with mRNA of 2.7 kilobases in length. The 55-kDa multifunctional thyroid hormone binding protein was identified in isolated sarcoplasmic reticulum vesicles using a monoclonal antibody specific to the 55-kDa thyroid hormone binding protein from rat liver endoplasmic reticulum. The mature protein of Mr 56,681 contains 95 acidic and 61 basic amino acids. The COOH-terminal amino acid sequence of the protein is highly enriched in acidic residues with 17 of the last 29 amino acids being negatively charged. Analysis of hydropathy of the mature protein suggests that there are no potential transmembrane segments. The COOH-terminal sequence of the protein, Arg-Asp-Glu-Leu (RDEL), is similar to but different from that proposed to be an endoplasmic reticulum retention signal; Lys-Asp-Glu-Leu (KDEL) (Munro, S., and Pelham, H.R.B. (1987) Cell 48, 899-907). This variant of the retention signal may function in a similar manner to the KDEL sequence, to localize the protein to the sarcoplasmic or endoplasmic reticulum. The positively charged amino acids Lys and Arg may thus interchange in this retention signal.

Purification and Properties of Recombinant Rat Catalase Produced in Escherichia coli1

Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.

Construction and phosphorylation of a fusion protein [formula omitted

A protein consisting of human (Hu)-IFN-αA to which the COOH-terminal 16 amino acids of Hu-IFN-γ were fused was prepared by constructing an expression vector by oligonucleotide-directed mutagenesis. The hybrid protein Hu-IFN- α A γ was expressed under the control of phage λ P L promoter. The protein was purified with the use of a monoclonal antibody against Hu-IFN-α or the COOH-terminal amino acid sequence of Hu-IFN-γ. The purified protein exhibited a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has antiviral activity on human and bovine cells. Unlike Hu-IFN-αA, but similar to Hu-IFN-γ, the hybrid Hu-IFN- α A γ can be phosphorylated by [γ- 32P]ATP and cAMP-dependent protein kinase. The phosphorylated molecule binds to the IFN- α β receptor. The introduction of a phosphorylation site into Hu-IFN-αA by fusion of the region of Hu-IFN-γ which contains the phosphorylation site provides a new reagent for studies of receptor binding, pharmacokinetics, and other studies where labeled interferons are useful. Furthermore, the introduction of phosphorylation sites into proteins provides a new principle for the preparation of a wide variety of reagents for many purposes.

Drosophila sn-glycerol-3-phosphate dehydrogenase isozymes are generated by alternate pathways of RNA processing resulting in different carboxyl-terminal amino acid sequences.

Glycerol-3-phosphate dehydrogenase (GPDH, Ec 1.1.1.8) in Drosophila melanogaster consists of a family of three isozymes designated as GPDH-1, 2, and 3 which exhibit a unique temporal and tissue-specific pattern of expression. While each isozyme is encoded by the same structural gene, they differ by the amino acid sequence at the COOH-terminal end, with GPDH-3 having the sequence Asn-His-Pro-Glu-His-Met-COOH and with GPDH-1 extended by the three amino acid sequence Glu-Asn-Leu-COOH. We have isolated both genomic and cDNA clones in order to examine the structure of the 3'-end of this gene and its transcriptional products. This analysis has demonstrated three classes of transcripts, each differing in the 3'-untranslated region and coding for an enzyme with a different COOH-terminal amino acid sequence. Each transcript is shown to arise through the differential expression of three isotype-specific exons at the 3'-end of the gene. We propose a model where the expression of each isotype-specific transcript is controlled through a developmentally regulated process of 3'-end formation and alternate splicing pathways of the pre-mRNA. Furthermore, since each transcript and its cognant isozyme is tissue-specific in expression, this model suggests a role for tissue-specific trans-acting factors in these processing events.

Existence of multiple forms of cytochrome P-450 scc purified from bovine adrenocortical mitochondria

Three fractions of cytochrome P-450 scc (denoted as fractions a, b, and c) were purified by a new procedure from bovine adrenocortical mitochondria. The amino-acid content analyses of these three fractions showed no difference. NH 2-terminal amino-acid sequences of cytochrome P-450 scc fractions, a and b agreed completely with the sequence deduced by nucleotide sequence of cDNA of cytochrome P-450 scc mRNA (Morohashi, K.; Fujii-Kuriyama, Y.; Okada, Y., Sogawa, K.; Hirose, T.; Inayama, S. and Omura, T. (1984) Proc. Natl. Acad. Sci. USA 81, 4647–4651), whereas t he sequence of fraction c showed a missing of isoleucine at the NH 2-terminal. COOH-terminal amino-acid sequences of fraction a, b and c were -Gln-Ala-COOH, identical with the deduced sequence from the cDNA. Measurements of the enzymatic activities of cholesterol side-chain cleavage reaction revealed no distinct difference among these three fractions. Although each of these fractions appeared as a single protein staining band upon SDS-polyacrylamide gel electrophoresis, these fractions showed heterogeneities upon two-dimensional electrophoresis and chromatofocusing. Fraction a contained the major form of cytochrome P-450 scc, and its isoelectric point was estimated to be pH 7.8 by isoelectric focusing under both native and denatured conditions, and this value was confirmed by chromatofocusing. Neither of the carbohydrate-specific stainings (such as periodic acid-Schiff staining and lectin-peroxidase stainings using concanavalin A, wheat-germ agglutinin, and soybean agglutinin) of purified cytochrome P-450 scc fractions after the electrophoretic resolution on SDS-polyacrylamide gel could show cytochrome P-450 scc fractions as glycoproteins, suggesting that the heterogeneities were not due to the glycosylation state.

Prothrombin fragment 1 X 2 X 3, a major product of prothrombin activation in human plasma.

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.

Bovine leukemia virus protease: purification, chemical analysis, and in vitro processing of gag precursor polyproteins.

Bovine leukemia virus protease was purified to homogeneity and assayed by using murine leukemia virus Pr65gag, a polyprotein precursor of the viral core structural proteins, as the substrate. A chemical analysis of the protease, including an amino acid composition and NH2- and COOH-terminal amino acid sequence analysis, revealed that it has an Mr of 14,000 and is encoded by a segment of the viral RNA located between the gag gene and the putative reverse transcriptase gene. As expected from the nucleotide sequence data (Rice et al., Virology 142:357-377, 1985), the reading frame for the protease is different from both the gag and reverse transcriptase reading frames. The 5' end of the protease open reading frame extends 38 codons upstream from the codon for the NH2-terminal residue of the mature viral protease and overlaps the gag open reading frame by 7 codons. The 3' end of the protease open reading frame extends 26 codons beyond the codon for the COOH-terminal residue of the mature protease and overlaps 8 codons of the reverse transcriptase open reading frame. Several lines of evidence, such as protein mapping of the gag polyprotein precursor, the characteristic structure of the mRNA, and promotion of the synthesis of a gag polyprotein precursor by lysine tRNA in vitro, suggest that the protease could be translated by frameshift suppression of the gag termination codon. In vitro synthesized bovine leukemia virus gag-related polyproteins were cleaved by the protease into fragments which were the same size as the known components of bovine leukemia virus, suggesting that the specificity of cleavage catalyzed in vitro by the purified protease is the same as the specificity of cleavage found in the virus.

Modification of a tyrosine residue in the carboxyterminal portion of human alpha 2-antiplasmin

Alpha*-antiplasmin, the main physiological plasmin inhibitor in human plasma, is a single chain glycoprotein with a molecular weight of 70,000, containing about 13 percent carbohydrate, about 500 amino acids and 3 disulfide bridges (1). antithrombin III, Alpha2-antiplasmin belongs to the same protein family as alpha d-antitrypsin and ovalbumin (2). Its COOH-terminal amino acid sequence was etermined and shown to extend at least 52 amino acids beyond the COOH-terminal end of antithrombin III (3). During the reaction between plasmin and alpha -antiplasmin a stable complex is formed following a plasmic attack at a specs ic Arg-Met peptide bond situated about 80 residues + from the C-terminus in alpha2-antiplasmin. This peptide bond is cleaved in the native complex indicating that most likely an ester bond is stabilizing the complex (4,5).

Brain-specific polypeptide 1B236 exists in multiple molecular forms.

The COOH-terminal amino acid sequence of the rat brain-specific polypeptide 1B236 was previously deduced from molecular cloning and nucleotide sequence determination of its mRNA and the 1B236 protein shown to exist in the rat brain. The amino acid sequence of 1B236 contained at least three peptide sequences demarcated by pairs of basic amino acids--a structure similar to known neuropeptide and hormone precursors--which suggested that the protein might be processed in vivo to generate peptides. We have developed radioimmunoassays specific for 1B236 with antibodies against three synthetic peptides corresponding to putative cleavage products of this protein and have used these assays to define the molecular forms of 1B236 in rat brain extracts. The most abundant form is of high molecular weight (ca. 100,000) and requires detergent for solubilization; hence, it is probably membrane-bound. However, a small fraction of the high molecular weight material is soluble in the absence of detergent. In addition, several low molecular weight species are detectable in brain extracts prepared under conditions preventing proteolysis. These molecules correspond in size to two of the possible products of proteolytic processing predicted from the amino acid sequence of 1B236. The multiplicity of 1B236 forms, together with other data, suggests that this protein undergoes extensive post-translational modification, including proteolytic processing to generate peptides that may be physiologically relevant.