A cDNA clone encoding a 15.501 Da photosystem I (PSI) subunit of barley was isolated using an oligonucleotide based on the NH2-terminal amino acid sequence of the isolated protein. The polypeptide, which migrates with an apparent molecular mass of 9.5 kDa on denaturing SDS-PAGE, has been designated PSI-N, and the corresponding gene is PsaN. Analysis of the deduced protein sequence indicates a mature protein of 85 amino acid residues and a molecular mass of 9818 Da. PSI-N is a hydrophilic, extrinsic protein with no predicted membrane-spanning regions. The transit peptide of 60 residues (5683 Da) contains a predicted hydrophobic -helix, suggesting that the protein is routed into the thylakoid lumen. Thus, PSI-N is the second known lumenal protein component associated with PSI, together with PSI-F. 相似文献
The complete amino acid sequence has been derived for the zymogen of streptococcal proteinase. The protein yielded a unique sequence containing 337 amino acids in a single polypeptide chain. The NH2-terminal residue of the zymogen is aspartic acid and the COOH terminus is proline. The signal peptide commonly associated with the intracellular form of many proteins secreted from eukaryotic cells was absent from the zymogen sequence. The transformation of the zymogen to the enzyme under controlled conditions of proteolysis by trypsin and by streptococcal protease itself involves the removal of 84 amino acid residues from the NH2 terminus of the zymogen. The zymogen-to-enzyme conversion is accompanied by a change in serological specificity. An intermediate, modified zymogen formed in the transformation process contains only 12 amino acid residues less than the zymogen but shows the serological reactivity of both the zymogen and the enzyme. 相似文献
ARGINYL-tRNA-protein transferase is a soluble enzyme from mammalian tissues which catalyses the transfer of arginine from arginyl-tRNA into peptide linkage specifically with NH2-terminal aspartic or glutamic acid residues of protein acceptors1,2. Molar equivalents of arginine are transferred to appropriate NH2-terminal residues2. The reaction differs in many respects from the transfer of amino-acids associated with protein synthesis de novo and is thought to be a mechanism for regulating the activity of acceptor proteins3. Many immunoglobulins possess aspartic or glutamic acid in the NH2-terminal position and it was interesting to examine whether arginylation of such residues might result in an alteration of activity of these proteins, for there is considerable evidence that the antibody-combining site is contained in the NH2-terminal region of both light4–7 and heavy8,9 chains. 相似文献
The complete amino acid sequence of rat liver microsomal cytochrome P-450d (induced by isosafrole) was deduced by microsequence analysis of the tryptic peptides after separation by reverse-phase HPLC and alignment by comparison to the cDNA sequence reported by K. Kawajiri, O. Gotoh, K. Sogawa, Y. Tagashira, M. Muramatsu, and Y. Fujii-Kuriyama (1984, Proc. Natl. Acad. Sci. USA, 81, 1649–1653). Results from the two approaches are in complete agreement with the exception of two residues, Lys-30 (Arg in cDNA) and Phe-261 (Ser in cDNA). As previously reported by us (L. H. Botelho, D. E. Ryan, P.-M. Yuan, R. Kutney, J. E. Shively, and W. Levin (1982, Biochemistry, 21, 1152–1155) the NH2-terminal sequence of the mature protein lacks the NH2-terminal Met residue. Comparison of the rat cytochrome P-450d sequence with the mouse cytochrome P3-450 cDNA sequence reported by S. Kimura, F. J. Gonzalez, and D. W. Nebert (1984, Nucl. Acids Res., 12, 2917–2928) reveals a high sequence homology with a total of 32 amino acid differences including six conferring charge changes. Prediction of the secondary structure of cytochrome P-450d yields a maximum of 17 helices, two of which may be poly(Pro)-like helices adjacent to potential membrane-spanning α-helices. Four of the α-helices are sufficiently hydrophobic to traverse the endoplasmic reticulum. The remaining helices are largely amphiphilic. Analysis of the helices in reference to predicted membrane topology suggests that cytochrome P-450d either (1) has one large and one small globular domain separated by a transmembrane domain and anchored by NH2-terminal and COOH-terminal transmembrane domains, or (2) has one large globular domain anchored at both ends by transmembrane domains. 相似文献
Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (Mr ca. 29,000), and the S-peptide (Mr 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH2-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH2-terminal 60-amino acid peptide. 相似文献
Protamine of the archaeogastropod mollusc Monodonta turbinata has been isolated and characterized. With a mass of 13,476 Da, it is the largest known prolamine. Amino acid sequence of this protamine (106 residues) was established from data provided by automated sequence analysis and mass spectrometry of the protein and of its fragments. The primary structure of the NH2-terminal region exhibits repetitive sequence motifs Basic-Ser (mainly R-S) and both central and COOH-terminal regions are composed by arginine clusters. The amino acid sequence of Monodonta turbinata protamine shows structural similarities with other protamines from invertebrates and from birds and mammals. 相似文献
Acyl-peptide hydrolase catalyzes the removal of an N alpha-acetylated amino acid residue from an N alpha-acetylated peptide. Two overlapping degenerate oligonucleotide probes based on the sequence of a CNBr tryptic peptide, derived from purified rat acyl-peptide hydrolase, were synthesized and used to screen a rat liver lambda gt11 cDNA library. A 2.5-kilobase cDNA was cloned and sequenced. This clone contained 2364 base pairs of rat acyl-peptide hydrolase sequence but lacked a translational initiation codon. Using a 220-base pair probe derived from near the 5'-end of this almost full-length cDNA to rescreen the library, full-length clones were isolated, which contained an in-frame ATG codon at nucleotides 6-8 and encoded the NH2-terminal sequence, Met-Glu-Arg-Gln.... The DNA sequence encoded a protein of 732 amino acid residues, 40% of which were confirmed by protein sequence data from 19 CNBr or CNBr tryptic peptides. The isolated enzyme is NH2-terminally blocked (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), and based on the NH2-terminal protein sequence deduced from the DNA sequence and the sequence of the most NH2-terminal CNBr peptide, it is likely that the NH2-terminal residue is an acetylated methionine residue, since such residues are frequently juxtaposed to glutamyl residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). The RNA blot analysis revealed a single message of 2.7 kilobases in various rat tissues examined. Although this enzyme is known to be inhibited by diisopropyl fluorophosphate and acetylalanine chloromethyl ketone (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), no strong similarity in protein sequence has been found with other serine proteases. This result suggests that acyl-peptide hydrolase may be a unique serine protease. 相似文献
The amino acid sequences of pyridoxal-binding tetrapeptide and the NH2-terminal portion of aspartate transaminase from B were analyzed and compared with those of the corresponding parts of the cytosolic and mitochondrial isozymes from pig heart. After borohydride reduction and chymotryptic digestion of the enzyme, a pyridoxal-containing peptide was isolated, showing the sequence, Ser-Lys(Pxy)-Asn-Phe, identical with that of the cytosolic isozyme. The NH2-terminal sequence was determined up to 33 residues with a liquid phase sequence analyzer. Nearly the same degree of homology was observed among the NH2-terminal sequences of the three aspartate transaminases. 相似文献
By reducing the amount of ginkgo water-soluble polysaccharides, which occupy about 35% of the wet seed mass and interfere with the extraction of RNA, cDNA-quality mRNA was obtained from developing seeds of Ginkgo biloba. Based on the NH2-terminal 17-amino acid sequence and an internal 12-amino acid sequence derived from the basic subunit of ginnacin, 11S-seed storage protein family of ginkgo, two degenerate oligonucleotide primers were synthesized and used for polymerase chain reaction (PCR). The resulting PCR product was used for screening the above endosperm cDNA library, and a plaque carrying the 1614 bp cDNA insert, which contained the entire coding region for a precursor of ginnacin was isolated. This is the first reported cloning of cDNA from ginkgo seeds. The deduced primary sequence is composed of a signal peptide segment (25 amino acid residues) and an acidic subunit (248 residues) followed by a basic subunit (187 residues). It was also found that the post-translational cleavage site in the ginnacin precursor is the Asn-Asn rather than the Asn-Gly bond found in a variety of the major subunit precursors in 11S seed protein family known to date. We showed that a purified soybean extract and an extract of ginkgo seeds can specifically hydrolyze-Asn248-Asn249- but not -Asn249-Val250-, in the heptapeptide Gly-Asn248-Asn-Val-Glu-Glu-Leu that corresponds to the ginnacin cleavage region.Abbreviations SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- PVDF
polyvinylidene difluoride
- CBB
Coomassie Brilliant Blue
- HPLC
high-performance liquid chromatography
- bp
base pair(s)
- PCR
polymerase chain reaction 相似文献
Porcine low Mr phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH2-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH2-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5-phosphate (K=21M) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low Mr PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5-phosphate inhibition has been proposed.Abbreviations used PTPase
phosphotyrosine protein phosphatase
- TFA
trifluoroacetic acid
- SDS
sodium dodecylsulfate
- T
tryptic peptides
- SP
endoproteinase Glu-C peptides
- FAB
fast atom bombardment
- Ac
acetyl
- HPLC
high-performance liquid chromatography
- OPA
o-phtaldialdehyde
- PMSF
phenylmethylsulfonyl fluoride
- CD45
leukocyte common-antigen PTPase
- LAR
leukocyte-antigen-related PTPase
- PTP IB
human placental PTPase 相似文献
Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys-Arg, Arg-Arg, and Lys-Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin-containing peptides, peptide E and BAM-22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM-22P at the Lys-Arg site between the two basic residues. The Arg-Arg site of both peptide E and BAM-22P was cleaved at the NH2-terminal side of the paired basic residues to generate [Met]-enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH2-terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys-Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH2-terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme. 相似文献
A serine protease inhibitor with a molecular mass of 6106±2Da (designated as InhVJ) was isolated from the tropical anemone Radianthus macrodactylus by a combination of liquid chromatography methods. The molecule of InhVJ consists of 57 amino acid residues, has three disulfide bonds, and contains no Met or Trp residues. The N-terminal amino acid sequence of the inhibitor (19 aa residues) was established. It was shown that this fragment has a high degree of homology with the N-terminal amino acid sequences of serine protease inhibitors from other anemone species, reptiles, and mammals. The spatial organization of the inhibitor at the levels of tertiary and secondary structures was studied by the methods of UV and CD spectroscopy. The specific and molar absorption coefficients of InhVJ were determined. The percentage of canonical secondary structure elements in the polypeptide was calculated. The inhibitor has a highly ordered tertiary structure and belongs to mixed α/β-or α + β polypeptides. It was established that InhVJ is highly specific toward trypsin (Ki 2.49 × 10?9 M) and α-chymotrypsin (Ki 2.17 × 10?8 M) and does not inhibit other proteases, such as thrombin, kallikrein, and papain. The inhibitor InhVJ was assigned to the family of the Kunitz inhibitor according to its physicochemical properties. 相似文献
An artificially inserted extra peptide (21 amino acid peptide) between the B. subtilisα-amylase signal peptide and the mature thermostable α-amylase was completely cleaved by B. subtilis alkaline protease in vitro. The cleavage to form a mature enzyme was observed between pH 7.5 and 10, but not between pH 6.0 and 6.5, although a similar protease activity toward Azocall was observed between pH 6.0 and 7.5. To analyze the effects of pH on the cleavage, CD spectra at pH 6, 8, and 11 of the NH2-terminally extended thermostable α-amylase were analyzed and the results were compared with those of the mature form of the α-amylase. It is suggesteded that the cleavage of the NH2-terminally extended peptide is controlled by the secondary and tertiary structure of the precursor enzyme. Similar cleavage of different NH2-terminally extended peptides by the alkaline protease was also found in other hybrid thermostable α-amylases obtained. 相似文献
The rate of reaction of glyceraldehyde with a series of peptides was found to be dependent on their amino acid composition, sequence, and chain length. The presence of a histidine near the NH2-terminal increased the rate of glycation, whereas the presence of a carboxyl group near the reaction site led to a decrease in reaction rate. In general, tripeptides reacted faster than dipeptides, and dipeptides reacted faster than amino acids. Sodium phosphate and 2,3-diphosphoglycerate enhanced the rate of reaction of glyceraldehyde with all the dipeptides tested. Sodium chloride inhibited the reaction in phosphate buffer, but not in HEPES buffer. The NH2-terminal heptapeptide from the -chain of human hemoglobin A (HbA), where histidine is the second residue, reacted with glyceraldehyde faster than the NH2-terminal hexapeptide from the -chain. The glycation of tetrameric human Hb by glyceraldehyde was found to be dependent on the ligation state of the protein since deoxy-HbA reacted about 50% more with glyceraldehyde than did liganded HbA. The enhanced glycation of deoxy HbA was mainly attributable to the more extensive reaction at the NH2-terminal of the -chain. The presence of a histidine adjacent to the NH2-terminal at this site may facilitate the Amadori rearrangement. The glycation of horse Hb in which the second residue is glutamine was not increased under anaerobic conditions. 相似文献
The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (Kd=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (Kd=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (Kd=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH2-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH2-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH2-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985. 相似文献
Automated Edman degradation of a testis-specific basic protein isolated from the rat gave the following NH2-terminal sequence of amino acids: Cleavage of the native protein with cyanogen bromide produced two fragments which were purified by gel filtration. Amino acid analysis of the smaller fragment revealed it to be the NH2-terminal undecapeptide resulting from cleavage at Met11. The partial sequence analysis of the intact protein coupled with compositional analyses of these cyanogen bromide peptides indicate that the basic testis protein contains 24 basic amino acids and a single methionine in a sequence of 54 amino acids. 相似文献
Digestion of rabbit liver fructose 1,6-bisphosphatase with subtilisin followed by denaturation of the protein yields a peptide containing 60 amino acid residues, including the blocked NH2-terminus. This peptide has the following sequence: Ac-Ala-Asp-Lys-Ala-Pr o-Phe-Asp-Thr-Asp-Ile-Ser-Thr-Met-Thr-Arg-Phe-Val-Met-Glu-Glu-Gly-Arg-Ly s-Ala-Gly-Gly-Thr-Gly-Glu-Met-Thr-Gln-Leu-Leu-Asn-Ser-Leu-Cys-Thr-Ala-Va l-Lys-Ala-Ile-Ser-Thr-Ala-Val-Arg-Lys-Ala-Gly-Ile-Ala-His-Leu-Tyr-Gly-Ile-Ala. 相似文献
CD53 is an N-glycosylated pan-leucocyte antigen of 35–42 000 Mr. The sequence of the CD53 polypeptide deduced from a cDNA clone is 219 amino acids in length. It appears to lack a conventional leader sequence because the deduced NH2-terminal amino acid sequence is very similar to the rat MRC OX-44 and human CD37 antigens. The CD53 molecule is likely to consist of four transmembrane regions and a major extracellular hydrophilic loop containing two potential N-glycosylation sites. It is suggested that the CD53 glycoprotein is the true human homologue of the rat OX-44 antigen, rather than the CD37 antigen of more restricted expression and lower NH2-terminal sequence similarity to OX-44.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M37033.
Offprint requests to: P. Angelisová. 相似文献
The sequence coding for a snake venom phospholipase A2 (PLA2), BJUPLA2, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA2's. A striking feature of the cDNA is the high sequence conservation of the 5 and 3 untranslated regions in cDNAs coding for PLA2's from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA2 with group II PLA2's in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA2's.The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X76289.Correspondence to: A.M. Moura-da-Silva 相似文献
Sequence analysis by the automated Edman degradation shows that dopamine -hydroxylase (dopamine -monooxygenase; EC 1.14.17.1) from bovine adrenal medulla contains equal amounts of NH2-terminal alanine and serine residues. The sequence data are in agreement with the proposal that this enzyme consists of two types of polypeptide chains which are identical in the NH2-terminal ends, except that one of the chains lack the NH2-terminal tripeptide Ser-Ala-Thr. 相似文献