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1.
Pancreatic RNAase (ribonuclease) from the pike whale (lesser rorqual, Balaenoptera acutorostrata) was isolated by affinity chromatography. The protein was digested with different proteolytic enzymes. Peptides were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. The amino acid sequence of peptides was determined by the dansyl-Edman method. Although we do not have an amino acid composition for the whole protein, all peptide bonds were overlapped by one or more peptides. Residues 85-96 are bridged by a peptide of unstaisfactory composition and the sequence here depends, at least in part, on homology for its confirmation. Another region in which a similar situation obtains is residues 39-40. This pancreatic RNAase differs at 24-33% of the positions from all other mammalian pancreatic RNAases sequenced to date, except for pig RNAase, from which it differs by 19%. This indicates that whale RNAase has evolved independently during the larger part of the evolution of the mammals. Lesser-rorqual pancreatic RNAase is partially glycosidated (30%) at asparagine-76 in an Asn-Ser-Thr sequence (residues 76-78). Pig RNAase also has carbohydrate attached to asparagine-76 and is identical with lesser-rorqual RNAase in residues 76-98. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50066 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms ginen in Biochem. J. (1976) 135, 5. 相似文献
2.
The amino-acid sequence of kangaroo pancreatic ribonuclease 总被引:3,自引:0,他引:3
Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found. 相似文献
3.
The complete amino acid sequence of a constitutive form of liver microsomal cytochrome P-450 总被引:9,自引:0,他引:9
The complete covalent structure of the constitutive cytochrome P-450, form 3b, from rabbit liver microsomes was determined. The apocytochrome contains 490 amino acid residues in a single polypeptide chain, Mr = 55,860. Peptides from selective chemical and proteolytic cleavages were isolated by a combination of gel filtration and high performance liquid chromatography and sequenced by automated Edman degradation. Overlapping peptide sequences were used to deduce the complete sequence. The constitutive form is only 46% homologous to the phenobarbital-induced cytochrome P-450 (Heinemann, F. S., and Ozols, J. (1983) J. Biol. Chem. 258, 4195-4201) and contains a deletion at position 22. Strongly conserved regions include Cys435 and a previously identified tryptic peptide, residues 345-357. The distribution of hydrophobic segments is used to predict the membrane topology of the protein, and four possible orientations of this protein in the membrane are presented. 相似文献
4.
Protein L19, a component of the Escherichia coli 50S ribosomal subunit implicated in 30S-50S subunit interaction was sequenced by the dansyl-Edman method. L19 consists of a single polypeptide chain of 114 amino acids giving a calculated molecular weight of 13 002. Peptides obtained from various enzymatic cleavages were isolated on thin-layer peptide maps or gel filtration. Automated Edman degradation using a liquid phase sequenator was carried out on the whole protein as well as on a large 58-residue fragment arising from digestion with Staphylococcus aureus protease. Every position in protein L19 was confirmed at least twice. Results of secondary structure estimation and homologies with other E. coli ribosomal protein sequences are presented. 相似文献
5.
Amyloid fibrils derived from V-region together with C-region fragments from a lambda II-immunoglobulin light chain (HAR) 总被引:3,自引:0,他引:3
Amyloid fibril proteins were isolated from the spleen of a patient with IgD(lambda)-plasmocytoma by extraction and gel filtration in 5M guanidine hydrochloride. The molecular mass of the predominant polypeptide chain was approximately 5000 Da. Its complete amino-acid sequence was elucidated by stepwise automated degradation of the carboxymethylated polypeptide chain and by structural studies of tryptic and thermolysinolytic cleavage products. The length of the polypeptide chain was 58 to 59 residues and it was homologous to the amino acids in positions 8 through 65 of the variable part of an lambda-type immunoglobulin light chain, which was most closely related to the lambda II subgroup. The N-terminal sequence of this amyloid fibril protein proved to be heterogeneous, indicating cleavage after the amino acids in positions 7 and 8. Peptides from the constant part of the lambda-chain were unexpectedly found in the tryptic digest of the denatured amyloid protein HAR. One polypeptide derived from the constant region was separated from the main component by high performance liquid chromatography. Its amino-acid sequence commenced at position 111 and could be traced in 41 steps. In this case, at least two constant region fragments were shown to be constituents of the amyloid fibril protein. The association of fragments from the variable as well as the constant region is discussed with respect to amyloid formation. 相似文献
6.
The covalent structure of hepatic microsomal epoxide hydrolase. II. The complete amino acid sequence 总被引:4,自引:0,他引:4
The complete amino acid sequence of hepatic microsomal epoxide hydrolase has been determined. The protein contains 455 amino acid residues in a single polypeptide chain and has Mr = 52,691. Peptides from selective chemical and proteolytic cleavages were isolated by a combination of gel filtration and high performance liquid chromatography and sequenced by automated Edman degradation. Overlapping peptide sequences were used to deduce the complete sequence. This is the first epoxide hydrolase and the third microsomal enzyme for which the complete sequence has been determined. 相似文献
7.
H G Pollock J W Hamilton J B Rouse K E Ebner A B Rawitch 《The Journal of biological chemistry》1988,263(20):9746-9751
Insulin, pancreatic polypeptide, glucagon, oxyntomodulin, and two distinct glucagon-like peptides were isolated from acidic ethanol extracts of bullfrog pancreas by gel filtration followed by high pressure liquid chromatography. The amino acid sequences of pancreatic polypeptide, oxyntomodulin, and both glucagon-like peptides were determined. Frog pancreatic polypeptide contains 36 amino acid residues and has a COOH-terminal phenylalaninamide. It is more homologous with human pancreatic polypeptide (61%) than other characterized members of this family of peptides. Frog glucagon has an amino acid composition identical to the NH2-terminal 29 residues of the larger, more abundant oxyntomodulin and was not sequenced. The finding of a single form of glucagon and oxyntomodulin, but two glucagon-like peptides in frog pancreas extract is similar to that found or deduced for mammals. 相似文献
8.
Amino- and carboxy-terminal sequence of mouse J chain and analysis of tryptic peptides 总被引:3,自引:0,他引:3
Mouse J chain was isolated from an IgM-producing hybridoma by gel filtration and ion-exchange chromatography. The sequence of the amino-terminal 25 residues was determined. At these positions, the results agree with the amino acid sequence deduced from the cDNA sequence determined previously by Koshland and co-workers and indicate that a leader sequence terminating in glycine is removed to form the mature J chain. Tryptic peptides of J chain were isolated by high pressure liquid chromatography and their amino acid compositions were compared with those expected from the cDNA sequence. The amino acid sequence of the carboxy-terminal peptide and a mixture of two other peptides was determined. The results were consistent with the cDNA sequence except that we found valine, not leucine, at position 67, and arginine, not glycine, at position 117. The presence of aspartic acid at the carboxy-terminus, as predicted from the cDNA, indicates that processing does not occur at this end of the polypeptide chain. Upon amino acid analysis, glucosamine was found in tryptic peptides 47-57 and 47-58. J chain was also cleaved at aspartylproline bonds with formic acid and the unfractionated digest was subjected to automated Edman degradation. The mixed sequence was consistent with the sequence deduced from the cDNA at positions 1 to 13, 28 to 40, 52 to 64, and 73 to 85. In conjunction with the results obtained previously by analysis of cDNA, these data show that mouse J chain is a polypeptide containing 137 amino acid residues, 93 of which are identical to residues in human J chain. 相似文献
9.
G O Levina L G Makevnina S F Sherstiuk T S Paskhina 《Biokhimii?a (Moscow, Russia)》1985,50(2):325-336
A highly purified preparation of low molecular weight kininogen (LMrK) was isolated from the plasminogen-free rabbit blood plasma, using chromatography on DEAE-Sepharose CL-6B, gel filtration on Ultrogel AcA 34 and Sephadex G-100 as well as gradient chromatography on a hydroxylapatite column. The yield of the 320-fold purified LMrK was 16%. Trypsin released 13-14 micrograms-eq. of bradykinin (BK) from 1 mg of LMrK or 0.85-0,95 mol of BK per mol of kininogen. Rabbit LMrK consists of one polypeptide chain of Mr 69 000 and pI 4.63. Porcine pancreatic kallikrein splits off kinin from the LMrK polypeptide chain by disrupting two peptide bonds resulting in the formation of S-S-bound two chain molecule. After reduction of the S-S bonds by dithioerithritol the latter is separated into a heavy (Mr 61 000) and light (Mr 6 800) chains. A biologically active peptide was isolated from the products of CNBr cleavage of LMrK. This peptide consists of Lys-BK elongated from the C-terminal with several amino acid residues. Rabbit LMrK closely resembles human LMrK in terms of Mr, pI and location of the kinin fragment in the protein molecule. 相似文献
10.
The RNAase inhibitor from rat testis has been purified to homogeneity. The purified protein appeared as a single spot after two-dimensional electrophoresis. The calculated Mr value is 48,000 which coincides with that obtained for the native protein on gel filtration chromatography, thus indicating a single polypeptide chain. The amino acid composition and the characteristics of the inhibitor activity are reported and compared to those of other RNAase inhibitors from mammalian tissues. The naturally occurring ribonuclease-RNAase inhibitor complex from rat testis has also been studied and compared with the rat testis inhibitor-RNAase A as model complex. The ribonuclease released from the natural rat testis complex showed heterogeneity of size. The significance of the rat testis ribonuclease/RNAase inhibitor system is discussed in terms of the important functionality of this organ. 相似文献
11.
Peptides isolated from several lactate dehydrogenases (EC 1.1.1.27) have been characterized and sequenced. These peptides include much of the substrate binding site as well as the loop of polypeptide chain which shows major conformational changes following coenzyme binding. Despite significant differences in catalytic properties, the amino acid sequence in these two active site regions of the molecule is highly conserved in most cases. A noteable exception is cysteine 165 which at one time was thought to be essential for enzymatic activity. The lactate dehydrogenases investigated were isolated from rabbit muscle, chicken heart, beef heart, and lobster tail. 相似文献
12.
The primary structures of the two isoforms of ribonuclease U2, RNAases U2-A and U2-B, were analysed and compared with each other. Among the chymotryptic peptides obtained from the reduced and S-carboxymethylated enzymes, only peptides C-3 were different from each other in terms of chromatographic behaviour on reverse-phase h.p.l.c. On the basis of chemical analyses of these peptides, it was shown that RNAase U2-B had an isopeptide bond in which Asp-32 was linked to Gly-33 through the beta-carboxy group in its side chain instead of the alpha-carboxy group. Deamidation of Asn-32 in RNAase U2-A led to the formation of this unusual linkage. The previously reported sequence of RNAase U2 [Sato & Uchida (1975) Biochem. J. 145, 353-360] was corrected by changing amino acid residues at eight different positions and by inserting an asparagine residue at position 32. The numbering of the positions of amino acid residues located downstream of Asn-32 was therefore shifted by 1. Accordingly, RNAase U2-A was shown to be composed of 114 amino acid residues. 相似文献
13.
The primary structure of a 1,4-β-glucan cellobiohydrolase from the fungus Trichoderma reesei QM 9414
The sequence of the approx. 490 amino acid residues of the main 1,4-β-glucan cellobiohydrolase (CBH I) (EC 3.2.1.91) from culture filtrates of the fungus Trichoderma reesei QM 9414 has been established by automatic liquid phase Edman degradation. Peptides obtained by chemical and enzymatic cleavage of the reduced and S-carboxymethylated protein were isolated by a combination of gel filtration and high-performance liquid chromatography. The amino-terminus of the single polypeptide chain is blocked by a pyroglutamyl residue. Most of the neutral carbohydrate present in the glycoprotein is bound within a short region near the carboxyl-terminus. Three attachment sites of glucosamine residues have also been established. 相似文献
14.
The role of ribosomal ribonucleic acid in the structure and function of mammalian brain ribosomes 总被引:3,自引:1,他引:2
In order to resolve the functional role of intact rRNA in polypeptide chain elongation mouse brain ribosomes were treated with dilute pancreatic or T(1) RNAase (ribonuclease). After RNAase treatment, several physical-chemical properties as well as the functional activity of the ribosomes were measured. RNAase treatment resulted in the extensive hydrolysis of both 18S and 28S rRNA; however, the sedimentation properties of mono-ribosomes were unaltered and more than 90% of the relatively low-molecular-weight RNA fragments remained associated with ribosome particles. Analysis of the ability of RNAase-treated ribosomes to participate in cell-free protein synthesis showed that ribosomes with less than 2% intact rRNA retained more than 85% of their activity in polyphenylalanine incorporation. Proof that the incorporation of phenylalanine by ribosomes with hydrolysed rRNA actually represented active translocation was obtained by the effective inhibition of incorporation by diphtheria toxin. In addition, the oligopeptide products of protein synthesis could be identified by BD (benzoylated diethylaminoethyl)-cellulose column chromatography. Analysis of the size distribution of oligopeptides synthesized by normal and RNAase-treated ribosomes showed no significant differences which indicated that there was no change in the proportion of ribosomes engaged in protein synthesis. Thus strong RNA-protein and protein-protein interactions must serve to maintain the functional integrity of ribosomes even when the rRNA is extensively degraded. The ability of the enzyme-treated ribosomes to efficiently incorporate amino acids clearly demonstrated that ;intact' rRNA is not required for protein-synthetic activity. 相似文献
15.
Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein. 总被引:6,自引:1,他引:5
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G Montalto J Bonicel L Multigner M Rovery H Sarles A De Caro 《The Biochemical journal》1986,238(1):227-232
Pancreatic stone protein (PSP) is the major organic component of human pancreatic stones. With the use of monoclonal antibody immunoadsorbents, five immunoreactive forms (PSP-S) with close Mr values (14,000-19,000) were isolated from normal pancreatic juice. By CM-Trisacryl M chromatography the lowest-Mr form (PSP-S1) was separated from the others and some of its molecular characteristics were investigated. The Mr of the PSP-S1 polypeptide chain calculated from the amino acid composition was about 16,100. The N-terminal sequences (40 residues) of PSP and PSP-S1 are identical, which suggests that the peptide backbone is the same for both of these polypeptides. The PSP-S1 sequence was determined up to residue 65 and was found to be different from all other known protein sequences. 相似文献
16.
Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain 总被引:5,自引:0,他引:5
Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the nucleotide binding site by the substrate photoaffinity analogue [gamma-32P]-8-azido-dATP. The alpha and beta polypeptides of photolabeled terminal transferase were resolved by high-performance liquid chromatography. The beta polypeptide was digested with trypsin and fractionated by reverse-phase chromatography. Two 32P-containing fractions were isolated and subjected to amino acid sequence analysis. Peptides were identified as Ile209-Lys232 (B26) and Val233-Lys239 (B27). Peptide B26 was further resolved into two overlapping species; one contained an additional lysine residue at the N-terminus which resulted from tryptic cleavage between Lys207 and Lys208. In order to ensure that the sequenced peptides corresponded to the photolabeled species, we devised an anion-exchange procedure to isolate photolabeled peptides from the mixture. Analysis of photolabeled peptides from terminal transferase alpha beta using DEAE-cellulose chromatography followed by reverse-phase HPLC confirmed that the photolabeled species were peptides B26 and B27. Peptide B26, the major photolabeled species, contained a conserved octapeptide region found in several eucaryotic DNA polymerases. In addition, peptide B27 was flanked by a sequence that has been implicated in triphosphate binding in other proteins. Structure predictions, based on sequence data, place the two peptides identified by photolabeling in spatial proximity consistent with the participation of both in the nucleotide binding domain. 相似文献
17.
Purification, chain separation and sequence of the MRC OX-8 antigen, a marker of rat cytotoxic T lymphocytes. 总被引:13,自引:4,他引:9
The MRC OX-8 antigen is a marker of the rat cytotoxic T lymphocytes that consists of disulphide-linked chains of mol. wts. 37 and 32 kd. It is thought to be equivalent to the human T8 and mouse Lyt2,3 antigens (all now called CD8 antigens). MRC OX-8 antigen was purified from thymocytes using a monoclonal antibody column and because antigenicity was retained after reduction and alkylation the two polypeptide chains could be separated by a subsequent affinity chromatography step. Peptides were isolated from each chain and their sequences determined. A cDNA probe coding for the mouse CD8 antigen (pLY2C-1 provided by Dr L. A. Herzenberg) was used to obtain rat cDNA clones from which the sequence of the equivalent rat molecule was determined. Peptides from the 32-kd chain were identified in this translated sequence whereas peptides from the 37-kd chain were not. The 32-kd polypeptide sequence consisted of 210 amino acids and had one possible N-linked glycosylation site. The N-terminal part of the sequence was surprisingly different from both its mouse and human counterparts but, as in the other two species, it showed a clear relationship to Ig V domains. 相似文献
18.
R M Magee C Shaw I Fairweather L Thim C F Johnston D W Halton 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1991,100(3):507-511
1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica. 2. Gel permeation chromatography of an acid ethanol extract of cattle flukes showed that the peptide is similar in size to mammalian (bovine) PP. 3. The Fasciola peptide was purified to homogeneity by means of reverse-phase HPLC, employing different column chemistries. 4. The purified peptide was sequenced using automated gas-phase Edman degradation and the first 24 amino acid residues determined. 相似文献
19.
Dromedary pancreatic PLA2 (DrPLA2) was purified from delipidated pancreases. Pure protein was obtained after heat and acidic treatment (70 degrees C; pH 3.0), precipitation by ammonium sulphate and ethanol respectively, followed by sequential column chromatographies on Sephadex G-50, MonoS Sepharose, MonoQ Sepharose and C-8 reverse phase high pressure liquid chromatography. Purified DrPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 13748.55 Da. A specific activity of 600 U/mg for purified DrPLA2 was measured at optimal conditions (pH 8.0 and 37 degrees C) in the presence of 3 mM NaTDC and 7 mM CaCl(2) using PC as substrate. The sequence of the first fourteen amino-acid residues at the N-terminal extremity of DrPLA2 was determined by automatic Edman degradation. One single sequence was obtained and shows a close similarity with all other known pancreatic secreted phospholipases A2. 相似文献
20.
E Fioretti I Binotti D Barra G Citro F Ascoli E Antonini 《European journal of biochemistry》1983,130(1):13-18
Four protein protease inhibitors (I, II, III, IV) having low molecular weights (10 600-6500) and basic isoelectric points were isolated by affinity chromatography from bovine spleen. Inhibitor IV was identified as the basic pancreatic trypsin inhibitor (Kunitz inhibitor); the presence and distribution of components I, II and III vary in the different bovine organs. Spleen inhibitors I, II, III and IV were purified by ion-exchange chromatography; they form 1:1 complexes with trypsin and inhibit enzymatic activity of trypsin, chymotrypsin and kallikrein. Inhibitors I, II and III contain carbohydrate moieties (7-4%) covalently bound to the polypeptide chain. Specific basic pancreatic trypsin inhibitor antiserum has shown the complete identity between inhibitor IV and the basic pancreatic trypsin inhibitor, while partial cross-reactivity between the basic pancreatic trypsin inhibitor and inhibitors I, II and III can be seen from a double immunodiffusion test. 相似文献