Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Globin prepared from hemoglobin of adult tupai (Tupaia glis) was separated into alpha and beta polypeptide chains by CM-cellulose column chromatography. The S-aminoethylated alpha polypeptide chain and S-carboxymethylated beta polypeptide chain were each digested with trypsin, and the sequences of all the peptides thus obtained were established. The ordering of these tryptic peptides in the alpha and beta polypeptide chains was deduced from the homology of their primary structures with that of human adult hemoglobin. In this way the primary structures of the alpha and beta polypeptide chains of tupai hemoglobin were established; 27 amino acids in the alpha polypeptide chain and 26 in the beta chain differ from those in human adult hemoglobin.
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PMID:Amino acid sequences of the aplpha and beta chains of adult hemoglobin of the tupai, Tupaia glis. 41 Aug 1

Synthetic mRNAs (i.e. cRNA alpha and cRNA beta) were obtained by cell-free transcription of M13 KS(+) (Bluescript) expression vectors which contained the entire coding region of the alpha or beta subunits of lamb kidney Na,K-ATPase. Translation in reticulocyte lysates of cRNA alpha yielded full length alpha polypeptide, as well as a limited array of immunoprecipitable lower molecular weight products. cRNA beta yielded a single immunoprecipitable full length polypeptide. Association of the alpha polypeptide with the microsomal membranes was obtained only co-translationally. Fifteen to 50% of the membrane-associated alpha subunit was resistant to extraction with alkali. The resistance of a 29-kDa fragment to trypsinolysis indicated that the alpha subunit was inserted into microsomal membranes. In the presence of dog pancreatic microsomes, the beta polypeptide was glycosylated as indicated by the appearance of three higher molecular weight polypeptides that were sensitive to endoglycosidase H and bound to Concanavalin A. The beta subunit was predominantly translocated into the lumen of the endoplasmic reticulum since 90% of the mass of the membrane-associated beta polypeptide was resistant to trypsin (i.e. reduced in size from 40 kDa to 37.5 kDa), and 95% of all of the beta chains were resistant to extraction with alkali. Neither the alpha nor the beta subunits have NH2-terminal leader signal sequences, but both may require the signal recognition receptor for membrane insertion, as evidenced by inhibition of incorporation of both subunits into microsomes pretreated with N-ethylmaleimide. Simultaneous translation of cRNA alpha and cRNA beta did not enhance membrane insertion of either the alpha or beta polypeptide.
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PMID:Cell-free transcription and translation of Na,K-ATPase alpha and beta subunit cDNAs. 169 72

Analysis of the transforming growth factor alpha (TGF alpha) cDNA predicts that the mature TGF alpha polypeptide is cleaved from the extracellular domain of its precursor, which is an integral membrane protein. Furthermore, the cleavage sites for the release of this mitogen are compatible with the participation of an elastaselike protease. We have immunohistochemically localized TGF alpha to the vascular smooth muscle cells in the arterioles. To investigate whether polymorphonuclear (PMN) leukocytic elastase, a blood-borne protease, could process the cell surface TGF alpha, NR6 cells were transfected with the rat TGF alpha cDNA. The cDNA encoded the entire open reading frame, and its expression was under the control of the mouse metallothionein I promoter. A cloned transfectant, termed 1B2, synthesized the TGF alpha precursor in a zinc-inducible manner, and the precursor was localized to the cell surface. Western blot (immunoblot) analysis indicated that treatment of the zinc-induced 1B2 cells with either PMN leukocytic or pancreatic elastase resulted in the release of the mature TGF alpha polypeptide. The released TGF alpha was bioactive, as it was capable of both competing with epidermal growth factor for binding to its receptor and stimulating [3H]thymidine incorporation in the mitogenic assay. Formaldehyde fixation of the 1B2 cells eliminated basal release of TGF alpha but allowed normal processing by both PMN leukocytic and pancreatic elastase to occur. However, human cathepsin G, bovine pancreatic alpha 1-chymotrypsin, collagenase, trypsin, subtilisin, and plasmin failed to release any detectable fragments of the TGF alpha precursor from the fixed cells. The location of TGF alpha in the arterioles and ability of PMN leukocytic elastase to process the membrane-bound TGF alpha precursor suggests a novel role for this elastase at the wound site.
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PMID:Transforming growth factor alpha in arterioles: cell surface processing of its precursor by elastases. 220 95

Determinations of reaction stoichiometry demonstrate that the covalent incorporation of one molecule of 5'-isothiocyanatofluorescein can inactivate one molecule of sodium and potassium ion activated adenosinetriphosphatase in agreement with earlier determination of this stoichiometry. Several different modified peptides are produced, however, when the modified enzyme is digested with trypsin. One of these peptides has been identified as HLLVMK (thioureidylfluorescein)GAPER by use of a specific immunoadsorbent. The modified lysine is lysine 501 in the amino acid sequence of the alpha polypeptide of (Na+ + K+)-ATPase. This peptide has been previously isolated from such digests [Farley, R. A., Tran, C. M., Carilli, C. T., Hawke, D., & Shively, J. E. (1984) J. Biol. Chem. 259, 9532-9535]. The other specifically modified peptides have been purified and identified by amino acid sequencing. Their sequences identify lysine 480 and lysine 766 from the alpha polypeptide as amino acids modified by 5'-isothiocyanatofluorescein in reactions sensitive to the addition of ATP and responsible for inactivation of the enzyme.
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PMID:Any of several lysines can react with 5'-isothiocyanatofluorescein to inactivate sodium and potassium ion activated adenosinetriphosphatase. 255 64

The number of free cysteines in each polypeptide of acetylcholine receptor from the electric organ of Torpedo californica has been assessed by alkylating the native protein with N-ethylmaleimide and iodoacetamide during homogenization of the tissue and alkylating the polypeptides with N-ethylmaleimide as they were unfolded in solutions of dodecyl sulfate. The cysteines unavailable for alkylation could be accounted for as specific cystines, connecting positions in the amino acid sequences of the individual polypeptides. Unreduced, alkylated polypeptides of acetylcholine receptor were digested with thermolysin or trypsin. Cystine-containing peptides in the chromatograms of the digests were identified electrochemically by the use of a dual gold/mercury electrode. Three thermolytic peptides and three tryptic peptides have been isolated from these digests and shown to contain intact cystines that were originally present in the native protein. The majority of these peptides contained an intact, intramolecular cystine connecting two cysteines in locations homologous to cysteines 128 and 142 from the alpha polypeptide. Each of these cystines from each of the polypeptides of acetylcholine receptor was isolated in at least one peptide, respectively. Each of these cystine-containing peptides also contained glucosamine. It can be concluded that each asparagine in the sequence Asn-Cys-Thr/Ser, which occurs in the respective, homologous location in every polypeptide, is glycosylated even though a cystine sits between the asparagine and the threonine or serine. In addition, the existence of the cystine connecting the adjacent cysteines, alpha 192 and alpha 193, in the alpha subunit of acetylcholine receptor [Kao, P. N., & Karlin, A. (1986) J. Biol. Chem. 261, 8085-8088] has been confirmed.
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PMID:Assessment of the number of free cysteines and isolation and identification of cystine-containing peptides from acetylcholine receptor. 274 50

Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the Mr 10,000 psbE gene product (alpha subunit) of the chloroplast cytochrome b-559. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the alpha subunit to a slightly smaller polypeptide (delta Mr approximately -1000) as detected on Western blots, without loss of reactivity to COOH-terminal antibody. The disappearance of the parent Mr 10,000 polypeptide from thylakoids in the presence of trypsin correlated with the appearance of the smaller polypeptide with delta Mr = -750, the conversion having a half-time of approximately 15 min. Exposure of inside-out vesicles to trypsin resulted in almost complete loss of reactivity to the antibody, showing that the COOH terminus is exposed on the lumenal side of the membrane. Removal of the extrinsic polypeptides of the oxygen-evolving complex resulted in an increase of the accessibility of the alpha subunit to trypsin. These data establish that the alpha subunit of cytochrome b-559 crosses the membrane once, as predicted from its single, 26-residue, hydrophobic domain. The NH2 terminus of the alpha polypeptide is on the stromal side of the membrane, where it is accessible, most likely at Arg-7 or Glu-6/Asp-11, to trypsin or V8 protease, respectively. As a consequence of this orientation, the single histidine residue in the alpha subunit is located on the stromal side of the hydrophobic domain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thylakoid membrane protein topography: transmembrane orientation of the chloroplast cytochrome b-559 psbE gene product. 307 23

Purified B875 light-harvesting complex, chromatophores, and spheroplast-derived vesicles from wild-type Rhodobacter sphaeroides were treated with proteinase K or trypsin, and the alpha and beta polypeptides were analyzed by electrophoretic, immunochemical, and protein-sequencing methods. With the purified complex, proteinase K digested both polypeptides and completely eliminated the A875 peak. Trypsin digested the alpha polypeptide and reduced the A875 by 50%. Proteinase K cleaved the beta polypeptide of chromatophores and the alpha polypeptide of spheroplast-derived vesicles. Sequence analyses of polypeptides extracted from proteinase K-treated chromatophores revealed that the beta polypeptide was cleaved between amino acids 4 and 5 from the N terminus. The N terminus of the alpha polypeptide was intact. We concluded that the N terminus of the beta polypeptide is exposed on the cytoplasmic membrane surface, and the difference in the digestion patterns between the spheroplast-derived vesicles and chromatophores suggested that the C terminus of the alpha polypeptide is exposed on the periplasmic surface.
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PMID:Transverse membrane topography of the B875 light-harvesting polypeptides of wild-type Rhodobacter sphaeroides. 330 52

Proteinase K and trypsin were used to determine the orientation of the light-harvesting B800-850 alpha and beta polypeptides within the chromatophores (inside-out membrane vesicles) of the mutant strain Y5 of Rhodopseudomonas capsulata. With proteinase K 7 amino acid residues of the B800-850 alpha polypeptide were cleaved off up to position Trp-7--Thr-8 of the N terminus, and 11 residues were cleaved off up to position Leu-11-Ser-12 of the beta chain N terminus. The C termini of the B800-850 alpha and beta polypeptides, including the hydrophobic transmembrane portions, remained intact. It is proposed that the N termini of the alpha and beta subunits, each containing one transmembrane alpha-helical span, are exposed on the cytoplasmic membrane surface and the C termini are exposed to or directed toward the periplasm.
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PMID:Localization of the exposed N-terminal region of the B800-850 alpha and beta light-harvesting polypeptides on the cytoplasmic surface of Rhodopseudomonas capsulata chromatophores. 352 57

Five long, membrane-spanning tryptic peptides from the alpha polypeptide of sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase] have been purified. (Na+ + K+)-ATPase, isolated from canine kidney, was exposed to ultraviolet light in the presence of a high concentration of 1-tritiospiro[adamantane-4,3'-diazirine], a carbene precursor that partitions into the bilayer of the membrane. The alpha polypeptide, modified with 1.2 mol of [3H]adamantylidene (mol of polypeptide)-1, was isolated and digested with trypsin. Digestion with trypsin ensures that membrane-spanning sequences remain intact during the digestion, since lysine and arginine, being extremely hydrophilic, rarely appear in the membrane-embedded regions of membrane proteins. This digestion produced radioactive tryptic peptides greater than 25 residues in length. The tryptic digest of the labeled alpha polypeptide was chromatographed on Sephadex LH-60 in ethanol-formic acid, 4:1. The majority of the radioactivity (87%) eluted with distribution coefficients corresponding to peptides longer than melittin (26 residues), whereas 73% of the protein traveled with distribution coefficients corresponding to peptides less than 30 residues in length. Five radioactive peptides were further purified by high-pressure liquid chromatography, and each peptide displayed a unique, hydrophobic amino-terminal sequence. No other candidates could be found when a search for additional membrane-spanning peptides was conducted. Gel filtration of the tryptic peptides from the alpha polypeptide of (Na+ + K+)-ATPase labeled with 5-[125I]iodo-1-naphthyl azide, a lipophilic nitrene precursor, produced no additional radioactive components. Amino-terminal sequences and amino acid compositions of the five purified peptides are presented.
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PMID:Purification of the membrane-spanning tryptic peptides of the alpha polypeptide from sodium and potassium ion activated adenosinetriphosphatase labeled with 1-tritiospiro[adamantane-4,3'-diazirine]. 632 57

Pyruvate carboxylase from Pseudomonas citronellolis is composed of non-identical subunits which include a larger biotin-containing polypeptide (alpha) of Mr = 65,000, and a smaller biotin-free polypeptide (beta) of Mr = 54,000. We have investigated these two polypeptides by analyzing their amino acid composition, cyanogen bromide peptide maps, and immunochemistry. The results showed that the subunits of the enzyme have quite different properties. Antibodies prepared against the polypeptides were used as probes of the catalytic functions of the subunits. Immunotitration studies indicated that only anti-alpha inhibited enzyme activity. The antibiotin fraction of this antibody population was removed by passage through biotin-Sepharose (anti-alpha'). Titration curves using anti-alpha' showed identical inhibition when total pyruvate carboxylase activity, ATP/Pi exchange activity, and pyruvate/oxalacetate exchange activity were measured, suggesting that both active sites are located on the alpha polypeptide. The arrangement of the subunits in the quaternary structure was investigated by means of the surface probe carbonic anhydrase linked to toluene isocyanate, and by partial digestion experiments with trypsin, chymotrypsin, and pronase. The results indicated that the alpha polypeptides are on the outside of the molecule and the beta polypeptides are the internal subunits.
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PMID:Characterization of the subunit structure of pyruvate carboxylase from Pseudomonas citronellolis. 679 93


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