Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to obtain further information on the structure of D-amino acid oxidase (EC 1.4.3.3), limited proteolysis experiments have been carried out on its apo-, holo-, and holoenzyme-benzoate forms. The enzyme is unsensitive to 10% (w/w) chymotrypsin, while incubation with 10% (w/w) trypsin, under nondenaturating conditions, produces inactivation and proteolysis patterns which are different for the three forms of enzyme analyzed. These results confirm the previously reported conformational changes which occur upon binding of coenzyme to the apoprotein, and of benzoate to holoenzyme. The stable 37.0-kDa polypeptide, obtained from the apo- and holoenzyme-benzoate complex upon cleavage of a C-terminal 2.0-kDa fragment, retains full catalytic activity with unaltered kinetic parameters, and the coenzyme binding properties of the native enzyme. These results are in agreement with the tentative localization of the FAD-binding domain in the N-terminal region of the enzyme, and with the hypothesis that the function of the C-terminal region of D-amino acid oxidase could be related to the import of the enzyme into the peroxisomes, as suggested by Gould et al. (Gould, S. J., Keller, G. A., and Subramani, S. (1988) J. Cell. Biol. 107, 897-905).
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PMID:Characterization of a fully active N-terminal 37-kDa polypeptide obtained by limited tryptic cleavage of pig kidney D-amino acid oxidase. 197 77

We have studied the effects of pH, ionic strength, and hydrophobic fluorescence probes, 8-anilinonaphthalene-1-sulfonate (ANS) and bis-ANS, on the structure of intact (124-kDa) Avena phytochrome. The Pfr form of phytochrome forms oligomers in solution to a greater extent than the Pr form. Hydrophobic forces play a major role in the oligomerization of phytochrome, as suggested by fluorescence and monomerization by bis-ANS. However, electrostatic charges also take part in the phytochrome oligomerization. The partial proteolytic digestion patterns for the Pr and Pfr species are different, but binding of bis-ANS to the phytochrome abolishes this difference and yields an identical proteolytic peptide mapping for both spectral forms of phytochrome. This appears to result from bis-ANS binding at the carboxy-terminal domain, which induces monomerization of phytochrome oligomers. A second bis-ANS binding at an amino-terminal site blocks cleavage sites of trypsin and alpha-chymotrypsin. Bis-ANS especially blocks access of the proteases to the amino-terminal cleavage site that produces an early proteolytic product (114/118 kDa) on SDS gels. The bis-ANS binding does not, however, affect the proteolytic cleavage site that occurs in the hinge region between the two structural domains of phytochrome, the chromophore domain and the C-terminal non-chromophore domain. A chromophore binding site in the Pfr form is apparently exposed for preferential binding of bis-ANS, causing cyclization of the chromophore and bleaching of its absorbance at 730 nm. These observations have been discussed in terms of a photoreversible topographic change of the chromophore/apoprotein during the phototransformation of phytochrome.
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PMID:Spectral perturbations and oligomer/monomer formation in 124-kilodalton Avena phytochrome. 220 22

Prostaglandin H synthase has two distinct enzymatic activities: a cyclooxygenase that forms PGG2 from arachidonate and a peroxidase that can reduce hydroperoxides, such as PGG2, to the corresponding alcohols. The relative sensitivities of the two synthase activities to proteolytic attack have been examined, using trypsin, chymotrypsin, and proteinase K, all known to attack the native apoprotein in the arg 253 region. The relation between the specific activity of the synthase and the loss of the two activities and the cleavage of the synthase subunit during trypsin digestion was also examined. The cyclooxygenase and peroxidase activities declined in concert throughout room temperature digestions with each of the three proteases. There was no indication of a selective loss of either activity in any of the digestions. In separate digestions with the same preparation of synthase, 3.3% (w/w) proteinase K resulted in more extensive loss of activity (90% decrease after 90 min) than did 3% (w/w) trypsin (70% decrease after 120 min) or 5% (w/w) chymotrypsin (60% decrease after 135 min). In tryptic digestions of synthase preparations with cyclooxygenase specific activity between 16 and 125 k units/mg protein, the fractional loss of cyclooxygenase activity was, within experimental error, the same as that of peroxidase activity. The extent of cleavage of the 70 kDa synthase subunit was greater than the loss of enzymatic activity, with the discrepancy being larger for synthase preparations with lower specific activity. The presence of a variable amount of catalytically-inactive, protease-sensitive, synthase protein could account for the difference between surviving activity and intact subunit in six out of the seven synthase preparations examined. Thus, it is likely that the cyclooxygenase and peroxidase activities are destroyed together during proteolytic attack on the arg 253 region of the native synthase apoprotein.
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PMID:Concerted loss of cyclooxygenase and peroxidase activities from prostaglandin H synthase upon proteolytic attack. 250 12

The expression of cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) was examined in Escherichia coli transformed with either of two plasmids, pJ8 and pJ81. The former has an 840 bp insert of D. vulgaris DNA, containing the structural gene for cytochrome c3 (387 bp) and its promoter region. Plasmid pJ81 was generated from pJ8 by deoxyoligonucleotide-directed mutagenesis to direct the synthesis of a protein with an altered signal peptidase cleavage site [Ala(-1)----Asp(-1)]. Synthesis of the 14 kDa precursor, which was partly processed to the 12 kDa mature protein, was observed in cells of E. coli TG2(pJ8) by SDS gel electrophoresis and Western blotting. Analysis of spheroplasts revealed that the processed polypeptide was present in the periplasm while the precursor was found only in the membrane/cytoplasmic fraction. No processing was observed in E. coli TG2(pJ81) cells, due to the mutation of the signal peptide cleavage site. No insertion of haem into the E. coli product could be detected in E. coli TG2(pJ8) cells by post-electrophoretic protohaem fluorescence analysis. The sensitivity of the cytochrome c3 synthesized in E. coli TG2(pJ8) to digestion by chymotrypsin also indicated that the apoprotein was formed. The results indicate that E. coli is capable of synthesizing and exporting the cytochrome c3 polypeptide, but fails to insert the haems.
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PMID:Expression of the gene encoding cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) in Escherichia coli: export and processing of the apoprotein. 256 Dec 88

Plasmin preferentially cleaves rabbit hemopexin at a single site, generating two nondisulfide-linked carbohydrate-containing fragments. In contrast, heme-hemopexin is almost totally resistant to this enzyme and is more resistant than the apoprotein to digestion by trypsin, chymotrypsin, papain, subtilisin, and proteinase K as well. Plasmin digestion dramatically shortens the plasma clearance time of the molecule. The larger glycopeptide (I), shown to be derived from the amino terminus of the parent molecule by sequence analysis, has a molecular weight near 35,000 with a pI of 5.0. It binds 1 mol of heme per mol in a manner analogous to intact hemopexin, molecular weight near 60,000 and pI 5.8. The smaller glycopeptide (II) has a molecular weight near 25,000, a pI of 6.4, and does not bind heme. Of the four oligosaccharides of rabbit hemopexin, peptide I contains three oligosaccharides and peptide II contains one. At micromolar concentrations, the two peptides migrate together during centrifugation through sucrose gradients in the presence, but not in the absence, of heme. Peptide I has a far UV circular dichroism spectrum indicating it has some alpha-helical and extensive nonrepeating peptide structures whereas peptide II appears to be almost exclusively in a beta-sheet conformation. Peptide II is responsible for most of the positive ellipticity at 231 nm of native apohemopexin, but the increase in ellipticity at 231 nm characteristic of heme-hemopexin is not seen when peptide I binds heme, even in the presence of peptide II.
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PMID:Domain structure of rabbit hemopexin. Isolation and characterization of a heme-binding glycopeptide. 623 5

The amino acid sequence of the soluble monohaem cytochrome c-556 from Agrobacterium tumefaciens, strain B2a, has been determined. The sequence was derived from peptides obtained by digestion of the apoprotein with trypsin and chymotrypsin, and by subdigestion of some of the peptides with Staphylococcus aureus protease and thermolysin. Sequencing of the various peptides was achieved by a combination of manual dansyl-Edman degradation and automatic liquid-phase sequence analysis. The main characteristic of this cytochrome is that the haem-binding sequence Cys-Xaa-Yaa-Cys-His occurs in the C-terminal region of the polypeptide chain, the first cysteine being located 11 residues ahead of the C-terminal lysine-122. As such, the protein belongs to cytochrome c sequence class II (sensu Ambler). The cytochrome c-556 is the first example known of a class II cytochrome of the low-spin type isolated from an obligate aerobic organism.
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PMID:The complete amino-acid sequence of the low-spin class II cytochrome c-556 from Agrobacterium tumefaciens strain B2a. 629 89

The amino acid sequence has been determined for the insecticyanin from the hemolymph of the fifth instar larvae of the tobacco hornworm, Manduca sexta. The apoprotein is a single polypeptide chain of 189 amino acids, molecular weight 21,378, containing two disulfide bridges, 9-119 and 42-176. The sequence analysis was performed by automated Edman degradation of reduced and carboxymethylated insecticyanin and fragments generated therefrom by cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus proteinase. Most of the peptides were purified by reverse-phase high-performance liquid chromatography. A purification procedure for the isolation of insecticyanin in high yields and a simple method of determining disulfide linkages are also reported.
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PMID:The covalent protein structure of insecticyanin, a blue biliprotein from the hemolymph of the tobacco hornworm, Manduca sexta L. 638 9

Human plasma low density lipoproteins (LDL) contain one major apoprotein of apparent Mr = 550,000 designated apolipoprotein B-100 (apo-B-100) and in some LDL preparations, minor components termed apo-B-74 (Mr = 410,000) and apo-B-26 (Mr = 145,000). The structural and metabolic relationships among these LDL apoproteins remain obscure. In the present study, we show that the mixing of proteolytic inhibitors with blood at the moment of collection prevents the appearance of apo-B-74 and -26 in plasma LDL indicating that these peptides are derived by proteolytic degradation of apo-B-100. In order to simulate the degradation in vitro, LDL were digested with plasmin, trypsin, chymotrypsin, thrombin, and tissue and plasma kallikreins and the degradation products analyzed by polyacrylamide gradient gel electrophoresis. While plasmin, trypsin, and chymotrypsin caused extensive degradation of apo-B-100, thrombin, and tissue and plasma kallikreins generated limited cleavage patterns. LDL digested with thrombin contained stoichiometric amounts of two peptides with apparent Mr = 385,000 and 170,000. Mixing experiments showed that the thrombin-derived peptides of apo-B-100 did not co-migrate with apo-B-74 and B-26 during electrophoresis indicating that these peptides were different. In contrast, LDL digested with kallikrein contained stoichiometric amounts of two peptides with apparent molecular weights identical to apo-B-74 and -26. Together, the above results indicate that apo-B-74 and -26 are degradation products of apo-B-100 and are not produced by the action of thrombin. Whether the expression of a kallikrein-like activity in vivo accounts for the specific degradation of LDL B-100 to yield LDL B-74 and -26 remains to be determined.
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PMID:Degradation of apolipoprotein B-100 of human plasma low density lipoproteins by tissue and plasma kallikreins. 656 30

The chemical cleavage of lipophilin (proteolipid apoprotein) from bovine brain white matter with HBr/dimethyl sulfoxide at the tryptophan residues, under conditions adapted to this hydrophobic protein, releases four fragments with approximate molecular masses 14 kDa (Trp I), 6.8 kDa (Trp IV), 5.2 kDa (Trp III) and 2.1 kDa (Trp II). These fragments have been separated and purified by a combination of solvent distribution, molecular sieve chromatography (Bio-Gel P-150) and high-performance liquid chromatography for automated Edman degradation and combined gas-liquid chromatography/mass spectroscopy. The complete amino acid sequences of Trp II and III and large sequences of Trp I are reported in this communication. The amino acid sequence of Trp IV and the sequences of peptides releasable from lipophilin by proteolytic enzymes (trypsin, thermolysin, subtilisin, chymotrypsin) have been described in previous reports from this laboratory. Despite two small gaps in the complete primary structure of lipophilin from myelin of central nervous system, our sequence data suggest the arrangement of four long hydrophobic sequences (30-40 apolar amino acid residues) within the hydrophobic core of the myelin lipid bilayer, linked by three hydrophilic regions at the aqueous membrane interphase. These features lend lipophilin the properties of a polytopic membrane protein.
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PMID:Lipophilin (proteolipid apoprotein) of brain white matter. Purification and amino acid sequence studies of the four tryptophan fragments. 717 28

In the green alga Chlamydomonas reinhardtii, the copper-dependent accumulation of plastocyanin is effected via the altered stability of the protein in copper-deficient versus copper-sufficient medium (t1/2) < 20 min versus several hours). To understand the mechanism of plastocyanin degradation in vivo, the purified apoprotein was characterized relative to the holoprotein with respect to conformation and protease susceptibility. Circular dichroism spectroscopy revealed that the apoprotein in solution did not display the characteristic secondary structure displayed by the native or reconstituted holoprotein. The apoprotein was also susceptible to digestion in vitro by chymotrypsin whereas the holoprotein was resistant. High ionic conditions, which stabilize the folded structure of apoplastocyanin, also inhibit its degradation by chymotrypsin. These results suggest that one explanation for plastocyanin degradation in copper-deficient cells in vivo might be the increased susceptibility of the apo form to a lumenal protease. Since apoplastocyanin is a normal biosynthetic intermediate for the formation of holoplastocyanin, the increased susceptibility of apoplastocyanin to proteolysis implies that degradative and biosynthetic activities would compete for the same substrate. However, characterization of an apoplastocyanin-accumulating mutant suggests that a plastocyanin-degrading protease is active only in copper-deficient cells. Thus, apoplastocyanin is rapidly degraded in copper-deficient cells, whereas its major fate in copper-supplemented cells is holoplastocyanin formation.
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PMID:Degradation of plastocyanin in copper-deficient Chlamydomonas reinhardtii. Evidence for a protease-susceptible conformation of the apoprotein and regulated proteolysis. 755 14


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