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)

We have confirmed the propensity of fragments of cytochrome c to form complexes that reproduce the structure and, in part, the functionality, of the native protein by preparing four novel complexes. We have used trypsin under three different sets of conditions in sequence to prepare a contiguous two-fragment complex (1-55).(56-104). One of the intermediates is a stable overlapping complex (1-65).(56-104). Conditions for limited acid hydrolysis of peptide bonds in cytochrome c have been developed that optimize the yield of fragments (1-50) and (51-104). These two fragments also form a stable association, as do (1-50) and (56-104). These complexes are potentially useful for the semisynthesis of analogues modified in the region of the cleavage sites, which include a number of highly conserved amino acid residues, and are being used for studies of protein folding, interactions with oxidase, cytochrome c immunogenicity and of artificially induced spontaneous resyntheses between complexing fragments. Like other known two-fragment complexes of cytochrome c, they exhibit normal visible spectra, including the presence of the 695 nm band, indicative of a functional haem crevice. Studies of their biological activities and redox potentials lead to a number of conclusions on structure-function relationships in cytochrome c. Most significantly there is a linear relationship between the logarithm of electron-transfer rates from cytochrome c reductase and redox potential in this series of analogues, indicating that such transfer is thermodynamically controlled. This discovery contributes to our understanding of the interaction of cytochrome and reductase. Since the relationship is obeyed by other types of analogues, except for those that involve modification of the active site of cytochrome c, we have a useful diagnostic for those residues that participate directly in electron transfer.
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PMID:On the relationship between oxidation-reduction potential and biological activity in cytochrome c analogues. Results from four novel two-fragment complexes. 282 30

Co-translational insertion of liver microsomal cytochrome P-450 into the endoplasmic reticulum membrane is mediated by the signal recognition particle (SRP) and the presence in the cytochrome molecule of a signal sequence that can be recognized by SRP has been postulated. To locate this signal sequence, six hybrid cDNAs were constructed in which various segments of a cDNA for a rabbit liver cytochrome P-450 are fused with a cDNA or its fragment encoding yeast porin (an outer mitochondrial membrane protein) or with a cDNA for pre-interleukin 2 (a secretory protein) from which the 5'-terminal portion encoding most of its signal sequence had been removed. These hybrid cDNAs were inserted into an SP-6 transcription vector and transcribed in vitro. The mRNAs thus synthesized were translated in a cell-free system in the presence of rough microsomes. It was thus found that only those chimeric proteins containing (at their amino-terminal end) the amino-terminal cytochrome P-450 segments consisting of greater than or equal to 29 amino acid residues were co-translationally inserted into the membrane in an SRP-dependent fashion. These proteins were, however, neither processed nor translocated across the membrane. These findings, coupled with the observation that the major portion of these proteins, when inserted into the membrane, was degraded by trypsin, led to the conclusion that a short amino-terminal segment (less than 29 residues) of the cytochrome P-450 functions not only as an insertion signal but also as a stop-transfer sequence. This segment is, therefore, similar to the internal signal of type II plasma membrane proteins, but differs from the latter in the topogenic function.
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PMID:A short amino-terminal segment of microsomal cytochrome P-450 functions both as an insertion signal and as a stop-transfer sequence. 282 91

Cytochrome c1 is an amphiphilic protein which binds to the mitochondrial inner membrane, presumably through a hydrophobic region near the carboxyl (C)-terminus. In the preceding study (Hase, T., et al. (1987) J. Biochem. 102, 401-410), two cytochrome c1 mutations were constructed: delta 1 and delta 2 cytochromes c1, in which the C-terminal segments of 17 and 71 residues were replaced by foreign sequences of 20 and 15 residues, respectively. delta 2 cytochrome c1 had lost the putative membrane anchor. The two cytochrome c1 mutants were localized in mitochondria, but succinate-cytochrome c1 reductase activity was detected only in the mitochondria containing delta 1 cytochrome c1. The membrane association of the two mutant molecules as well as that of authentic cytochrome c1 was investigated. These three molecules were firmly attached to mitochondrial membranes and not solubilized on either sonication or sodium carbonate (pH 11) treatment. However, when the membranes were solubilized with Triton X-100, both the delta 1 and authentic cytochromes c1 were extracted from the membranes more easily than delta 2 cytochrome c1. By fractionating cholate extracts of mitochondrial membranes with ammonium sulfate, delta 1 cytochrome c1 was cofractionated with the enzymatic activity of complex III, but delta 2 cytochrome c1 was clearly separated from the complex III fraction. Trypsin treatment of mitochondria and mitoplasts showed that delta 2 cytochrome c1 was exposed to the intermembrane space, with such a topology that its trypsin susceptibility became much higher than that of the authentic molecule.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A carboxyl-terminal hydrophobic region of yeast cytochrome c1 is necessary for functional assembly into complex III of the respiratory chain. 282 89

The cytochrome d complex is one of two membrane-bound terminal oxidases of the Escherichia coli aerobic respiratory chain. Previous studies have shown that this enzyme reconstituted into proteoliposomes rapidly oxidizes ubiquinol-8 as well as the soluble homologue, ubiquinol-1, and that quinol oxidase activity is accompanied by the formation of a transmembrane H+ electrochemical gradient. The enzyme also oxidizes the artificial reductant, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) with the generation of a H+ electrochemical gradient. In this work, it is established that trypsin digestion of the purified cytochrome d complex cleaves subunit I while subunit II is unaffected. Proteolysis of subunit I is correlated with loss of ubiquinol-8 and ubiquinol-1 oxidase activities. Trypsin digestion has no effect on TMPD oxidase activity. The cytochrome d complex is concluded to possess three distinct active sites for 1) ubiquinol oxidation, 2) TMPD oxidation, and 3) oxygen binding and reduction. Data also suggest that both sites of ubiquinol and TMPD oxidations are located on the periplasmic side of the E. coli membrane while the site of oxygen reduction is on the opposite side.
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PMID:Trypsin proteolysis of the cytochrome d complex of Escherichia coli selectively inhibits ubiquinol oxidase activity while not affecting N,N,N',N'-tetramethyl-p-phenylenediamine oxidase activity. 283 3

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.
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PMID:Identification of the binding site on cytochrome c1 for cytochrome c. 298 91

In a previous publication (Narhi, L. O. and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a soluble 119,000-dalton P-450 cytochrome (P-450BM-3) that was induced by barbiturates in Bacillus megaterium. This single polypeptide contained 1 mol each of FAD and FMN/mol of heme and, in the presence of NADPH and O2, catalyzed the oxygenation of long-chain fatty acids without the aid of any other protein. We have now utilized limited trypsin proteolysis in the presence of substrate to cleave P-450BM-3 into two polypeptides (domains) of about 66,000 and 55,000 daltons. The 66-kDa domain contains both FAD and FMN but no heme, reduces cytochrome c in the presence of NADPH, and is derived from the C-terminal portion of P-450BM-3. The 55-kDa domain is actually a mixture of three discrete peptides (T-I, T-II, and T-III) separable by high performance liquid chromatography. All three contain heme and show a P-450 absorption peak in the presence of CO and dithionite. The major component, T-I (Mr = 55 kDa), binds fatty acid substrate and has an N-terminal amino acid sequence identical to that of intact P-450BM-3, an indication that this domain constitutes the N-terminal portion of the 119-kDa protein. T-II (54 kDa) is the same as T-I except that it is missing the first nine N-terminal amino acids and does not bind substrate. T-III (Mr = 53.5 kDa) has lost the first 15 N-terminal residues and does not bind substrate. Since trypsin digestion of P-450BM-3 carried out in the absence of substrate yields T-II and T-III but no T-I, it appears that 1 or more residues of the first nine N-terminal amino acids of this protein are intimately involved in substrate binding. Although both the heme- and flavin-containing tryptic peptides retain their original half-reactions, fatty acid monooxygenase activity cannot be reconstituted after proteolysis, and the two domains, once separated, show no affinity for each other. In most respects, the reductase domain of P-450BM-3 more closely resembles the mammalian microsomal P-450 reductases than it does any known bacterial protein.
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PMID:Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium. 310 60

Treatment of Chlamydomonas reinhardtii thylakoids with cross-linking reagents including glutaraldehyde causes polymerization of all thylakoid polypeptides, but not of the reaction center II polypeptide D1 unless the thylakoids are presolubilized by octyl beta-D-glucoside (Adir, N., and Ohad, I. (1986) Biochim. Biophys. Acta 850, 264-274). The results presented here show that this is a general property of D1 as it can be demonstrated in thylakoids of cyanophytes, Dasicladaceae, green algae, and C3 and C4 plants. Solubilization of the membranes by ionic detergents, deoxycholate, lauryl sucrose, or dodecyl beta-D-maltoside is not effective in inducing cross-linking of the D1 polypeptides by glutaraldehyde. The most effective alkyl glucosides were those with 7-9 carbon alkyl chains. The same behavior toward glutaraldehyde was exhibited by the unprocessed D1 precursor and by the palmitoylated D1 protein. Based on the refractility of the D1 protein to cross-linking reagents, a procedure was developed for its isolation from cross-linked thylakoids by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Isolated D1 retained its behavior toward cross-linking by glutaraldehyde and generated tryptic fragments similar to those obtained following trypsin treatment of intact thylakoids. Denaturation of isolated D1 protein by acetone facilitates cross-linking by glutaraldehyde and extensive degradation by trypsin. The photosystem II polypeptides are differentially cross-linked with increasing concentrations of glutaraldehyde, the most susceptible being the 28- and 23-kDa components of the light-harvesting chlorophyll a-b protein complex and the core complex 44- and 51-kDa polypeptides, and the least affected being the cytochrome b559, the D2 protein, and a 24-kDa component of the light-harvesting chlorophyll a-b protein complex. These results reflect the relative position and interaction of the photosystem II polypeptides within the complex and suggest that strong and specific hydrophobic interactions may be responsible for the tight and stable conformation of D1. This may be based mostly on the conserved amino acid sequences of D1 and possibly plays a role in the process of D1 integration and removal from the reaction center during its light-dependent turnover.
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PMID:Structural properties of the D1 and surrounding photosystem II polypeptides as revealed by their interaction with cross-linking reagents. 312 10

In two previous reports (Narhi LO, Fulco AJ, J. Biol. Chem. 261: 7160-7169, 1986; Ibid., 262: 6683-6690, 1987) we described the characterization of a catalytically self-sufficient 119,000-dalton P-450 cytochrome that was induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O2, this polypeptide (cytochrome P-450BM-3) catalyzed the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase was cloned into Escherichia coli and the clone harboring the recombinant plasmid produced a protein that behaved electrophoretically and immunochemically like the B. megaterium enzyme (Wen LP, Fulco AJ, J. Biol. Chem. 262: 6676-6682, 1987). We have now compared authentic P-450BM-3 from B. megaterium and putative P-450BM-3 isolated from transformed E. coli and have found them to be indistinguishable with respect to chromatographic and electrophoretic behavior, reaction with specific antibody, prosthetic group (heme, FAD and FMN) analyses, spectra, enzymology, limited trypsin proteolysis and partial amino acid sequencing. We thus conclude that the P-450 cytochrome expressed by the transformed E. coli is essentially identical to native P-450BM-3 induced by barbiturates in B. megaterium. The evidence furthermore suggests that the primary amino acid sequence of this complex protein is alone sufficient to direct the proper integration of the three prosthetic groups and to specify folding of the polypeptide into the correct tertiary structure.
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PMID:Characterization of the protein expressed in Escherichia coli by a recombinant plasmid containing the Bacillus megaterium cytochrome P-450BM-3 gene. 313 61

Immunocytochemical studies with a monoclonal antibody (MAb-HL3), which recognises a major isozyme of human hepatic cytochrome P-450, have demonstrated this cytochrome in both cryostat and formalin-fixed paraffin-embedded sections of normal human adult liver. Prior trypsin digestion of the formalin-fixed sections prevented staining. There was a zonal distribution of immunoreactive cytochrome P-450, with localization predominantly in the hepatocytes of zone 3 of the hepatic acinus (the centrilobular region). Cytochrome P-450 was also demonstrated in foetal liver, but all foetal hepatocytes contained immunoreactive cytochrome P-450 and there was no zonal distribution of the protein. The biliary epithelium of adult liver contained a small amount of immunoreactive cytochrome P-450 whereas there was no immunoreactivity in the epithelium of foetal bile ducts.
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PMID:Cytochrome P-450 localization in normal human adult and foetal liver by immunocytochemistry using a monoclonal antibody against human cytochrome P-450. 344 Jul 54

Cytochrome b5 holoenzyme was bound asymmetrically in the tightly bound form to small unilamellar dimyristoylphosphatidylcholine vesicles. [3H]Taurine, a membrane-impermeant nucleophile, was added to the external medium and was then cross-linked to cytochrome carboxyl residues by the addition of a water-soluble carbodiimide. Nonpolar peptide was isolated after trypsin digestion of taurine-labeled apocytochrome b5 and contained 1.7-1.9 residues of taurine. The C-terminal tetrapeptide containing residues Thr130-Asn133 was generated by chymotryptic hydrolysis of radiolabeled nonpolar peptide and was purified by gel filtration and ion exchange chromatography. Amino acid analysis of the C-terminal tetrapeptide showed that about 1.6 mol of taurine was cross-linked per mol of peptide. When the experiment was performed with taurine trapped inside the vesicles, no cross-linking was observed. The results suggest that when cytochrome b5 holoenzyme is bound to vesicles in the tight binding form, the C terminus is located on the external surface of the vesicles.
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PMID:Topography of the C terminus of cytochrome b5 tightly bound to dimyristoylphosphatidylcholine vesicles. 368 Feb 11


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