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)

A noncovalent complex of the apoprotein (1-104) and cyanogen bromide heme fragment containing residues 1 to 65, (1-65) H, has been prepared from horse heart cytochrome c. Conditions under which the redundant portions of the ferrous complex can be removed by limited trypsin digestion have been devised. The complementing fragments have been isolated from the derived complexes and four apofragments and one heme fragment have been identified in the amino acid sequence of cytochrome c. They are (39-104), (40-104), (54-104), (56-104), and (1-53)H. The formation of an ordered ferric complex composed of one heme fragment and one apofragment for the cases (1-53)H (39-104), (1-53)H-(40-104), (1-53)H-(54-104), and (1-53)H-(56-104) has been demonstrated by the quenching of the tryptophan 59 fluorescence and the regain of biological activity in a cytochrome b2 assay. The apparent dissociation constant has been estimated as less than 3 X 10(-7) M in all the aforementioned cases. Thus, the region (between residues 38 and 57) of the amino acid sequence permissible for cleavage without disruption of the ordered structure indicated by the present in vitro experiments corresponds to that (between residues 38 and 57) evolutionally deleted in the three-dimensional structure of Pseudomonas aeruginosa cytochrome c551 discovered by Dickerson et al. (Dickerson, R.E., Timkovich, R., and Almassy, R.J. (1976) J. Mol. Biol. 100, 473-491).
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PMID:Formation of a biologically active, ordered complex from two overlapping fragments of cytochrome c. 19 Feb 31

The transverse distribution of enzyme proteins and phospholipids within microsomal membranes was studied by analyzing membrane composition after treatment with proteases and phospholipases. Upon trypsin treatment of closed microsomal vesicles, NADH- and NADPH-cytochrome c reductases as well as cytochrome b5 were solubilized or inactivated, while cytochrome P-450 was partially inactivated. When microsomes were exposed to a concentration of deoxycholate which makes them permeable to macromolecules but does not disrupt the membrane, the detergent alone was sufficient to release four enzymes: nucleoside diphosphatase, esterase, beta-glucuronidase, and a portion of the DT-diaphorase. Introduction of trypsin into the vesicle lumen inactivated glucose-6-phosphatase completely and cytochrome P-450 partially. The rest of this cytochrome, ATPase, AMPase, UDP-glucuronyltransferase, and the remaining 50% of DT-diaphorase activity were not affected by proteolysis from either side of the membrane. Phospholipase A treatment of intact microsomes in the presence of albumin hydrolyzed all of the phosphatidylethanolamine, phosphatidylserine, and 55% of the phosphatidylcholine. From this observation, it was concluded that these lipids are localized in the outer half of the bilayer of the microsomal membrane; Phosphatidylinositol, 45% of the phosphatidylcholine, and sphingomyelin are tentatively assigned to the inner half of this bilayer. It appears that the various enzyme proteins and phospholipids of the microsomal membrane display an asymmetric distribution in the transverse plane.
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PMID:Enzyme and phospholipid asymmetry in liver microsomal membranes. 19 Feb 41

The L-(+)-Lactate:cytochrome c oxidoreductase or cytochrome b2 from the yeasts Saccharomyces cerevisiae and Hansenula anomala were partially hydrolysed in various concentrations of trypsin. Conditions were found which allowed the isolation from the Hansenula enzyme of a 140 000 +/- 10 000-dalton flavoprotein. The prosthetic flavin groups were still reducible by substrate (spectroscopic evidence) but the flavoprotein was unable to form a complex with cytochrome c, the physiological acceptor in the enzymatic reaction. No such flavoprotein units could be found during proteolysis of the Saccharomyces enzyme. The heme prosthetic group of the Hansenula enzyme remained bound to a 15 500 +/- 1000-dalton protein unit which was larger than, but very similar to, the well known 'cytochrome b2 core' of the Saccharomyces enzyme. Moreover, the degradation of different enzyme samples by contaminated proteases allowed the isolation of a particular form of Hansenula enzyme: each tetramer had, on the mean, four bound flavins and only two heme groups. These molecules completely retained their ability to form a complex with cytochrome c.
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PMID:Proteolysis of L-(+)-lactate cytochrome c oxidoreductase (cytochrome b2) extracted from Saccharomyces cerevisiae and Hansenula anomala yeasts. 19 7

Parameters of oxidative phosphorylation, the rate of respiration in various metabolic conditions as well as an activity and stability of mitochondrial polyenzyme systems were altered in rats with experimental hepatitis, treated with CCl4. Proteins and phospholipids from mitochondrial membranes proved to be more susceptible to the effect of trypsin and phospholipase D. Kinetics of cytochrome C desorption from membranes and mitochondria demonstrated that capacity of these membranes to confine the exogenous proteins was altered in hepatitis.
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PMID:[Functioning of rat liver mitochondria in hepatitis]. 21 41

The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria were studied by using controlled digestion with trypsin of purified inner mitochondrial membranes. Inhibition of steroid hydroxylase activity by trypsin was only observed in inner mitochondrial membranes which had been disrupted by various techniques. The steroid hydroxylase activity of intact inner membranes was not inhibited by trypsin. The effect of tryptic digestion was monitored by measuring 11 beta-hydroxylase and cholesterol side chain cleavage activities, as well as cytochrome P-450 reduction. The effect of trypsin on the steroid-induced difference spectra using pregnenolone, 20 alpha-hydroxycholesterol, and deoxycorticosterone was also measured. The results were similar regardless of which procedure was utilized and strongly suggest that both cytochrome P-45011 beta and cytochrome P-450scc are located on the matrix side of the mitochondrial inner membrane.
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PMID:Topological studies of cytochromes P-450scc and P-45011 beta in bovine adrenocortical inner mitochondrial membranes. Effects of controlled tryptic digestion. 48 6

Rabbit cytochrome b5 was incorporated into single-walled phosphatidylcholine liposomes, and the cytochrome b5-liposome complex thus formed was digested with trypsin. Protein chemical characterization indicated that the main products formed were 1) a hydrophilic (heme-containing) fragment of the cytochrome corresponding to the sequence consisting of the masked NH2-terminus through residue 88, 2) a hydrophobic peptide which spans residue 91 to the COOH-terminus (residue 133), and 3) a dipeptide, seryl-lysine, derived from residues 89 and 90. The hydrophobic peptide was obtained in the form of its complex with liposomes. It was concluded that trypsin cleaved rather specifically the peptide bonds between residues 88 and 89 (Arg-Ser) and between residues 90 and 91 (Lys-Leu). Tryptic digestion of free, unbound cytochrome b5 also resulted in the cleavage of the same peptide bonds. These results are not consistent with the proposal of Visser et al. (Visser, L., Robinson, N.C., & Tanford, C. (1975) Biochemistry 14, 1194-1199) that the hydrophilic and hydrophobic domains of cytochrome b5 are connected to each other by a link peptide consisting of some 15 amino acid residues and that this link peptide can be cut out by the action of trypsin. The circular dichroism spectrum of intact cytochrome b5 or its complex with liposomes in the far-ultraviolet region was closely similar to the sum of the spectra of the hydrophilic fragment and the hydrophobic peptide (or its complex with liposomes). This indicates that the tryptic cleavage of the cytochrome molecule does not induce any significant changes in the conformations of the hydrophilic and hydrophobic moieties of the molecule and thus provides further evidence that the three-dimensional structures of the two domains are independent of each other.
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PMID:Nature of tryptic attack on cytochrome b5 and further evidence for the two-domain structure of the cytochrome molecule. 73 4

Rat liver microsomes have previously been shown to contain hemoproteins having molecular weights of 53,000, 50,000, and 45,000. The 45,000-dalton hemoprotein, which is induced in rat liver microsomes by pretreatment of animals with phenobarbital, is resistant to proteolysis by trypsin. This characteristic was used to purify it from the other microsomal hemoproteins. In the procedure used, a sodium cholate-solubilized microsomal fraction from phenobarbital-pretreated rats was treated with trypsin and chromatographed on Sephadex G-100 to separate the hemoprotein from preolytic degradation products. The hemoprotein thus isolated was homogenous on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified spectrally as a cytochrome P-420 hemoprotein. This hemoprotein was free of cytochrome b5 and NADPH-cytochrome c reductase activity. Antibody prepared against the protease-treated cytochrome P-420 hemoprotein will not cross-react with the 53,000- and 50,000-dalton hemoproteins. This was assessed by three criteria. First, immunoprecipitation studies were conducted with detergent-solubilized partially purified cytochrome P-450 preparations isolated from the liver microsomes of control and phenobarbital- and 3-methylcholathrene-retreated rats. The antibody immunoprecipitated only the 45,000-dalton hemoprotein from these partially purified cytochrome P-450 preparations, each of which contains all three hemoproteins. Second, the antibody demonstrated specificity with regard to the microsomal hydroxylation reactions it would inhibit in a reconstituted hydroxylation system containing partially purified cytochrome P-450 (448) fractions isolated from the liver microsomes from phenobarbital- or 3-methylcholathrene-pretreated rats. The antibody would inhibit benzphetamine-N-demethylation catalyzed by both cytochrome P-450 fractions but would not inhibit benzpyrene hydroxylation catalyzed by either. Third, agglutination and complement fixation assays were performed to assess the binding of the antibody to liver microsomes isolated from control and phenobarbital- or 3- methylcholanthrene-pretreated rats. These studies demonstrated that the antibody binds preferentially to the liver microsomes isolated from phenobarbital-pretreated rats, in which the 45,000-dalton hemoprotein has been shown to be induced. It is hypothesized that there are very significant structural and catalytic differences among the cytochrome P-450 hemoproteins.
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PMID:Multiplicity of cytochrome P-450 hemoproteins in rat liver microsomes. Preparation and specificity of an antibody to the hemoprotein induced by phenobarbital. 80 94

An automated method for the optimal placement of polar hydrogens in a protein structure is described. This method treats the polar, side chain hydrogens of lysine, serine, threonine, and tyrosine and the amino terminus of a protein. The program, called NETWORK, divides the potential hydrogen-bonding pairs of a protein into groups of interacting donors and acceptors. A search is conducted on each of the local groups to find an arrangement which forms the most hydrogen bonds. If two or more arrangements have the same number of hydrogen bonds, the arrangement with the shortest set of hydrogen bonds is selected. The polar hydrogens of the histidyl side chain are specifically treated, and the ionization state of this residue is allowed to change, if this change results in additional hydrogen bonds for the local group. The program will accept Protein Data Bank as well as Biosym-format coordinate files. Input and output routines can be easily modified to accept other coordinate file formats. The predictions from this method are compared to known hydrogen positions for bovine pancreatic trypsin inhibitor, insulin, RNase-A, and trypsin for which the neutron diffraction structures have been determined. The usefulness of this program is further demonstrated by a comparison of molecular dynamics simulations for the enzyme cytochrome P-450cam with and without using NETWORK.
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PMID:A method for determining the positions of polar hydrogens added to a protein structure that maximizes protein hydrogen bonding. 137 79

In a recent study [Wang & Beattie (1991) Arch. Biochem. Biophys. 291, 363-370], we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in the cytochrome bf complex reconstituted into proteoliposomes and was bound selectively to cytochrome b6. To establish the site of binding of DCCD on cytochrome b6, the cytochrome bf complex labeled with [14C]DCCD was selectively digested with chymotrypsin and trypsin. A 17-kDa fragment containing radioactive DCCD and the heme moiety was obtained after chymotrypsin digestion, while a 12.5-kDa fragment containing both radioactive DCCD and the heme moiety was obtained after trypsin digestion, suggesting that the site of DCCD binding might be on aspartate-140, aspartate-155, or glutamate-166. Extensive digestion of cytochrome b6 isolated from a [14C]DCCD-labeled cytochrome bf complex with trypsin followed by isolation and sequencing of two radioactive peptides obtained revealed that DCCD is bound at either residue aspartate-155 or residue glutamate-166 localized in amphipathic extramembranous helix IV. In addition, the cytochrome bf complex labeled with [14C]DCCD was reconstituted into liposomes and digested with trypsin. Three fragments of 9.3, 10.5, and 11.5 kDa were obtained, suggesting that the four-helix model for the topography of cytochrome b6 in the membrane is correct.
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PMID:Binding of dicyclohexylcarbodiimide to aspartate-155 or glutamate-166 of cytochrome b6 in a cytochrome bf complex isolated from spinach thylakoids. 139 Jun 29

Sera from patients with halothane hepatitis contain immunoglobulin G (IgG) antibodies to trifluoroacetylated liver microsomal proteins of 100, 76, 59, 57 and 54 kDa, which are produced as a consequence of metabolism of halothane to trifluoroacetyl halide by cytochrome(s) P450. In the present study, the membrane topographies of the various antigens in rat liver microsomal fractions were investigated. Liver microsomal fractions from rats treated with halothane in vivo, and rat liver microsomal fractions which had been incubated with halothane in vitro, were used as the source of trifluoroacetyl antigens. The antigens were detected by immunoblotting. Whereas the 100, 76, 59 and 57 kDa antigens were solubilized from the microsomal membrane by either 0.1 M sodium carbonate or 0.1% (w/v) sodium deoxycholate, the 54 kDa antigen was not solubilized by 0.1% (w/v) sodium deoxycholate. In intact microsomal fractions, the 100, 76, 59 and 57 kDa antigens were not degraded appreciably by trypsin unless detergent was added to permeabilize the microsomal membrane. These results indicate that the 54 kDa antigen is an integral membrane protein, whereas the 100, 76, 59 and 57 kDa antigens are peripheral membrane proteins situated within the lumen of microsomal vesicles, and hence presumably located within the lumen of the endoplasmic reticulum in vivo.
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PMID:The topography of trifluoroacetylated protein antigens in liver microsomal fractions from halothane treated rats. 151 Jul 11


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