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)

The mitochondrial proteins involved in adrenocortical steroidogenesis are synthesized as higher molecular weight precursors which require processing by the mitochondria to their mature sizes. The post-translational maturation of two of these proteins has been examined: the cholesterol side chain cleavage cytochrome P-450 (P-450scc) and the iron-sulfur protein, adrenodoxin. Total translation products synthesized in a cell-free system programmed by bovine adrenocortical poly(A+) RNA were incubated with isolated bovine adrenocortical or heart mitochondria followed by immunoisolation of radiolabeled P-450scc or adrenodoxin. In the presence of adrenocortical mitochondria, the precursor form of P-450scc was converted into a trypsin-resistant form that had the same molecular weight as mature P-450scc. Unlike adrenocortical mitochondria, heart mitochondria were unable to process the P-450scc precursor which remained unaltered and trypsin-sensitive. In addition, a matrix fraction of heart mitochondria did not cleave the P-450scc precursor. In contrast, the adrenodoxin precursor did not exhibit similar specificity as it was processed to the mature form by both adrenocortical and heart mitochondria. Also, the adrenocortical mitochondria were not restricted to processing endogenous proteins as they imported and cleaved the precursor to ornithine transcarbamylase. The results indicate that some mitochondrial precursor proteins have tertiary structures which allow them to be recognized by all mitochondria while other mitochondrial precursor proteins have structures recognizable by only specialized mitochondria.
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PMID:Discriminatory processing of the precursor forms of cytochrome P-450scc and adrenodoxin by adrenocortical and heart mitochondria. 673 44

Detergent-solubilized liver microsomal NADPH-cytochrome P-450 reductase is known to retain the ability to transfer electrons to cytochrome P-450, whereas the trypsin-solubilized reductase transfers electrons only to artificial acceptors. Due to the loss of a hydrophobic fragment by the action of trypsin, the altered reductase is no longer capable of binding cytochrome P-450. In the present study the primary tryptic attack on the rabbit reductase was shown to be at the Lys 44-Ile 45 bond to liberate the hydrophilic domain (molecular weight, 71,000) from the intact enzyme (molecular weight, 77,000). The other fragment (molecular weight, 4,800) undergoes tryptic attack at the Lys 34-Lys 35-Lys 36 sequence to yield a polypeptide representing the hydrophobic domain of the reductase and a nona- or decapeptide (Lys 35 or Lys 36 through Lys 44) which serves as the connecting region. The hydrophobic peptide, which is derived from the NH2-terminal end of the reductase, has an acetylated NH2 terminus and a region (Val 16 through Phe 32) which is exceptionally hydrophobic, with a predicted beta-sheet structure, and is believed to be involved in the binding of cytochrome P-450 and phospholipid. The site of attack on the reductase by various proteases is different, but the cleavage points are localized within a short segment of the polypeptide chain. A comparison of the tryptic forms (representing the hydrophilic domains) of the rabbit and rat reductases by terminal sequence analysis showed a high degree of similarity, with about 80% of the residues in exact correspondence and only a short variable region near the Ile NH2 terminus.
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PMID:Structural features of liver microsomal NADPH-cytochrome P-450 reductase. Hydrophobic domain, hydrophilic domain, and connecting region. 680 23

Bilirubin diglucuronide and bilirubin monoglucuronide are formed on incubation of microsomal preparations from rat liver with bilirubin and UDPglucuronate. Microsomal diglucuronide formation is a two-step reaction: first monoglucuronide is formed and this is subsequently converted to diglucuronide. Both steps require UDPglucuronate and have a similar pH optimum at pH 7.8. Albumin inhibits the conversion of monoto diglucuronide. Factors favouring diglucuronide formation are: (a) low bilirubin concentration; (b) relatively high UDPglucuronate concentration; (c) complete removal of UDPglucuronyltransferase latency. For the latter, trypsin-treatment appeared superior over digitonin or UDP-N-acetylglucosamine. Trypsin-treatment had to be done under strictly anaerobic conditions. If trypsin treatment was done under aerobic conditions, reactive molecules were formed which initiated the rapid oxidation of bilirubin and its glucuronides. Microsomal oxidation of bilirubin and glucuronides also occurred in untreated and digitonin-treated microsomes and was stimulated by NADPH and by the cytochrome P-450 inhibitor, metyrapone. This suggests that lipid peroxides act as initiators of bilirubin oxidation. Indirect evidence was found that trypsin inactivates nucleotide pyrophosphatase. This is an active UDPglucuronate-consuming enzyme in microsomal preparations which must be inactivated before meaningful kinetic studies can be done. With trypsin-treated microsomal preparations the Vmax for bilirubin monoglucuronide formation was 1.7 X 10(-9) mol . mg protein-1 . min-1 and KUDPglucuronatem 43 X 10(-6) M. For bilirubin diglucoronide formation the apparent Vmax was 0.7 X 10(-9) mol . mg protein-1 . min-1 and the apparent KUDPglucuronate m 1.0 X 10(-3) M.
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PMID:Microsomal conjugation and oxidation of bilirubin. 687 Dec 45

The effects of trypsin, phospholipase A, and chymotrypsin on NADPH-cytochrome c reductase and cytochrome P-450 of microsomes from cryptorchid mouse testes and liver were compared. Trypsin released both enzymes almost completely from testis microsomes, while it readily released only NADPH-cytochrome c reductase from liver microsomes. Chymotrypsin alone, even under conditions where 30-40% of the microsomal protein was hydrolyzed, had little effect on localization or activity of either enzyme in either tissue. Phospholipase A destroyed cytochrome P-450 in testicular microsomes but had little effect on this enzyme in hepatic microsomes or on NADPH-cytochrome c reductase in either preparation. When, however, the microsomes were incubated with chymotrypsin in the presence of a detergent, the effects were similar to those of trypsin alone; testicular cytochrome P-450 was destroyed, while hepatic cytochrome P-450 was only slightly solubilized, and NADPH-cytochrome c reductase from both types of microsomes was both solubilized and activated. From these results we conclude that arginyl and/or lysyl bonds may play a significant role in the junction between the hydrophobic region of the membrane and the anchor region of the reductase molecule and that cytochrome P-450 of testicular microsomes is more superficially located in the lipid bilayer than is hepatic microsomal cytochrome P-450.
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PMID:The environment of cytochrome P-450 in testicular microsomes. 720 24

A method for biomembrane reconstitution from microsomal proteins and lipids solubilized by sodium cholate consisting in a removal of the detergent by its dialysis followed by treatment with 10% albumin has been developed. A comparison of the original and reconstituted membranes showed that the phospholipid, protein and enzymatic composition of the latter is similar or only slightly different from that of the original ghosts. The reconstituted membranes contained 1.5 times more cytochrome b5 and an equal amount of cytochrome P-450. No more than 20% of cytochrome P-450 was represented by the inactive form. The inactivation rate of the reduced hemoprotein in the reconstituted membranes was lower than in the ghosts. Both in the reconstituted and original membranes the similarity of solubilization patterns of microsomal electron carries upon trypsin treatment was indicative of identical topography of these proteins. The most effective was the reconstitution of NADH and cumole hydroperoxide-dependent N-demethylase, whereas the p-hydroxylase and O-dealkylase activities of the reactivated P-450 were not retained. Hence, no complete reconstitution of the properties of the microsomal membrane and its redox chain was observed in spite of the effective removal of the detergent, similar localization of microsomal electron carriers in the reconstituted membranes and ghosts and the reconversion of cytochrome P-420 into cytochrome P-450.
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PMID:[Comparative study of original and reconstituted by self-assembly endoplasmic reticulum membranes]. 729 23

Two cytochrome P-450 preparations, a constitutive isozyme, form 3b, and a phenobarbital-induced isozyme, form 2, were isolated from rabbit liver microsomes and compared by peptide mapping following digestion with trypsin and by partial sequence analysis. The NH2-terminal sequence of form 3b differed from form 2 in 15 out of 18 amino acids, but both forms have an NH2-terminal methionine residue followed by an acidic residue. Comparisons of many of the tryptic peptides of the two forms by means of high pressure liquid chromatography, as well as amino acid composition and sequence analysis, indicated that peptides from these forms, with one exception, are different. A tridecapeptide, differing only in a methionine (form 3b)/isoleucine (form 2) replacement was isolated from both forms. The amino acid sequence of this peptide is as follows: Met-Pro-Tyr-Thr-Asp-Ala-Val-Ile/Met-His-Glu-Ile-Gln-Arg. Taken together, these data indicate that forms 2 and 3 represent dissimilar gene products. The observation that these two cytochromes share an analogous peptide suggests that this tridecapeptide may contribute structural information necessary for common functional properties.
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PMID:Amino acid sequence of an analogous peptide from two forms of cytochrome P-450. 729 9

1. Cytochrome P-450 has been detected in preparations of golgi apparatus from the livers of phenobarbital-induced rats. 2. Newly biosynthesized cytochrome P-450 is also present in preparations of golgi apparatus. By using three different techniques to fractionate the golgi into vesicle contents and membrane components it was found that newly biosynthesized cytochrome P-450 is associated solely with the membrane fraction. 3. By investigating the susceptibility of cytochrome P-450, present in the golgi apparatus, to the action of trypsin it was found that the majority was oriented on the cytosolic face of the membrane.
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PMID:Newly biosynthesized cytochrome P-450 associated with the golgi apparatus from livers of rats induced with phenobarbital. 730 18

Noninbred male guinea-pigs (b. mass 250-300 g) were kept for 35 days on diets in which 50% of standard proteins were replaced for cow milk protein (CMP) 20/80 and its enzymatic hydrolyzate (EH) 20/80 obtained by ultrafiltration. CMP 20/80 and EH 20/80 have been examined for the effect on proteolytic activity of gastric mucosa, small intestinal activity of trypsin and chymotrypsin, hepatic cytochrome P-450 and metabolism of excreted 17-OCS. The animals given EH 20/80 exhibited enhanced total proteolytic activity of trypsin and chymotrypsin, higher total levels of cytochromes P-450 and P-450B in the liver, increased urine excretion of 17-OCS, reduced amount of polar forms in all the glucocorticoid fractions. Potential mechanisms securing the action of enzymatic hydrolyzates on the systems responsible for nonspecific resistance to food allergens are suggested for discussion.
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PMID:[Effect of milk proteins and their enzymatic hydrolyzates on non-specific resistance of some systems to food allergens]. 797 5

Various reports have described that amino acid substitutions can alter substrate, positional, inhibitory, and target gene specificities of proteins. By using the method of Chou and Fasman, the present work predicts that critical amino acids for converting these substrate specificities of trypsin, L-lactate dehydrogenase, aspartate aminotransferase, beta-lactamase, and cytochrome P-450 are found to exist within regions predicted as beta-turns. The ratios of hydroxylation and oxygenation positions of substrates by cytochrome P-450 and lipoxygenase, respectively, are varied by changes of the protein structures, probably around turn conformations. Inhibitory specificities of bovine pancreatic trypsin inhibitor and alpha 1-antitrypsin and target gene specificity of glucocorticoid receptor are converted by changing turn structures. Occurrence of beta-turn probabilities can be predicted around the amino acid alteration positions of an evolutionally antecedent protein of a nylon degradation enzyme. These findings will have relevance to work on protein engineering and enzyme evolution.
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PMID:Critical amino acids responsible for converting specificities of proteins and for enhancing enzyme evolution are located around beta-turn potentials: data-based prediction. 813 29

Methyltrienolone, a synthetic steroid, was used as a photoaffinity ligand for steroid-binding proteins. The enzymatic activity of bovine adrenocortical cytochrome P-450(11) beta was inhibited by methyltrienolone in a competitive manner without exposure to light and cytochrome P-450(11) beta was photolabeled with methyltrienolone after irradiation with UV light. The addition of 11-deoxycorticosterone during photolabeling protected cytochrome P-450(11) beta from photolabeling. Photolabeled cytochrome P-450(11) beta was digested with TPCK-treated trypsin and the peptide fragments were separated with a reverse-phase HPLC system. The labeled peptide was analyzed and its amino acid sequence was determined to be Trp428-Leu429-Asp430-Arg431. Alignment of the primary structure of cytochrome P-450(11) beta with that of cytochrome P-450cam revealed that the identified sequence corresponds to the region between the beta 3-sheet and L-helix of cytochrome P-450cam. This region of mammalian cytochromes P-450 shows poor homology with that of cytochrome P-450cam, but is well-conserved, especially at Trp-428 and preceding amino acids, as the aromatic region. The present results demonstrate that the labeled sequence contributes in part to the formation of the substrate binding pocket of cytochrome P-450(11) beta which was not expected from the results of the primary sequence alignment with cytochrome P-450cam.
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PMID:Photoaffinity labeling of cytochrome P-45011 beta with methyltrienolone as a probe for the substrate binding region. 843 74


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