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)

Adrenodoxin is an iron-sulfur protein which functions as a carrier of reducing equivalents in steroid hydroxylation reactions catalyzed by specific cytochromes P-450 in steroidogenic tissues such as adrenal cortex. Purified bovine adrenocortical adrenodoxin was shown to be selectively phosphorylated upon incubation with purified cAMP-dependent protein kinase, whereas other protein kinases were ineffective. The phosphorylation reaction was completed within 45 min at 30 degrees C and resulted in the optimal incorporation of 1 mol phosphate/mol adrenodoxin. Apoadrenodoxin, lacking the iron-sulfur cluster, was also phosphorylated under similar conditions. An apparent Km of 55 microM with a Vmax of 0.3 pmol 32P incorporated min-1 mg adrenodoxin-1 was calculated. Phosphorylation resulted in a striking change in several molecular properties of adrenodoxin, such as electrophoretic behavior and hydroxyapatite affinity, thus providing the possibility of clearly separating phosphorylated from unphosphorylated adrenodoxin. In addition, phosphoadrenodoxin became refractory to mild trypsin degradation, whereas this was not the case with apoadrenodoxin. The phosphorylated site of adrenodoxin was identified as a serine residue; study of peptide products resulting from CNBr and proteolytic cleavages of phosphoadrenodoxin suggested that Ser-88 was the target of the phosphorylation reaction. The influence of phosphorylation upon adrenodoxin activity was examined using cholesterol side-chain cleavage and 11 beta-hydroxylase (11 beta) systems, reconstituted from purified components. Phosphorylation of adrenodoxin resulted in an average twofold decrease in its Km values for the two specific cytochromes P-450 involved. This effect was paralleled by a positive relationship between the degree of adrenodoxin phosphorylation and its ability to support the overall activity of reconstituted side-chain cleavage and 11 beta-hydroxylase systems. Although it remains to be examined whether adrenodoxin is phosphorylated in the intact cell, the present observations suggest that it represents a potential target in the hormonal regulation of the adrenocortical differentiated functions, especially by stimulatory agents acting through a cyclic-AMP-dependent mechanism, such as adrenocorticotropin.
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PMID:Phosphorylation of bovine adrenodoxin. Structural study and enzymatic activity. 282 99

Adrenodoxin, purified from bovine adrenal cortex, was subjected to trypsin cleavage to yield a trypsin-resistant form, designated TT-adrenodoxin. Sequencing with carboxypeptidase Y identified the trypsin cleavage site as Arg-115, while Edman degradation indicated no NH2-terminal cleavage. Native adrenodoxin and TT-adrenodoxin exhibited similar affinity for adrenodoxin reductase as determined in cytochrome c reductase assays. In side chain cleavage assays using cytochrome P-450scc, however, TT-adrenodoxin demonstrated greater activity than adrenodoxin with cholesterol, (22R)-22-hydroxycholesterol, or (20R,22R)-20,22-dihydroxycholesterol as substrate. This enhanced activity is due to increased affinity of TT-adrenodoxin for cytochrome P-450scc; TT-adrenodoxin exhibits a 3.8-fold lower apparent Km for the conversion of cholesterol to pregnenolone. TT-Adrenodoxin was also more effective in coupling with cytochrome P-450(11) beta, exhibiting a 3.5-fold lower apparent Km for the 11 beta-hydroxylation of deoxycorticosterone. In the presence of partially saturating cholesterol, TT-adrenodoxin elicited a type I spectral shift with cytochrome P-450scc similar to that induced by adrenodoxin, and spectral titrations showed that oxidized TT-adrenodoxin exhibited a 1.5-fold higher affinity for cytochrome P-450scc. These results establish that COOH-terminal residues 116-128 are not essential for the electron transfer activity of bovine adrenodoxin, and the differential effects of truncation at Arg-115 on interactions with adrenodoxin reductase and cytochromes P-450 suggest that the residues involved in the interactions are not identical.
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PMID:Adrenodoxin with a COOH-terminal deletion (des 116-128) exhibits enhanced activity. 291 75

Chloroplast fructose-1,6-bisphosphatase is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars and the properties of this enzyme are clearly distinct from cytosolic gluconeogenic fructose-1,6-bisphosphatase. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. In the present study, purified spinach chloroplast fructose-1,6-bisphosphatase was reduced, S-carboxymethylated with iodoacetic acid, and cleaved with either cyanogen bromide or trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography. Automated Edman degradation of some of the purified peptides showed amino acid sequences highly homologous to residues 72-86, 180-199, and 277-319 of pig kidney fructose-1,6-bisphosphatase. These findings suggest a common evolutionary origin for mammalian gluconeogenic and chloroplast fructose-1,6-bisphosphatase, enzymes catalyzing the same reaction but having different functions and modes of regulation.
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PMID:Amino acid sequence similarity between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase. 300 49

Modification of carboxyl groups on putidaredoxin with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) resulted in loss of putidaredoxin reductase activity. The modification did not affect the visible absorption spectrum of putidaredoxin, indicating that the iron-sulfur center was not perturbed. In order to identify the carboxyl groups labeled by EDC, native and EDC-treated putidaredoxin were digested with a combination of trypsin and Staphylococcus aureus protease, and the resulting peptides were separated by high pressure liquid chromatography. The most heavily modified carboxyl groups were found to be those at residues 58, 65, 67, 72, and 77. These carboxyl groups are located in the same general region of the protein as those on adrenodoxin that have been shown to be involved in binding to both adrenodoxin reductase and cytochrome P-450scc. Chemical modification was also used to compare the role of lysine, arginine, and histidine residues on putidaredoxin and adrenodoxin. Modification of lysine and arginine residues had no effect on the reductase activity of either protein. The reductase activity of adrenodoxin was unaffected by labeling with 1 eq of diethyl pyrocarbonate/histidine residue, but labeling with a second equivalent completely abolished both activity and the iron-sulfur center spectrum. In contrast, modification of the 2 histidines in putidaredoxin with 1 eq each resulted in nearly complete loss of reductase activity. There was no significant activity for adrenodoxin in the putidaredoxin reductase assay or for putidaredoxin in the adrenodoxin reductase assay, demonstrating that, in spite of the structural similarity between the two proteins, they are not interchangeable functionally.
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PMID:The involvement of carboxylate groups of putidaredoxin in the reaction with putidaredoxin reductase. 309 90

The ferredoxin was purified from the green alga, Chlamydomonas reinhardtii. The protein showed typical absorption and circular dichroism spectra of a [2Fe-2S] ferredoxin. When compared with spinach ferredoxin, the C. reinhardtii protein was less effective in the catalysis of NADP+ photoreduction, but its activity was higher in the light activation of C. reinhardtii malate dehydrogenase (NADP). The complete amino acid sequence was determined by automated Edman degradation of the whole protein and of peptides obtained by trypsin and chymotrypsin digestions and by CNBr cleavage. The protein consists of 94 residues, with Tyr at both NH2 and COOH termini. The positions of the four cysteines binding the two iron atoms are similar to those found in other [2Fe-2S] ferredoxins. The primary structure of C. reinhardtii ferredoxin showed a great homology (about 80%) with ferredoxins from two other green algae.
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PMID:Purification, properties and complete amino acid sequence of the ferredoxin from a green alga, Chlamydomonas reinhardtii. 335 5

Mature bovine adrenocortical ferredoxin (adreno-ferredoxin) was extracted from fresh adrenal glands at pH 9.0. Extraction and purification at this alkaline pH protected the mature adreno-ferredoxin molecule from proteolytic degradation. The mature adreno-ferredoxin was extensively purified by a rapid procedure including two kinds of column chromatography, hydrophobic and ion exchange. The purified adreno-ferredoxin was homogeneous on the basis of two HPLC analyses, hydrophobic and ion exchange, and had the highest purity so far reported. Then it was digested by trypsin and the carboxyl-terminal peptide was isolated from the tryptic digest by a novel column chromatographic method using a cation-exchange HPLC column, TSK-gel SP-5PW. The carboxyl-terminal amino acid was isoleucine, so the adreno-ferredoxin had 127 amino acid residues, the longest polypeptide so far determined chemically for bovine adreno-ferredoxin. Only Glu-128 was lacking within the carboxyl-terminal elongated peptide that was found by nucleotide sequencing of the adreno-ferredoxin gene. There was no evidence obtained on whether the deletion of Glu-128 was due to so-called carboxyl-terminal processing or to proteolytic degradation during storage and purification.
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PMID:Isolation and purification of mature bovine adrenocortical ferredoxin with an elongated carboxyl end. 339 21

Cytochrome P-450scc consists of two domains linked with a short loop of the polypeptide chain; under its hydrolysis with trypsin the domains retain their associated state due to rigid noncovalent interactions. The structural characteristics of the individual domains have been investigated. It is established that domain I containing the haem and the adrenodoxin-binding site is the N-terminal, and domain II the C-terminal moiety of the polypeptide chain of cytochrome P-450scc.
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PMID:Domain structure of adrenocortical cytochrome P-450scc: localization of functionally important sites in the polypeptide chain. 406 13

The ferredoxin (Fd) molecule is a small non-mammalian immunogenic protein containing 55 amino acid residues with only two major antigenic determinants located with the NH2-terminal heptapeptide and the COOH-terminal pentapeptide. Selective enzyme cleavages of Fd with either trypsin or carboxypeptidase A result in the inactivation of the antigenic determinants by the removal of a tripeptide at the NH2-terminal and two amino acid residues at the COOH-terminal, effectively leaving 52 and 53 amino acid fragments respectively, each containing a single antigenic determinant. Fd digested with both enzymes yielded a 50 amino acid peptide with both determinants inactivated. Purity of these digests was assessed using monoclonal antibodies in standard and antigen-blocking ELISAs. The doubly digested peptide had virtually no reactivity with anti-Fd sera, reconfirming that the central cysteine-rich region is serologically silent. It was found that the sum of the reactivities of the N- and C-determinant-bearing peptides as equal to that of the native Fd and that the ratio of the reactivities could be used to assess determinant selectivity in the response to Fd in congenic recombinant strains of mice. This method was used in mapping the determinant selectivity in the antibody response to the MHC of mice to the left of the I-B subregion. Use of the B10.HTT strain indicated that separate genes mapping to the same subregion code for the magnitude of the antibody response and the determinant selectivity of the response.
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PMID:The use of unideterminant fragments of ferredoxin in the genetic mapping of determinant specificity of the immune response. 618 Mar 12

Modification of bovine adrenodoxin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) dramatically inhibited the reaction with adrenodoxin reductase (EC 1.18.1.2). The modification did not cause any change in the visible spectrum of adrenodoxin, indicating that the iron-sulfur center was not perturbed. Furthermore, the anomalous fluorescence of Tyr 82 was not changed in either intensity or wavelength. The inhibition was accompanied by the covalent incorporation of 14C-labeled EDC into adrenodoxin. The sites modified by EDC were determined by hydrolyzing adrenodoxin with either trypsin or Staphylococcus aureus protease and separating the resulting peptides by reverse phase high pressure liquid chromatography. The major carboxyl groups modified were found to be at Glu 74, Asp 79, and Asp 86, which are located in a sequence containing a high negative charge density. We propose that the conversion of negatively charged carboxylate groups at these residues to bulky, positively charged EDC-carboxyl groups inhibits the reaction with the reductase. EDC was also found to cross-link adrenodoxin to cytochrome c in yields up to 90%. The cross-links were found to involve the formation of amide linkages between carboxyl groups on adrenodoxin and the lysine amino groups surrounding the heme crevice of cytochrome c.
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PMID:Identification of specific carboxylate groups on adrenodoxin that are involved in the interaction with adrenodoxin reductase. 636 5

Highly specific antibodies against hemeprotein were obtained by immunizing rabbits with a highly purified cholesterol-hydroxylating cytochrome P-450scc from adrenocortical mitochondria. The antibodies do not specifically interact with other components of the adrenocortical electron transport chain, e. g., adrenodoxin reductase and adrenodoxin. Using double immunodiffusion technique (Ouchterlony method), it was shown that the antibodies did not precipitate the microsomal cytochromes P-450 LM2 and LM4, cytochrome b5 and 11 beta-hydroxylating cytochrome P-450 from adrenocortical mitochondria. Antibodies against cytochrome P-450scc inhibited the cholesterol side chain cleavage activity of cytochrome P-450scc in a reconstituted system. Limited proteolysis with trypsin and immunoelectrophoresis in the presence of specific antibodies revealed that antigenic determinants are present of the heme-containing catalytic domain of cytochrome P-450scc (F1) as well as on the domain responsible for the interaction with the phospholipid membrane (F2).
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PMID:[Immunochemical characteristics of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria]. 644 2


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