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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli cAMP receptor protein (CRP) is a homodimer in which each subunit is composed of two domains. The C-terminal domain is responsible for DNA recognition, whereas the larger N-terminal domain is involved in cAMP binding. Biochemical and genetic evidence suggests that both intersubunit and interdomain interactions play important roles in the regulatory mechanism of this protein. Essentially all intersubunit contacts occur via a long C-helix which is a part of the N-terminal domain. In this work, intersubunit interactions in CRP were studied with the use of two proteolytic fragments of the protein. Subtilisin digestion produces a fragment (S-CRP) which includes residues 1-117 and in which about 85% of the C-helix is removed, whereas chymotrypsin digestion produces a fragment (CH-CRP) consisting of residues 1-136, in which the whole C-helix is preserved. Both fragments were purified and subjected to functional tests which included cAMP binding, subunit assembly, and hydrodynamic properties in the presence and absence of cAMP. S-CRP binds cAMP with a similar affinity to that of native CRP but with reduced cooperativity. CH-CRP exhibits about 1 order of magnitude tighter binding of cAMP than S-CRP or CRP and the highest degree of negative cooperativity. Both fragments are dimeric with dimerization constants around 10(8) M-1. Ligand binding promotes dimerization and induces a small contraction of both S-CRP and CH-CRP. There is no apparent correlation between dimer stability and cooperativity of ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Intersubunit communications in Escherichia coli cyclic AMP receptor protein: studies of the ligand binding domain. 131 47

Using specific anti-BiP/Kar2 antibody as the probe, we have developed an efficient purification method of BiP/Kar2 protein from the total cell extract of Saccharomyces cerevisiae. Overproduction of BiP/Kar2 protein was achieved by the cloning of the KAR2 gene on multicopy plasmids and the treatment of cells harboring the cloned KAR2 gene with tunicamycin. Freeze-thaw treatment, hydroxyapatite high pressure liquid chromatography, and ATP-agarose column chromatography of crude extract yielded homogeneous BiP/Kar2 protein (including less than 0.2% of degradative derivative) with a 430-fold purification and 28% recovery. Edman degradation of purified BiP/Kar2 suggests that the mature protein corresponds to a processed product with the removal of a 42-amino acid presequence. It is active as a homodimer and exhibits ATPase activity with a specific activity of 2 pmol/min/micrograms of protein. Protease susceptibility indicated that the ADP form of BiP/Kar2 is more resistant than the ATP form to the chymotrypsin digestion and that BiP/Kar2 required the presence of ATP to avoid the irreversible denaturation. Synthesis of BiP/Kar2 was induced by the inducible expression of an aberrant heterologous protein, yeast killer prepro-signal mouse alpha-amylase fusion protein.
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PMID:Purification and characterization of BiP/Kar2 protein from Saccharomyces cerevisiae. 132 40

A novel class alpha glutathione S-transferase (GST) isozyme is expressed in the hepatic cytosol of rabbits treated with 4-picoline. SDS-PAGE analysis revealed the presence of a new 28-kDa band which cross-reacted with class alpha GST-specific IgG. This new GST isozyme was isolated from the hepatic cytosol of 4-picoline-treated rabbits and purified to homogeneity using S-hexylglutathione-agarose, CM-Sepharose, and PBE118 chromatofocusing chromatography. The isozyme was determined by SDS-PAGE and gel filtration analyses to be a homodimer of approximately 28 kDa with blocked N-terminus. A heterodimer consisting of 25 and 28 kDa subunits with activity toward the substrate 1-chloro-2,4-dinitrobenzene was also purified. Immunoblot analysis revealed that the 25, 26.5, and 28 kDa bands cross-reacted with class alpha GST-specific IgG and failed to react with either class mu or class pi GST-specific antibodies. The 28 kDa enzyme had a pI of 8.2 as determined by nonequilibrium pH gel electrophoresis. The purified 28 kDa enzyme exhibited activity toward 1-chloro-2,4-dinitrobenzene (Km = 1.60 mM and Vmax = 73.5 mumol/min/mg) and cumene hydroperoxide (Km = 1.02 mM and Vmax = 6.92 mumol/min/mg). Amino acid sequence analysis of several fragments resulting from cyanogen bromide cleavage of the 28 kDa GST isozyme revealed a class alpha GST consensus sequence. In addition, proteolytic digestion with alpha-chymotrypsin yielded peptide maps which showed distinct differences between the purified 28 kDa GST and another purified class alpha GST isozyme present in rabbit liver. These results provide evidence that class alpha GST isozymes containing a novel 28 kDa subunit are expressed following treatment with 4-picoline.
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PMID:Enhanced expression, purification, and characterization of a novel class alpha glutathione S-transferase isozyme appearing in rabbit hepatic cytosol following treatment with 4-picoline. 153 65

Studies of cGMP binding to both the native cyclic GMP-stimulated phosphodiesterase and to two unique isolated chymotryptic fragments lacking the catalytic domain suggest that the enzyme contains two noncatalytic cGMP-binding sites/homodimer. In the presence of high concentrations of ammonium sulfate, 2 mol of cGMP are bound/mol of cGMP-stimulated phosphodiesterase homodimer. Under these conditions, linear Scatchard plots of binding are obtained that give an apparent Kd of approximately 2 microM. The inclusion of 3-isobutyl-1-methylxanthine produces a curvilinear plot. In the absence of ammonium sulfate, the dissociation of cGMP from the holoenzyme is rapid, having a t1/2 of less than 10 s, and addition of ammonium sulfate to the incubation greatly decreases this rate of dissociation. The native enzyme is resistant to degradation by chymotrypsin in the absence of cGMP; however, in its presence, chymotrypsin treatment produces several discrete fragments. Similarly, in the presence but not in the absence of cGMP, dicyclohexylcarbodiimide causes an irreversible activation of the enzyme without cross-linking the nucleotide to the phosphodiesterase. Both observations provide evidence that a different conformation in the enzyme results from cGMP binding. Only the conformation formed upon cGMP binding is easily attacked by chymotrypsin or permanently activated by treatment with dicyclohexylcarbodiimide. One major chymotryptic cleavage site exposed by cGMP binding is at tyrosine 553, implying that this region takes part in the conformational change. Limited proteolysis experiments indicate that these noncatalytic binding sites are located within a region of internal sequence homology previously proposed to include the cGMP-binding site(s) and that they retain a high affinity and specificity for cGMP independent of the catalytic domain of the enzyme. The products formed by partial proteolysis can be separated into individual catalytically active and cGMP-binding fractions by anion exchange chromatography. Gel filtration and electrophoresis analysis of the isolated fractions suggest that the cGMP-binding peak has a dimeric structure. Moreover, it can be further resolved by polyethyleneimine high performance liquid chromatography into two peaks (Peaks IIIA and IIIB). Peak IIIA binds 2 mol of cGMP/mol of dimer with an apparent Kd of 0.2 microM. Peak IIIB, however, has greatly reduced cGMP binding. Further digestion of these fragments with cyanogen bromide show that the differences between Peaks IIIA and IIIB are due to one or more additional proteolytic nicks in IIIB that remove a few residues near its C terminus, most probably residues 523-550 or 534-550. This in turn suggests that this region is essential for cGMP-binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structure and function studies of the cGMP-stimulated phosphodiesterase. 172 Oct 55

A green flavoprotein (GFP) was isolated and purified to homogeneity from Photobacterium leiognathi, strain 208. GFP is a homodimer of molecular weight 54,000 and contains two molecules of an unusual flavin per molecule of protein. Various biochemical characteristics including isoelectric point, trypsin and chymotrypsin degradation, SDS and temperature influence on subunit dissociation and the dissociation of the flavin chromophore, were investigated. The sequence of 23 N-terminal amino acids was determined and found to be concurrent with the N-terminal amino acid sequence encoded by the lux G(N) gene of P. leiognathi. This fact suggests that GFP is a structural component of the Photobacterium luminescence system.
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PMID:Green flavoprotein from P. leiognathi: purification, characterization and identification as the product of the lux G(N) gene. 174 16

The structural organization of the oxysterol receptor, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol biosynthesis in mammalian cells, has been explored by limited proteolysis with trypsin, alpha-chymotrypsin, and endoproteinase GluC. Treatment with each of these proteases converts the receptor from a homodimer of approximately 95 kDa subunits to a 44-kDa form, based on hydrodynamic measurements by sucrose density gradient centrifugation and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of photoaffinity-labeled preparations indicates that the oxysterol binding site is on a 28-kDa fragment within the 44-kDa limit form of the receptor. The limit proteolytic form exhibits the high affinity and structural specificity for oxysterols of the native dimeric receptor with an increase in the rate constant of association for 25-hydroxycholesterol. The proteolytic form also shows an increased binding affinity for nonspecific DNA, but no sequence specificity for the oxysterol regulatory element from the reductase gene was detected.
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PMID:A proteolytic fragment of the oxysterol receptor which retains oxysterol binding activity. 189 49

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase is a homodimer of monomer Mr = 109,228. Hydropathy analysis of its cDNA-deduced amino acid sequence (1043 residues) has indicated that the molecule is composed of 3 domains: a 430-residue-long hydrophilic N-terminal domain which binds NAD(H), a 200-residue-long hydrophilic C-terminal domain which binds NADP(H), and a 400-residue-long hydrophobic central domain which appears to be made up mainly of about 14 hydrophobic clusters of approximately 20 residues each. In this study, antibodies were raised to the hydrophilic N- and C-terminal domains cleaved from the isolated transhydrogenase by proteolytic digestion, and to a synthetic, hydrophilic pentadecapeptide, which corresponded to position 540-554 within the central hydrophobic domain. Immunochemical experiments with mitoplasts (mitochondria denuded of outer membrane) and submitochondrial particles (inside-out inner membrane vesicles) as sources of antigens showed that essentially the entire N- and C-terminal hydrophilic domains of the transhydrogenase, as well as epitopes from the central pentadecapeptide, protrude from the inner membrane into the mitochondrial matrix, where the N- and C-terminal domains would be expected to come together to form the enzyme's catalytic site. Treatment of mitoplasts with several proteolytic enzymes indicated that large protease-sensitive masses of the transhydrogenase are not exposed on the cytosolic side of the inner membrane, which agreed with the exception that the central highly hydrophobic domain of the molecule should be largely membrane-intercalated. Trypsin, alpha-chymotrypsin, and papain had little or no effect on the mitoplast-embedded transhydrogenase. Proteinase K, subtilisin (Nagarse), thermolysin, and pronase E each split the mitoplast-embedded enzyme into two fragments only, a fragment of approximately 70 kDa containing the N-terminal hydrophilic domain, and one of approximately 40 kDa bearing the C-terminal hydrophilic domain. The cleavage site of proteinase K was determined to be A690 -A691, which is located in a small hydrophilic segment within the central hydrophobic domain. This protease-sensitive loop appears to be exposed on the cytosolic side of the inner membrane. The proteinase K-nicked enzyme containing two peptides of 71 and 39 kDa was isolated from mitoplasts and shown to have high transhydrogenase activity.
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PMID:Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase. Membrane topography of the bovine enzyme. 200 10

We have investigated the influence of the N-terminal domain of the 94-kDa glucocorticoid receptor on the DNA:receptor interaction. An alpha-chymotrypsin-induced 39-kDa receptor fragment, containing the hormone and DNA binding domains, binds DNA with a reduced specificity compared to the intact 94-kDa receptor. Various footprinting assays did not reveal any qualitative differences when comparing the DNA contact points made by the two different receptor entities. Like the intact receptor, the 39-kDa receptor fragment binds as a dimer to DNA. Glutaraldehyde cross-linking demonstrated a difference in the protein:protein contacts of the two homodimers. Furthermore, the dimeric 94-kDa receptor did not recognize a half-DNA site, while the dissociated 94-kDa receptor dimer and the dimeric 39-kDa receptor fragment allowed binding to such a site. These results suggest that the loss of the N-terminal domain of the receptor affects the steric arrangement and/or rigidity of the two DNA binding domains of the receptor homodimer, resulting in a decreased DNA binding specificity of the 39-kDa receptor fragment.
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PMID:Protein-protein contacts in the glucocorticoid receptor homodimer influence its DNA binding properties. 230 60

Bacterially expressed recombinant HIV-1 reverse transcriptase is active as both a homodimer of Mr 66,000 subunits and a heterodimer of Mr 66,000 and 51,000 subunits. The heterodimer is formed by cleavage of a C-terminal fragment from one Mr 66,000 polypeptide, which occurs during purification and crystallization of reverse transcriptase. Thus, crystals obtained from purified Mr 66,000 polypeptide preparations consisted of an apparently equimolar mixture of Mr 66,000 and 51,000 polypeptides, which were apparently analogous to the Mr 66,000 and 51,000 polypeptides detected in HIV-infected cells and in virions. Limited proteolysis of the homodimer with alpha-chymotrypsin also resulted in cleavage to a stable Mr 66,000/51,000 mixture, and proteolysis with trypsin resulted in the transient formation of some Mr 51,000 polypeptide. These results are consistent with the reverse transcriptase molecule having a protease-sensitive linker region following a structured domain of Mr 51,000. Further digestion with trypsin resulted in cleavage of the Mr 51,000 polypeptide after residue 223, yielding peptides of apparent Mr 29,000 and 30,000. A minor peptide of Mr 40,000 was also produced by cleavage of the Mr 66,000 polypeptide after residue 223. About half the original Mr 66,000 polypeptides remained resistant to proteolysis and existed in complex with the above peptides in solution. During both chymotrypsin and trypsin digestion there was an increase in the reverse transcriptase activity caused by a doubling of Vmax with little change in Km for dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis. 246 81

Earlier histological studies have demonstrated that copper deficiency results in a selective and progressive atrophy of pancreatic acinar tissue. The present study examined both biochemical and morphological changes of the exocrine pancreas in nutritional copper deficiency. Groups of mature female rats were fed a purified diet either deficient (less than 0.5 micrograms/g) or sufficient (6.2 micrograms/g) in copper for 6 wk. Copper deficiency resulted in distinct ultrastructural changes in acinar cells, including marked variability in zymogen granule content, autophagic vacuoles and dilation of acinar lumen. Pancreatic weight and total DNA, RNA and protein content of the pancreas were similar in both groups of rats, whereas pancreatic amylase, trypsin and chymotrypsin activity was significantly lower in the copper-deficient group. In addition, secretagogue-induced release of these enzymes from dispersed acini isolated from copper-deficient rats was significantly reduced in comparison to enzyme secretion from normal controls. Pancreatic Cu-Zn and Mn superoxide dismutase activity was also found to be significantly lower in the copper-deficient rats than in normal controls. We conclude that nutritional copper deficiency in adult female rats reduces the responsiveness of the pancreas to secretagogues and may increase the susceptibility of the pancreas to oxidative damage.
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PMID:Morphological and biochemical changes in the pancreas of the copper-deficient female rat. 247 34


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