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)

The highly specific ligand [125I]bovine (b) PTH-(1-34) and a chemical cross-linking technique were used to explore structural features of the canine renal cortical PTH receptor. Membranes isolated under conditions designed to inhibit endogenous proteolysis displayed a major 85K labeled PTH receptor moiety on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-linked receptors were solubilized with Lubrol-PX and partially purified by affinity chromatography on wheat germ agglutinin-agarose, and their hydrodynamic properties were assessed [Stokes radius = 7.3 +/- 0.1 nm; sedimentation coefficient = 6.4 +/- 0.2S; partial specific volume = 0.758 +/- 0.01 ml/g; frictional coefficient = 1.68 +/- 0.04; mol wt (Mr) = 216,000 +/- 14,000]. Corrections for detergent binding and for the presence of carbohydrate yielded an estimated Mr of 166,000 +/- 11,000 for the solubilized PTH receptor. Thus, the renal PTH receptor is oligomeric, with a Mr approximating that expected of a homodimer of 85K subunits. Peptide-mapping experiments revealed the presence within the 85K PTH receptor subunit of at least two major regions sensitive to proteolytic attack. Both elastase and an endogenous renal protease(s) cleaved the PTH receptor to a 70K form that is fully functional with respect to high affinity, guanyl nucleotide-sensitive PTH binding. Cleavage in a second domain by elastase, S. aureus V8 protease, or chymotrypsin generated a 50K labeled PTH receptor fragment. Cleavage at this second site was prevented by prior occupancy of the receptor with [125I]bPTH-(1-34), suggesting that this domain may be functionally important. Reduction of receptor disulfide bonds with dithiothreitol and beta-mercaptoethanol released a low Mr (less than or equal to 14K) labeled PTH receptor component, similar treatment of renal membranes abolished specific PTH binding, indicating that an intact disulfide bond(s) is essential for receptor function. These results provide new insights into the structural basis of PTH receptor function.
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PMID:Structural properties of the renal parathyroid hormone receptor: hydrodynamic analysis and protease sensitivity. 284 81

Purified calmodulin-stimulated cyclic nucleotide phosphodiesterase from brain, a homodimer of 59-kDa subunits, was activated by limited proteolysis with trypsin, alpha-chymotrypsin, Pronase, or papain and could not be further stimulated by addition of Ca2+ and calmodulin. Proteolysis increased Vmax and had little effect on the Km for cGMP. Treatment with alpha-chymotrypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) produced, sequentially, 57- and 45-kDa peptides from the bovine and 55-, 53-, and 38-kDa peptides from the ovine enzyme. This protease-treated phosphodiesterase exhibited a Stokes radius of 3.9 nm and an S20,w value of 4.55; comparison with the hydrodynamic properties observed for native enzyme (4.3 nm, 5.95 S) strongly suggests a dimeric protein of Mr approximately 80,000-90,000. The proteolyzed species does not interact significantly with calmodulin immobilized on agarose, nor does it show complex formation with 2-dimethylaminonaphthalene-1-sulfonyl-calmodulin even at micromolar concentrations of protein. Proteolysis, in the presence of calmodulin plus Ca2+, fully activated phosphodiesterase, producing the same intermediate peptides; however, final peptides from the bovine and ovine enzymes were 47 and 42 kDa, respectively, indicating a new, specific conformation of the enzyme. When EGTA was added to such incubations, these peptides were cleaved to those of the size seen when proteolysis was carried out entirely in the presence of EGTA. The initial rate of activation was increased by the presence of Ca2+ and calmodulin, suggesting that, in complex, phosphodiesterase exhibits a site with increased susceptibility to proteolysis. Since calmodulin can still interact with a fully activated form of the enzyme, it appears that retention of calmodulin binding can occur concomitantly with damage to that portion of the phosphodiesterase molecule responsible for suppression of its basal catalytic activity.
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PMID:Proteolytic activation of calmodulin-dependent cyclic nucleotide phosphodiesterase. 299 Dec 33

H1 histone homodimer has been purified from nuclei treated with either long (dimethylsuberimidate, dimethyl dithiobisproprionimidate) or short (ethyl dimethylaminocarbodiimide) cross-linking reagents. When such H1 dimers were cleaved with N-bromosuccinimide, chymotrypsin, or staphylococcal protease, analysis of the resulting fragments suggests that the major cross-links were formed between the COOH-terminal portions of neighboring H1 molecules, and that substantial bridging can also be made between the NH2-terminal portion of one H1 molecule and the COOH-terminal portion of another. NH2-terminal to NH2-terminal cross-links were not seen at significant levels, suggesting that the position of the NH2-terminal portion of H1 in chromatin is more restricted.
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PMID:Close contacts between H1 histone molecules in nuclei. 636 Oct 33

A protein capable of inhibiting trypsin and other pancreatic proteases has been purified to homogeneity from Escherichia coli by conventional procedures and affinity chromatography. It is stable for at least 30 min at 100 degrees C and pH 1.0, but it is inactivated by digestion with pepsin. The inhibitor has an apparent molecular weight of 38,000 as determined by gel filtration and must be a homodimer since it contains a single 18,000-dalton subunit upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor has an isoelectric point of 6.1. One dimeric molecule of the inhibitor can bind two trypsin molecules to form a mixed tetrameric complex, in which trypsin molecules are completely inhibited. The inhibitor is not digested by the trypsin. When N-benzoyl-DL-arginine-p-nitroanilide was used as a trypsin substrate, half-maximal inhibition was observed at 22 nM. This protein also inhibits chymotrypsin, pancreatic elastase, rat mast cell chymase, and human serosal urokinase, but it does not inhibit human pulmonary tryptase, kallikrein, papain, pepsin, Staphylococcus aureus V8 protease, subtilisin, and thermolysin. Surprisingly, it did not inhibit any of the eight soluble endoproteases recently isolated from E. coli (i.e. proteases Do, Re, Mi, Fa, So, La, Ci, and Pi) nor the chymotrypsin-like (protease I) and trypsin-like (protease II) esterases in E. coli. The inhibitor is localized to the periplasmic space and its level did not change with different growth media or stages of cell growth. The physiological function of this E. coli trypsin inhibitor is unknown. We suggest that E. coli trypsin inhibitor be named "Ecotin."
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PMID:Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases. 641 24

This study describes the mechanism of homodimer formation of the 90-kDa heat-shock protein (HSP90). In eukaryotic cells, there are two HSP90 isoforms, alpha and beta, encoded by two separate genes. HSP90 alpha exists predominantly as a homodimer, HSP90 beta mainly as a monomer. Analysis by native PAGE revealed that bacterially expressed HSP90 alpha fused to glutathione S-transferase (GST) existed as a high-molecular-mass oligomer, and was converted to a homodimer following removal of the fusion enzyme by thrombin cleavage. A deletion mutant, HSP90 alpha D44-603, formed a monomer and an N-terminal truncated mutant, HSP90 alpha 533-732, existed as a dimer, indicating that the dimer-forming ability resides somewhere in the C-terminal 200 amino acids. Limited proteolysis of the C-terminal 200 amino acids of HSP90 alpha with chymotrypsin produced the C-terminal 16-kDa fragment (Met628/Ala629-Asp732) and its adjacent more N-terminal 13-kDa fragment (Val542-Tyr627/Met628). Size-exclusion HPLC and two-dimensional PAGE analyses demonstrated that these two chymotryptic fragments bound each other. The C-terminal 198 amino acids as well as the full-length form of HSP90 beta revealed a lower dimer-forming activity than HSP90 alpha. Expression of the chimeric proteins at the C-terminal 198 amino acids of the alpha and beta isoforms further indicated that the 16 amino acid substitutions locating between amino acids 561 and 685 account for the impeded dimerization of HSP90 beta. A leucine zipper motif (Met402-Leu423) was unlikely to be involved in the dimer formation. Taken together, these results indicate that the dimeric structure of HSP90 alpha is mediated by the C-terminal 191 amino acids and consists of duplicate interactions of the C-terminal region (Met628/Ala629-Asp732) of one subunit and the adjacent more N-terminal region (Val542-Try627/Met628) of the other subunit.
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PMID:Mechanism of dimer formation of the 90-kDa heat-shock protein. 758 31

The coding region of copper/zinc-superoxide dismutase (Cu/Zn-SOD) cDNA from sweet potato, Ipomoea batatas (L.) Lam. cv. Tainong 57, was introduced into an expression vector, pET-20b(+). The Cu/Zn-SOD purified by His-tagged technique showed two active forms (dimer and monomer). The amount of proteins of dimer and monomer appeared to be equal, but the activity of dimeric form was seven times higher than that of monomeric form. The enzyme was dissociated into monomer by imidazole buffer above 1.0 M, acidic pH (below 3.0), or SDS (above 1%). The enzyme is quite stable. The enzyme activity is not affected at 85 degrees C for 20 min, in alkali pH 11.2, or in 0.1 M EDTA and also quite resistant to proteolytic attack. Dimer is more stable than monomer. The thermal inactivation rate constant kd calculated for the monomer at 85 degrees C was 0.029 min-1 and the half-life for inactivation was about 28 min. In contrast, there is no significant change of dimer activity after 40 min at 85 degrees C. The enzyme dimer and monomer retained 83% and 58% of original activity, respectively, after 3 h incubation with trypsin at 37 degrees C, while those retained 100% and 31% of original activity with chymotrypsin under the same condition. These results suggest subunit interaction might change the enzyme conformation and greatly improve the catalytic activity and stability of the enzyme. It is also possible that the intersubunit contacts stabilize a particular optimal conformation of the protein or the dimeric structure enhances catalytic activity by increasing the electrostatic steering of substrate into the active site.
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PMID:Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method. 759 15

Chemical cross-linking has been used to determine the composition of the erythrocyte band 3 protein dimer in Southeast Asian ovalocytes (SAO). Individuals with SAO are heterozygous for a mutation in which residues 400-408 of band 3 are deleted. Normal and variant protein are present in equal amounts, but the SAO protein does not transport anions or bind stilbenedisulfonates with high affinity. We find that the rate constant for 35SO4(2-) efflux from SAO cells is about 50% that of normal cells, but the time course is a single exponential, indicating that there is no detectable heterogeneity in the distribution of SAO band 3 in the population of cells. Treatment of intact cells with the homobifunctional crosslinker BS3 (bis[sulfosuccinimido]suberate) produces similar amounts of covalent dimer in both normal and SAO cells. In SAO cells, copies of normal band 3 can be distinguished from SAO band 3 by treating with H2DIDS to form a crosslink between major chymotryptic fragments (60 kDa and 35 kDa) within one subunit. Successive treatment of cells with [3H]-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate ([3H]H2DIDS), BS3, and chymotrypsin gives 3H-labeled products that include homodimer of normal band 3 as well as products of crosslinking normal band 3 with the 60- and 35-kDa fragment of SAO band 3. The results are in semiquantitative agreement with a model in which covalent dimer forms between normal and SAO subunits with the same probability as between two normal subunits. These results indicate that the normal copy of band 3, complexed in a heterodimer with SAO band 3, reacts with H2DIDS as in normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Anion exchange protein in Southeast Asian ovalocytes: heterodimer formation between normal and variant subunits. 789 55

The hemagglutinin-neuraminidase (HN) glycoprotein of paramyxoviruses is anchored in the virion membrane near its amino terminus, protruding from the virion surface to mediate attachment to cellular receptors. Solubilization of HN spikes can be achieved by treatment of virions with detergent and high salt concentrations. When the solubilized HN protein from the Australia-Victoria (AV) isolate of the virus is incubated at 37 degrees C, a chymotrypsin-sensitive site between residues 112 and 113 is exposed. A chymotrypsin-cleaved soluble form of the protein, named CT-HN, has been prepared using this approach. It is membrane anchor-less, due to removal of a 14-kDa fragment from the NH2 terminus of HN. It retains all potential glycosylation sites and cysteines present in the ectodomain of the native protein. It migrates in nonreducing gels and sediments in sucrose gradients at the rate expected for homodimeric HN. The latter is also consistent with our demonstration by site-directed mutagenesis that cysteine residues at positions 6 and 123, respectively, mediate disulfide-linked homotetramer and homodimer formation. CT-HN retains almost total antigenicity, suggesting that it is conformationally very similar to the intact molecule, as well as receptor recognition function and, at low pH, neuraminidase activity. It should prove to be a useful tool for further studies of the structure and function of this important viral glycoprotein.
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PMID:Structure and function of a membrane anchor-less form of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus. 840 85

Talin, a putative homodimer of 230-kDa polypeptides, was cleaved into the N-terminal 47-kDa and C-terminal 190-kDa fragments with calpain II. The 190-kDa fragment, but not the 47-kDa fragment, was found to bind to actin. The 190-kDa fragment possessed similar levels of activities to stimulate both polymerization of G-actin and alpha-actinin-dependent gelation of F-actin as did intact talin. Limited digestions of the 190-kDa fragment with chymotrypsin and papain resulted in partial and complete reductions, respectively, of both activities, although these digests contained 95- and 46-kDa major polypeptides, respectively, which were able to bind to actin. Whereas the 190-kDa fragment generated fully cross-linked oligomeric polypeptides on treatment with 1-ethyl-3[3-(dimethylamino)-propyl]carbodiimide, the 95-kDa chymotryptic polypeptide generated heterologous polypeptides cross-linked partially with smaller polypeptides. The papain digest did not contain any cross-linkable polypeptide. Intact talin and the 47-kDa calpain fragment, but not the 190-kDa calpain fragment, were found to bind to phospholipid vesicles containing phosphatidylserine. These results indicate that the N-terminal and C-terminal domains play distinct roles in interacting with the membrane and cytoskeletal elements, respectively, and that the dimeric structure is also required for the latter interactions.
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PMID:Organization of the functional domains in membrane cytoskeletal protein talin. 858 16

beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli. To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The Kd of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding of the structure and function of beta protein.
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PMID:Characterization of the DNA-binding domain of beta protein, a component of phage lambda red-pathway, by UV catalyzed cross-linking. 898 71


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