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 protein moiety of squid (Watasenia scintillans) rhodopsin has been shown to have a molecular weight of 46 800 by means of amino acid analysis. This value was comparable to the value (51 000) obtained from SDS-polyacrylamide gel electrophoresis. After the squid eyes were incubated at 10 degrees C for 8 days, the rhodopsin showed a molecular weight of 39 000 on electrophoresis. The smaller molecular weight was ascertained by amino acid analysis of the rhodopsin; and may result from autolysis by the lysosomal enzyme. The rhodopsin in rhabdomeric membranes and in detergent solution was treated with chymotrypsin, papain or subtilisin. These enzymes first produced the 39 000 dalton rhodopsin and then cleaved this into the 25 000 and 14 000 dalton peptides without bleaching. The rhodopsin was attacked by proteases and readily lost an approx. 12 000 dalton peptide portion. This portion included the COOH-terminal and was rich in glutamic acid, proline, glycine, alanine and tyrosine residues.
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PMID:Molecular weight and structural studies on cephalopod rhodopsin. 46 26

A purified iodopsin was digested by CNBr or several proteolytic enzymes into fragments, the amino acid sequences of which were determined. A partial sequence of the C-terminal fragment was utilized for synthesizing an oligonucleotide probe which identified the iodopsin cDNA (1339 bases). The deduced amino acid sequence (362 residues) had 80%, 42%, or 43% homology to that of human red-sensitive cone pigment, cattle or chicken rhodospin, respectively. Although the hydropathy profile implies that iodopsin, like rhodopsin, has 7 transmembrane alpha-helical segments, iodopsin may have a hydrophilic pocket near the seventh segment on the basis of the unexpected cleavages in the middle of the segment VII by chymotrypsin under nondenaturing conditions.
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PMID:The primary structure of iodopsin, a chicken red-sensitive cone pigment. 222 24

Bovine photoreceptor membranes have been treated with proteases to determine the accessibility of rhodopsin to these large, water soluble molecules. The polypeptides that remain associated with the membranous structure after proteolysis were detected by sodium dodecyl sulfate gel electrophoresis. Thermolysin and chymotrypsin degraded rhodopsin (apparent mol wt 35,000-36,000) to fragments of 29,000 and 23,000 apparent mol wt, respectively, without affecting the chromophoric absorption of the molecule or removing the region of the polypeptide carrying carbohydrate. The two fragments were isolated and their amino acid compositions were determined. They do not appear to be more hydrophobic than rhodopsin. Subtilisin, at low concentration and temperature, produced a fragment with the same molecular weight as that produced by thermolysin. At higher concentrations, subtilisin yields major fragments of mol wt 23,000 and 20,000 without affecting the chromophoric absorption. Two intermediate fragments of apparent mol wt 29,000 and 26,000 were detected during the course of this degradation. Carbohydrate is retained by all but the smallest fragment. Bleaching of the photoreceptor pigment did not appreciably alter any of the fragmentation patterns. Trypsin did not alter the molecular weight of rhodopsin under the conditions of this study. Approximately 35-45% of rhodopsin appears to be accessible to the aqueous environment and can be removed without affecting the chromophoric properties of the retinaldehyde-carrying region which remains bound to the membrane.
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PMID:The accessibility of bovine rhodopsin in photoreceptor membranes. 441 32

Proteolysis of rhodopsin in disc membranes of right-side out orientation by thermolysin, papain and St. aureus V8 protease allowed to identify two highly exposed regions of polypeptide chain located on the cytoplasmic membrane surface: carboxyl terminal sequence 321-348 and the fragment 236-241. Incubation with chymotrypsin reveals the third site on the cytoplasmic surface, 146-147, accessible to proteolytic enzymes. Frozen-thawed membranes comprise a mixture of vesicles with normal and inverted orientation. Both thermolytic and chymotryptic digests of rhodopsin in these membranes contain the polypeptide which represents the amino terminal sequence lacking the first 30 amino acid residues. Thus at least 30 amino acids from the N-terminus must protrude into the intradiscal space. One additional site was located on the intradiscal surface: papain digests rhodopsin in the inverted membranes at the position 186-187. Localization of the proteolytic cleavage sites allowed to propose a model for rhodopsin topography in disc membrane: the polypeptide chain traverses the bilayer thickness seven times; each of seven transmembrane segments containing approximately 40 amino acid residues includes a sequence of approximately 30 hydrophobic amino acids; which are probably in close contact with hydrocarbon matrix of the membrane. Hydrophobic sequences are terminated with fragments containing clusters of hydrophilic amino acids, possibly interacting with lipid polar head groups and orienting each segment in the bilayer.
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PMID:[Study of the molecular organization of visual rhodopsin in photoreceptor membranes by limited proteolysis]. 667 81

The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop domains extending out of the hydrophobic core. The thermal stability of rhodopsin and its bleached form, opsin, was investigated using differential scanning calorimetry (DSC). The thermal transitions were asymmetric, and the temperatures of the thermal transitions were scan rate dependent. This dependence exhibited characteristics of a two-state irreversible denaturation in which intermediate states rapidly proceed to the final irreversible state. These studies suggest that the denaturation of both rhodopsin and opsin is kinetically controlled. The denaturation of the intact protein was compared to three proteolytically cleaved forms of the protein. Trypsin removed nine residues of the carboxyl terminus, papain removed 28 residues of the carboxyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments remained associated as a complex in the membrane. DSC studies were carried out on each of the fragmented proteins. In all of the samples the scan rate dependence of the Tm indicated that the transition was kinetically controlled. Trypsin-proteolyzed protein differed little from the intact protein. However, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both bleached and unbleached samples. In the presence of the chromophore, 11-cis-retinal, the noncovalent interactions among the proteolytic fragments produced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragments exhibited a single thermal transition. However, after bleaching, the chymotrypsin fragments exhibited two calorimetric transitions. These data suggest that the loops of rhodopsin exert a stabilizing effect on the protein.
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PMID:Thermal destabilization of rhodopsin and opsin by proteolytic cleavage in bovine rod outer segment disk membranes. 1155 Dec 16

The effects have been examined of chymotrypsin, pepsin, trypsin, and pancreatic lipase on cattle rhodopsin in digitonin solution. The digestion of rhodopsin by chymotrypsin was measured by the hydrolysis of peptide bonds (formol titration), changes in pH, and bleaching. The digestion proceeds in two stages: an initial rapid hydrolysis which exposes about 30 amino groups per molecule, without bleaching; superimposed on a slower hydrolysis which exposes about 50 additional amino groups, with proportionate bleaching. The chymotryptic action begins at pH about 6.0 and increases logarithmically in rate to pH 9.2. Trypsin and pepsin also bleach rhodopsin in solution. A preparation of pancreatic lipase bleached it slightly, but no more than could be explained by contamination with proteases. In digitonin solution each rhodopsin molecule is associated in a micelle with about 200 molecules of digitonin; yet the latter do not appear to hinder enzyme action. It is suggested that the digitonin sheath is sufficiently fluid to be penetrated on collision with an enzyme molecule; and that once together the enzyme and substrate are held together by intermolecular attractive forces, and by the "cage effect" of bombardment by surrounding solvent molecules. The two stages of chymotryptic digestion of rhodopsin may correspond to an initial rapid fragmentation, such as has been observed with many proteinases and substrates; superimposed upon a slower digestion of the fragments. Since the first phase involves no bleaching, this may mean that rhodopsin can be broken into considerably smaller fragments without loss of optical properties.
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PMID:The action of enzymes on rhodopsin. 1358 19

The CB1 and CB2 cannabinoid receptors belong to the GPCR superfamily and are associated with a variety of physiological and pathophysiological processes. Both receptors, with several lead compounds at different phases of development, are potentially useful targets for drug discovery. For this reason, fully elucidating the structural features of these membrane-associated proteins would be extremely valuable in designing more selective, novel therapeutic drug molecules. As a first step toward obtaining information on the structural features of the drug-receptor complex, we describe the full mass spectrometric (MS) analysis of the recombinant human cannabinoid CB2 receptor. This first complete proteomic characterization of a GPCR protein beyond rhodopsin was accomplished by a combination of several LC/MS approaches involving nanocapillary liquid chromatography, coupled with either a quadrupole-linear ion trap or linear ion trap-FTICR mass spectrometer. The CB2 receptor, with incorporated N-terminal FLAG and C-terminal HIS6 epitope tags, was functionally expressed in baculovirus cells and purified using a single step of anti-FLAG M2 affinity chromatography. To overcome the difficulties involved with in-gel digestion, due to the highly hydrophobic nature of this membrane-associated protein, we conducted in-solution trypsin and chymotrypsin digestions of purified and desalted samples in the presence of a low concentration of CYMAL5. This was followed by nanoLC peptide separation and analysis using a nanospray ESI source operated in the positive mode. The results can be reported confidently, based on the overlapping sequence data obtained using the highly mass accurate LTQ-FT and the 4000 Q-Trap mass spectrometers. Both instruments gave very similar patterns of identified peptides, with full coverage of all transmembrane helices, resulting in the complete characterization of the cannabinoid CB2 receptor. Mass spectrometric identification of all amino acid residues in the cannabinoid CB2 receptor is a key step toward the "Ligand Based Structural Biology" approach developed in our laboratory for characterizing ligand binding sites in GPCRs using a variety of covalent cannabinergic ligands.
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PMID:Comprehensive proteomic mass spectrometric characterization of human cannabinoid CB2 receptor. 1747 60

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