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)

A structural and functional homologue of vertebrate alpha 2-macroglobulin (alpha 2M) has been identified in the hemolymph and blood cells of the arthropod Limulus polyphemus, one of the oldest living fossil invertebrates (Quigley, J. P., and Armstrong, P. B. (1985) J. Biol. Chem. 260, 12715-12719). The subunit molecular mass is 185 kDa. The native molecular mass, determined by scanning transmission electron microscopy (STEM) under conditions in which the linear relationship between the STEM large angle detector signal and specimen mass thickness allows the determination of the total macromolecular mass, was 354 +/- 35 kDa. Sedimentation equilibrium measurements gave a value of 366 kDa, independent of solute concentration. Sedimentation velocity experiments indicated a homogeneous component with a frictional ratio of 1.41. Thus, the native structure appears to be a dimer, with a somewhat extended conformation. The behavior during gel permeation chromatography was anomalous, yielding an apparent molecular mass approximately half-way between that expected for the dimeric and tetrameric configurations. Transmission electron microscopy of negatively stained preparations revealed a dimeric butterfly-like structure that collapsed following reaction with chymotrypsin.
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PMID:Structure of alpha 2-macroglobulin from the arthropod Limulus polyphemus. 170 30

Proteolytic fragments of murine band 3 were produced by exposure to extracellular chymotrypsin and intracellular trypsin. The ensuing proteolytic fragments were isolated, their N-terminal sequences were determined and their locations in the known amino acid sequence of murine band 3 established. Equivalents of the human 60, 35 and 17 kDa fragments were obtained through the cleavage sites were situated at locations that are not strictly homologous to the corresponding cleavage sites in human band 3, although all of them were near such sites. Exposure of the intact murine red cell to chymotrypsin leads to the formation of two fragments of 67 kDa and 41 kDa, which are equivalent to the 60 kDa and the 35 kDa fragments of the human band 3. Internal trypsin cleaves the chymotryptic 67 kDa fragment while the 41 kDa fragment appears essentially unaffected. The 67 kDa fragment is first degraded to 64 kDa, then further to 22 kDa and finally to 19 kDa. The anion transport inhibitor H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) combines with murine band 3 protein as it does with human band 3. Anion transport is maximally inhibited when 5.10(5) H2DIDS molecules per cell are bound to band 3. As in the human red cell, after exposure to high pH (9.0-9.5) of the H2DIDS-labeled, chymotryptically cleaved band 3 intramolecular cross-linking takes place. This joins the 67 and 41 kDa chymotryptic pieces together to form a peptide of the original molecular mass of band 3 of 108 kDa. If cross-linking is performed after additional tryptic cleavage, the 19 and 22 kDa pieces join together with 41 kDa pieces to form overlapping bands that cover the molecular weight range from 60 to 63 kDa.
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PMID:Major proteolytic fragments of the murine band 3 protein as obtained after in situ proteolysis. 271 7

The effects of poly(L-aspartic acid) (PLAA) on microtubule assembly and microtubule-associated protein (MAP) 2-actin interaction were examined in vitro. PLAA inhibited assembly of rat brain microtubules and induced rapid disassembly of already formed microtubules. Inhibition was stronger by PLAA with a high molecular weight than that by low molecular weight. The ratios of 47 kDa PLAA to microtubule proteins causing 50% inhibition of the assembly and disassembly were 0.015 and 0.04 (w/w), respectively. Both MAP 1 and MAP 2 were bound to a PLAA-Sepharose 4B affinity column, while tubulin was not retained by the column. PLAA caused selective dissociation of MAP 1 and MAP 2 from microtubules polymerized by taxol. It is therefore concluded that PLAA interacts specifically with MAPs. PLAA also inhibited the MAP 2 induced cross-linking of actin filaments, showing an almost complete inhibition at a PLAA to MAP 2 ratio of 1:5,000 (w/w). Binding experiments of PLAA with digested MAP 2 by chymotrypsin using affinity chromatography and sedimentation experiments showed that PLAA was preferentially bound to a 35 kDa fragment which includes the microtubule- and actin-binding domain of the MAP 2 molecule. These results suggest that PLAA suppressed the functions of MAP 2 through a domain which is located in the 35 kDa fragment.
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PMID:Inhibitory effects of poly(L-aspartic acid) on the assembly of brain microtubules and the interaction of microtubule-associated protein 2 with F-actin in vitro. 277 57

The glycoprotein thrombospondin is distributed between the extracellular matrix and the platelet-sequestered pool in the resting state and it undergoes redistribution upon platelet stimulation. It is believed to play a role in matrix structure and in coagulation. We have studied the structural domains of endothelial cell (EC) thrombospondin by use of the serine proteases thrombin, trypsin and chymotrypsin and have characterized the heparin-binding domains of this molecule. For this purpose we used purified thrombospondin synthesized and secreted by bovine aortic endothelial cells grown in the presence of radiolabeled methionine. We find that the susceptibility of EC thrombospondin to proteolysis is five-fold smaller than that of platelet thrombospondin. In the presence of 2 mM Ca ions the molecule is cleaved by 20 U/ml thrombin at a single locus, to yield fragments of 160 kDa and 35 kDa. Trypsin digestion for 5 min at room temperature at an enzyme-to-substrate ratio of 1:20 produces a stable fragment of 140 kDa but not the 30-kDa fragment observed in platelet thrombospondin. Chymotrypsin, under identical conditions to those used for trypsin, cleaves EC thrombospondin into four stable fragments of 160 kDa, 140 kDa, 27 kDa and 18 kDa. Chelation of Ca by EDTA increases susceptibility of the molecule to proteolysis. Under the conditions used a cryptic thrombin-cleavage site, not hitherto observed in platelet thrombospondin, was observed in EC thrombospondin. The location of this site is near a chymotrypsin-susceptible site, which has been observed in the long connecting arm, which is particularly Ca-stabilized. Heparin-binding capacity of EC thrombospondin was observed in at least two separate loci. Both thrombin and chymotrypsin produced small fragments (35 kDa and 27 kDa respectively) which bound to heparin with high affinity, and large fragments (160 kDa for thrombin and 140 kDa for chymotrypsin) which had low affinity. Chelation of Ca substantially decreased the low-affinity binding of the large fragments but not the high-affinity binding of the small fragments. Two-dimensional gel electrophoresis of the chymotryptic heparin-binding fragments shows that each molecule gave rise to a heterogeneous array of fragments of high molecular mass bound by disulfide bonds, indicating that there is a difference in the rate of cleavage between the three subunits of EC thrombospondin. Trypsin, despite its limited degradation, completely eliminated the heparin-binding capacity of both high and low-affinity loci, in contrast to platelet thrombospondin where the high affinity remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The structure of endothelial cell thrombospondin. Characterization of the heparin-binding domains. 282 10

Using flow dialysis, we found two classes of calcium-binding sites on tubulin: high-affinity binding sites (1.56 +/- 0.38 per tubulin dimer) with a dissociation constant of (4.86 +/- 0.12).10(-6) M and low-affinity binding sites (5.82 +/- 0.50 per tubulin dimer) with a dissociation constant of (6.4 +/- 0.4).10(-5) M. In the presence of 6.10(-5) M MgSO4, we found 0.64 +/- 0.18 calcium-binding sites per tubulin dimer with a dissociation constant of (4.7 +/- 0.5).10(-6) M and 1.2 +/- 0.2 sites per dimer with a dissociation constant of (3.5 +/- 0.4).10(-5) M. Under controlled conditions, trypsin and chymotrypsin selectively cleaved alpha- and beta-subunits, respectively, forming major fragments of 35 kDa and 20 kDa from the alpha-subunit, and major fragments of 31 kDa and 22 kDa from the beta-subunit. The high-affinity calcium-binding sites were detected in the carboxyl-terminal region of each tubulin subunit. Computer analysis of the subunit amino-acid sequences suggested possible locations of the putative calcium-binding sites.
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PMID:Calcium binding to tubulin. 320 16

Trypsin and chymotrypsin were used as probes of structure-divalent cation relationships in G-actin molecule. The pattern of fragments produced has been analyzed by sodium dodecyl sulfate gel electrophoresis. The tryptic product of G-actin, 33 kDa is a protease-resistant fragment in the presence of divalent cations. However, once divalent cations are eliminated from the solution during the digestion, the 33 kDa fragment starts to degrade into smaller peptides via a 30 kDa fragment. On the other hand the chymotryptic product of G-actin, 35 kDa (precursor of 33 kDa) is rather stable even in the absence of divalent cations. In addition it is observed that the presence of divalent cation is necessary for the degradation of G-actin to the 33 kDa fragment by trypsin. The ultra violet and intrinsic tryptophan fluorescence spectra of G-actin are changed after the elimination of divalent cations. These results suggest that the structure of G-actin molecule depends on the presence or absence of divalent cations, and that the divalent cation-dependency of G-actin structure is still conserved even after the tryptic digestion.
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PMID:Effect of divalent cation on the structure of skeletal muscle G-actin molecule. 335 76

The 'native' Mg-ATP-dependent protein phosphatase was isolated from rabbit skeletal muscle by a procedure that avoided the use of organic solvents or heating at 90-100 degrees C. The purified enzyme was composed of two major proteins (molecular mass 37 kDa and 31 kDa) that were present in a 1:1 molar ratio, and accounted for 70-80% of the material. The 37-kDa component comigrated with the catalytic subunit of protein phosphatase-1, and its identity with this protein was established by peptide mapping, and by its cleavage to the characteristic 34-kDa and 33-kDa fragments following incubation with chymotrypsin. The 31-kDa protein comigrated with inhibitor-2, and its identity with this protein was established by its heat stability, ability to inhibit protein phosphatase-1 at nanomolar concentrations, and its phosphorylation on a threonine residue by glycogen synthase kinase 3. It is therefore concluded that the 'native' Mg-ATP-dependent protein phosphatase is composed of the catalytic subunit of protein phosphatase-1 (37 kDa) and inhibitor-2 (31 kDa) in a 1:1 molar ratio. The 'native' Mg-ATP-dependent protein phosphatase had virtually identical properties to the enzyme reconstituted from inhibitor-2 and the 37-kDa catalytic subunit of protein phosphatase-1. Each preparation had a similar specific activity and was inhibited by identical concentrations of inhibitor-1. Both enzymes could be activated by incubation with glycogen synthase kinase-3 and Mg-ATP, or by Mn2+ and trypsin (or chymotrypsin). However, Mn2+ alone, or proteinase digestion in the absence of Mn2+, failed to activate either preparation. Incubation with glycogen synthase kinase-3 and Mg-ATP did not dissociate the 'native' or 'reconstituted' enzymes, whereas treatment with Mn2+ and trypsin decreased their apparent molecular masses from 70 kDa to 35 kDa. Incubation with chymotrypsin converted the 'native' and 'reconstituted' enzymes to forms that required preincubation with glycogen synthase kinase-3, Mg-ATP and inhibitor-2, in order to exhibit catalytic activity. The Mg-ATP-dependent protein phosphatase reconstituted from the 'nicked' 33-kDa catalytic subunit dissociated upon activation, in contrast to the enzyme reconstituted from the undegraded 37-kDa catalytic subunit. The results suggest that a 3-4-kDa fragment at one end of the polypeptide is involved in strengthening interaction between the undegraded 37-kDa catalytic subunit and the phosphorylated form of inhibitor-2.
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PMID:The protein phosphatases involved in cellular regulation. Comparison of native and reconstituted Mg-ATP-dependent protein phosphatases from rabbit skeletal muscle. 609 83

Flufenamate, non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte (I50 = 6 . 10(-7) M). The concentration dependence of the binding to ghosts reveals two saturable components. [14C]Flufenamate binds with high affinity (Kd1 = 1.2 . 10(-7) M) to 8.5 . 10(5) sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity (Kd2 = 10(-4) M) to a second set of sites (4.6 . 10(7) per cell). Pretreatment of cells with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [14C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport of [14C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [14C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [14C]flufenamate. Thus it appears that 35 kDa peptide is necessary is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.
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PMID:Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds. I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein. 704 2

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

We employed two in vitro buffer systems to determine the potential pathogenic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). Specifically, this study characterized the oyster plasma protein targets of P. marinus proteases. Additionally, protease-specific inhibitory activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated when a standard buffer (PBS) was used as a diluent; however, this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marinus proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteolytic activity for C. virginica plasma proteins, the anti-proteases could specifically inhibit only P. marinus proteases. Such specificity of anti-protease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoelectrically isolated for further characterization by N-terminal amino acid sequencing.
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PMID:Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the Eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). 1048 30


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