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 iron responsive element binding protein (IRE-BP) regulates iron storage and uptake in response to iron. This control results from the interaction of the IRE-BP with the iron responsive element (IRE), a conserved sequence/structure element located near the 5' end of all ferritin mRNAs and in the 3' UTR of transferrin receptor mRNAs. Proteolysis was used to probe for functional elements of the IRE-BP. Partial chymotrypsin digestion generates a simple digestion pattern yielding fragments of 68, 56, 41, and 30 kDa. The 68 and 30 kDa fragments are derived from a single cleavage at Trp623. Further cleavages of the 68 kDa polypeptide yield the 56 and 41 kDa peptides. A combination of UV-crosslinking and chymotrypsin digestion was used to localize an RNA binding element within the C-terminus of the 68 kDa fragment, between amino acid residues 480 and 623. This region includes cysteine residues 503 and 506 which have been shown to be required for iron-sulfur cluster assembly and for iron regulation of the IRE-BP. Proteolytic fragments of the IRE-BP that contain this RNA binding region can be crosslinked to the IRE but do not bind with high affinity, suggesting that elements within the IRE-BP, in addition to those located between residues 480 and 623, are required for high affinity binding to the IRE.
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PMID:Localization of an RNA binding element of the iron responsive element binding protein within a proteolytic fragment containing iron coordination ligands. 751 18

The brain is the most compartmentalized organ. It is also highly aerobic. Because nerve cells grow but do not regenerate, the brain is the organ best suited for the accumulation of metabolic errors colocalized in specific areas of the brain over an extended period. Alzheimer's disease (AD) is primarily a neurological disorder of the elderly. It is suggested that this disorder results from the accumulation of such errors, and that AD onset aluminum and iron contribute to but do not necessarily initiate the onset of the disease. In vitro and in vivo evidence summarized here suggests that this is effected by interfering in the utilization of glucose and glucose-6-phosphate, and sequestration of iron by ferritin. beta-amyloid precusor proteins (beta-APPs) are normal components of the human brain and some other tissues. Proteolysis of these, presumably by serine proteases, generates a 39 to 42 amino acid long peptide, the alpha-amyloid (beta-AP). In AD brains, beta-AP aggregates into plaque, the hallmark of AD brains. Some of the alpha-APPs also contain a 56 amino acid long segment which inhibits serine proteases. We show that in vitro, at pH 6.5, aluminum activates beta-chymotrypsin 2-fold and makes it dramatically resistant to protease inhibitors such as bovine pancreatic trypsin inhibitor (bPTI) or its mimic present in the beta-amyloid precursor proteins (beta-APPs). Iron and oxygen are reported to favor cross-linking of beta-AP in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Iron and aluminum homeostasis in neural disorders. 784 99

Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, was subjected to limited proteolysis using trypsin and chymotrypsin, in the absence or presence of its substrates or their analogs. Time-dependent degradation of glutamate synthase alpha and beta subunits, to yield several fragments of different stability, was observed, the alpha subunit being more sensitive than the beta to proteolytic attack. The main sites of proteolytic cleavage were determined by densitometric analysis of the electrophoretic patterns obtained under denaturing conditions and by N-terminal sequencing of the major proteolytic products. These analyses showed that most of the peptide bonds sensitive to the proteases are clustered in two regions of the alpha subunit, outside the proposed substrate and cofactor binding regions of glutamate synthase [Pelanda, R., Vanoni, M. A., Perego, M., Piubelli, L., Galizzi, A., Curti, B. & Zanetti, G. (1993) J. Biol. Chem. 268, 3099-3106]. Therefore, these protease-sensitive sites can be identified as flexible loops, exposed to solvent, connecting adjacent domains of the protein. The presence of the enzyme substrates or their analogs caused significant changes in the proteolytic patterns. NADP+ protected the C-terminal region of glutamate synthase beta subunit from tryptic cleavage, supporting the proposal that it contains the pyridine-nucleotide-binding site. Furthermore, NADP+, and to a lesser extent the glutamine analog L-methionine sulfone, which binds presumably to the N-terminal region of the alpha subunit, altered the sensitivity to proteolysis of the sites of the alpha subunit proposed to be part of links between domains of glutamate synthase. These results show that long-range conformational changes of glutamate synthase occur on binding of its substrates. The study of several NADPH-dependent diaphorase activities of glutamate synthase was also undertaken in order to test if proteolytic fragments of the enzyme retained their ability to transfer electrons from NADPH to synthetic electron acceptors. Although proteolysis yielded partial loss of all enzyme NADPH-dependent reactions, the kinetic analysis showed that the rates of reduction of iodonitrotetrazolium, ferricyanide and dichlorophenolindophenol were at least twofold faster than the rate of the physiological glutamate synthase reaction. These results indicate that enzyme reduction and intramolecular electron transfer are not rate limiting during catalysis of the physiological glutamate synthase reaction.
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PMID:Interdomain loops and conformational changes of glutamate synthase as detected by limited proteolysis. 800 67

Iron-saturated human transferrin was digested with either chymotrypsin or trypsin to produce C-lobe and N-lobe protein fragments. Individual protein fragments were purified by a combination of gel filtration and Concanavalin A affinity chromatographic procedures. The C-lobe and N-lobe fragments of human transferrin were then used in binding assays to assess their ability in binding to the bacterial transferrin receptors. Competitive binding assays demonstrated that the C-lobe fragment of human transferrin binds as well as intact human transferrin to bacterial transferrin receptors from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae. Using isogenic mutants of N. meningitidis deficient in either of the transferrin-binding proteins (Tbps), we demonstrated that both transferrin-binding proteins were able to bind to the C-lobe fragment of human transferrin.
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PMID:The region of human transferrin involved in binding to bacterial transferrin receptors is localized in the C-lobe. 836 58

Photoaffinity labeling with [alpha-32P]8N3GTP and [gamma-32P]8N3GTP was used to identify the guanine binding domain of the GTP regulatory site within glutamate dehydrogenase (GDH). Without photolysis, 8N3GTP mimicked the regulatory properties of GTP on GDH activity with 8N3GTP exhibiting a Ki of 5 microM while the Ki for GTP was about 0.6 microM. Under optimal photolabeling conditions saturation of photoinsertion with 1 microgram of GDH revealed an apparent Kd of 9 +/- 4 microM for [gamma-32P]8N3GTP. Photolabeling with this analog could be competitively inhibited with GTP with an apparent Kd of 12 +/- 2 microM. Other nucleotides such as ATP and NAD(P)H could not reduce the amount of photoinsertion as effectively as GTP. ADP could decrease photoinsertion, but only at much higher concentrations. NAD(P)+, GDP, AMP, and GMP had little effect on photoinsertion. Divalent cations Mg2+ and Ca2+ also reduced photoinsertion significantly while the monovalent K+ and Na+ ions had no effect. Aluminum(III)-chelate or iron(III)-chelate affinity chromatography and reversed-phase HPLC were used to purify photolabel-containing peptides generated with either trypsin or chymotrypsin. This identified a portion of the guanine binding domain within the GTP regulatory site as the region containing the sequence Ile439 to Tyr454. Photolabeling of this peptide was prevented 91% by the presence of 300 microM GTP during photolysis. Lys445 was not identified in sequence analyses of the photolabeled peptides. Also, trypsin was unable to cleave the photolabeled peptide at this site. These results suggest that Lys445 may be the residue modified by [alpha-32P]8N3GTP.
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PMID:Identification of a guanine binding domain peptide of the GTP binding site of glutamate dehydrogenase: isolation with metal-chelate affinity chromatography. 843 45

Lipoxygenase-1 (LOX1) from soybeans was cleaved with chymotrypsin (Ramachandran et al., 31 (1992) 7700-7706). The domains were separated on a Sephadex G-50 column by minimising domain interactions at pH 4.0. The molecular weight and apparent homogeneity of the domains were established by SDS-PAGE. The solution conformation of the 60 kDa and 30 kDa fragments was compared with that of native LOX1. 1-Anilino-8-naphthalene sulphonate (ANS) binding measurements confirmed the exposure of large hydrophobic residues on the surface of the 60 kDa due to separation of the domains. The monomeric nature of the 60 kDa fragment was confirmed by HPLC gel filtration. The increased number of binding sites and magnitude of binding constant suggested the involvement of extensive hydrophobic interactions between the two domains. The essential cofactor iron was with the C-terminal domain. The attempts to resolve and reconstitute the catalytic activity of isolated domains were not successful.
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PMID:Rapid method to separate the domains of soybean lipoxygenase-1: identification of the interdomain interactions. 910 14

Bovine lactoferrin binds to a 60 kDa heat shock protein of Helicobacter pylori. Binding ability was related to human immunoglobulin G because bovine lactoferrin binding proteins were isolated by extraction of cell surface associated proteins with distilled water, applied on IgG-Sepharose and nickel sulphate chelate affinity chromatography. Binding was demonstrated by Western blot after purified protein was digested with alpha-chymotrypsin and incubated with peroxidase-labeled bovine lactoferrin. Binding was inhibited by bovine lactoferrin, lactose, rhamnose, galactose, and two iron-containing proteins, ferritin and haptoglobin. Helicobacter pylori binds ferritin and haptoglobin via charge or hydrophobic interactions because this binding was not inhibited by specific and various glycoproteins or carbohydrates. Carbohydrate moieties of bovine lactoferrin molecules seem to be involved in binding because glycoproteins with similar carbohydrate structures strongly inhibited binding. Scatchard plot analysis of the binding of peroxidase-labeled bovine lactoferrin to H. pylori cells yielded a kd 2.88 x 10(-6) M. In addition, binding of H. pylori cells to bovine lactoferrin was enhanced when bacteria treated with pepsin or alpha-chymotrypsin after isolation from iron-restricted and iron-containing media.
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PMID:Cryptic domains of a 60 kDa heat shock protein of Helicobacter pylori bound to bovine lactoferrin. 911 43

Lactoferrin is an iron binding glycoprotein with a molecular weight of 80 kDa. The molecule is divided into two lobes representing the N-terminal and C-terminal halves of the polypeptide chain, each containing an iron binding site. The serine proteinases such as trypsin, chymotrypsin, and pepsin hydrolyze lactoferrin into two unequal halves while proteinase K divides this protein into two equal halves. In the first step of hydrolysis by proteinase K, the C- and N-lobes, each having a molecular weight of approximately 40 kDa, are generated. In the next step, the lobes are further hydrolyzed into small molecular weight peptides. The proteinase K isolated from the hydrolyzed product does not show enzymatic activity suggesting that the enzyme is inhibited. Furthermore, the hydrolysis experiments on N-lobe and C-lobe showed that the inhibitory fragment came from the C-lobe. The purified lactoferrin fragment was found to be a decapeptide with an amino acid sequence of H2N-Val-Ala-Gln-Gly-Ala-Ala-Gly-Leu-Ala-COOH. The complex formed between proteinase K and lactoferrin fragment was crystallized by microdialysis. The crystals belonged to the monoclinic space group P2(1) with cell dimensions a = 44.4 A, b = 38.6 A, c = 79.2 A, beta = 105.8 degrees and Z = 2. The crystal structure has been determined at 2.4 A resolution. It has been refined to an R factor of 0.163 for 9044 reflections. The Lf-fragment forms several intermolecular interactions with proteinase K. The Ser-224 Ogamma and His-57 N epsilon2 move away to a distance of 3.68 A in the complex. In the crystal structure, Gln-3I (I indicates inhibitor i.e., lactoferrin fragment) is involved in a direct intermolecular interaction with a symmetry related proteinase K molecule through a strong hydrogen bond with Asp-254. The mode of intermolecular interactions in the complex conformational features of the enzyme and placement of the fragment with respect to the enzyme resemble with the molecular complex of proteinase K with its natural inhibitor PKI3 from wheat.
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PMID:Crystal structure of a complex formed between proteolytically-generated lactoferrin fragment and proteinase K. 974 42

This study was mainly aimed to investigate the efficacy of trypsin:chymotrypsin to elicit anti-oxidant properties. In our earlier studies it was observed that the enzyme preparation exhibited an anti-inflammatory action as there was a remarkable reduction in oedema formation and tissue destruction. This led to further study on the amount of lipid peroxidation products formed and the levels of enzymatic and non-enzymatic anti-oxidants and relative trace element contents of copper, selenium, iron and zinc during administration of the enzyme preparation. Decreased formation of lipid peroxidation products was observed in treated group in comparison with the untreated group. Higher levels of enzymatic anti-oxidants mainly super oxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase and non-enzymatic antioxidant namely ceruloplasmin persisted for a longer period of time in the treated group than in the untreated group. No statistical significance was observed in non-enzymatic antioxidants viz. ascorbic acid and tocopherol levels in both the groups. Increased serum copper and selenium levels in the treated group could be related to higher levels of the ceruloplasmin and glutathione peroxidase observed in the treated group. The above studies support the finding that treatment with the enzyme preparation reduced tissue destruction leading to decreased formation of free radicals and subsequent effective scavenging of free radicals by the higher levels of enzymatic and non-enzymatic anti-oxidants.
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PMID:The efficacy of trypsin: chymotrypsin preparation in the reduction of oxidative damage during burn injury. 977 92

The two glycosylated N- and C-terminal lobes of buffalo lactoferrin have been produced by limited proteolysis using proteinase K. Lactoferrin is a single chain glycoprotein of molecular mass 80 kDa with two iron-binding sites and two structural lobes connected by a short peptide. Purified samples of lactoferrin, isolated from buffalo colostrum, were subjected to hydrolysis using trypsin, chymotrypsin, pepsin, subtilisin and proteinase K. The first three proteinases produced two major fragments of approximately 35 and 23 kDa together with small molecular mass peptides. Trypsin and chymotrypsin partly digested lactoferrin, while pepsin converted all the intact lactoferrin into fragments. Subtilisin hydrolysis produced fragments of 40 and 26 kDa together with low molecular mass peptides. However, SDS-PAGE of the proteinase K hydrolysis product gave a clear band at 40 kDa together with a band indicating a substantial quantity of low molecular mass peptides (< 14.4 kDa). Upon ion-exchange chromatography this product gave two major fractions, which were further purified by gel filtration and identified as the C and N lobes from their N-terminal sequences. Thus, the 40 kDa band in SDS-PAGE of the proteinase K hydrolysis product contained two fragments of equal molecular mass. On further hydrolysis with proteinase K, the N lobe was completely hydrolysed into low molecular mass peptides, while only a small fraction of the C lobe was converted into small products. This suggested that an inhibitory fragment was present in the C lobe that was released on hydrolysis to small fragments and prevented complete digestion of the C lobe by high-affinity binding to the active site of proteinase K. This fragment was isolated from the lactoferrin-proteinase K complex and its sequence determined to be Val-Ala-Gln-Gly-Gly-Ala-Ala-Gly-Leu-Ala. Circular dichroism studies indicated a high alpha-helical content in the native lactoferrin while comparatively lower helical structures were present in the N and C lobes. In addition, the iron saturations of the N and C lobes appeared to be lower than that of the native protein.
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PMID:Preparation and characterization of the N and C monoferric lobes of buffalo lactoferrin produced by proteolysis using proteinase K. 1019 76


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