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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Insecticidal crystal proteins (delta-endotoxins), CryIA(a) and CryIA(c), from Bacillus thuringiensis are 82% homologous. Despite this homology, CryIA(c) was determined to have 10-fold more insecticidal activity toward Heliothis virescens and Trichoplusia ni than CryIA(a). Reciprocal recombinations between these two genes were performed by the homolog-scanning technique. The resultant mutants had different segments of their primary sequences exchanged. Bioassays with toxin proteins from these mutants revealed that amino acids 335-450 on CryIA(c) are associated with the activity against T. ni, whereas amino acids 335-615 on the same toxin are required to exchange full H. virescens specificity. One chimeric protein toxin, involving residues 450-612 from CryIA(c), demonstrated 30 times more activity against H. virescens than the native parental toxin, indicating that this region plays an important role in H. virescens specificity. The structural integrity of mutant toxin proteins was assessed by treatment with bovine trypsin. All actively toxic proteins formed a 65-kDA trypsin-resistant active toxic core, similar to the parental CryIA(c) toxin, indicating that toxin protein structure was not altered significantly. Contrarily, certain inactive mutant proteins were susceptible to complete protease hydrolysis, indicating that their lack of toxicity may have been due to structural alterations.
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PMID:Functional domains of Bacillus thuringiensis insecticidal crystal proteins. Refinement of Heliothis virescens and Trichoplusia ni specificity domains on CryIA(c). 191 34

Abrin B chain and trypsin inhibitor isolated from Acacia confusa (ACTI) were covalently linked to form a chimeric protein (ANB-ACTI) with N-succinimidyl-3-(-2-pyridyldithio)propionate. The chimeric protein had 31% of trypsin inhibitory activity of ACTI and 7% of hemagglutinating activity of abrin B chain, but no inhibition on protein biosynthesis. ANB-ACTI had strong inhibitory effects on the growth of sarcoma 180 cells and Hela cell culture while the mixture of an equivalent amount of free abrin B chain and ACTI did not. The results suggests that abrin B chain of chimeric protein may act as a vector to carry ACTI into the tumor cells. ACTI into the tumor cells. ACTI in the chimeric protein potentiates its antitumor activity as well as its resistance to tryptic digestion.
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PMID:Chimeric protein: abrin B chain-trypsin inhibitor conjugate as a new antitumor agent. 281 99

DNA sequences encoding Ile-Glu-Gly-Arg and human prorenin were joined and placed under the transcriptional control of the Escherichia coli trp promoter-operator in the expression vector pTR501. E. coli cells transformed with pTR501 expressed high levels (30% of total cell protein) of prorenin as part of a hybrid protein with the trp E gene product. The chimeric protein, accumulated in a sedimentable form, was dissolved in 6 M guanidine X HCl, purified to near homogeneity, and renatured by dialysis. The complete prorenin sequence was then excised from the renatured hybrid protein using blood coagulation factor Xa, a proteinase which is highly specific for the tetrapeptide insert Ile-Glu-Gly-Arg introduced between the 9 amino-terminal residues of the trp E gene product and the first amino acid (Thr 1) of prorenin. Human prorenin thus obtained was readily activatable with trypsin and showed close similarities to naturally occurring prorenin in its biochemical and immunochemical properties.
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PMID:Synthesis and characterization of human prorenin in Escherichia coli. 353 94

Due to the great length of apolipoprotein (apo) B-100, the localization of lipid-associating domains in this protein has been difficult. To address this question, we developed a computer program called Locate that searches amino acid sequences to identify potential amphipathic alpha-helixes and beta-strands by using sets of rules for helix and strand termination. A series of model chimeric protein test datasets were created by tandem linking of amino acid sequences of multiple proteins containing four different secondary structural motifs: motif A (exchangeable plasma apolipoproteins); motif G (globular alpha-helical proteins); motif C (coiled-coil alpha-helical proteins); and motif B (beta pleated-sheet proteins). These four test datasets, as well as randomly scrambled sequences of each dataset, were analyzed by Locate using increasingly stringent parameters. Using intermediately stringent parameters under which significant numbers of amphipathic helixes were found only in the unscrambled motif A, two dense clusters of putative lipid-associating amphipathic helixes were located precisely in the middle and at the C-terminal end of apoB-100 (a sparse cluster of class G* helixes is located at the N-terminus). The dense clusters are located between residues 2103 through 2560 and 4061 through 4338 and have densities of 2.4 and 2.2 amphipathic helixes per 100 residues, respectively; under these conditions, motif A has a density of 1.4 amphipathic helixes per 100 residues. These two domains correspond closely to the two major apoB-100 lipid-associated domains at residues 2100 through 2700 and 4100 through 4500 using the principle of releasability of tryptic peptides from trypsin-treated intact low-density lipoprotein. The classes of amphipathic helixes identified within these two putative lipid-associating domains are considerably more diverse than those found in the exchangeable plasma apolipoproteins. Interestingly, apoB-48 terminates at the N-terminal edge of the middle cluster. By using a similar strategy for analysis of amphipathic beta-strands, we discovered that the two gap regions between the three amphipathic helix clusters are highly enriched in putative amphipathic beta-strands, while the three amphipathic helical domains are essentially devoid of this putative lipid-associating motif. We propose, therefore, that apoB-100 has a pentapartite structure, NH2-alpha 1-beta 1-alpha 2-beta 2-alpha 3-COOH, with alpha 1 representing a globular domain.
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PMID:apoB-100 has a pentapartite structure composed of three amphipathic alpha-helical domains alternating with two amphipathic beta-strand domains. Detection by the computer program LOCATE. 791 18

The synthesis, maturation, and localization of the human homolog of the bacterial ATP-dependent Lon protease have been studied in cultured cells and in a cell-free system. Immunofluorescence microscopy of cells transfected with a recombinant human Lon protease-FLAG epitope chimeric protein confirmed the mitochondrial location of human Lon protease. The primary product of transcription and translation directed by a human LON cDNA in vitro had an apparent mass of approximately 107 kDa, and incubation of the translation product with isolated rat liver mitochondria converted the precursor into the approximately 100-kDa mature form. The latter pelleted with mitochondria and was resistant to trypsin digestion. Maturation of human Lon protease in vitro was dependent on mitochondrial inner membrane potential, as the uncoupling agent 2,4-dinitrophenol (DNP) completely blocked this process. In intact cultured cells treated with DNP, newly synthesized Lon protease also accumulated as the precursor form, and subsequent removal of DNP allowed the precursor to be translocated into mitochondria and cleaved to its mature form. A pulse-chase experiment in the absence of DNP showed that the Lon protease precursor is converted to the mature form with a half-time of < 2 min and that the mature human Lon protease is indefinitely stable in intact cells. Submitochondrial fractionation and immunoblot analysis of rat liver mitochondrial proteins using the polyclonal anti-human Lon protease antiserum indicated that the protease is located exclusively in the matrix. These data demonstrate that the maturation of human Lon protease is characterized by steps similar to those reported for other mitochondrial matrix proteins.
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PMID:Synthesis, processing, and localization of human Lon protease. 796 1

Sin a 1, the major yellow mustard allergen, is a seed storage protein that belongs to the 2S albumin family. It is composed of two disulfide-bonded polypeptide chains. The cloning of this allergen has been carried out by means of the polymerase chain reaction using non-degenerate oligonucleotides encoding the N-terminal and C-terminal regions of the mature protein as primers. Five genomic nucleotide sequences have been analyzed, encoding both mature polypeptide chains linked by the internal processed fragment. The sequence data show the existence of microheterogeneities at ten positions, demonstrating the polymorphism exhibited by the natural protein. One of the genomic clones was expressed in Escherichia coli by fusion to glutathione S-transferase from Schistosoma japonicum. The resulting chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B matrix, and digested with thrombin to release the recombinant allergen. The recombinant Sin a 1 is recognized by rabbit polyclonal and mouse monoclonal antisera raised against natural Sin a 1, as well as by the IgE of mustard-sensitive human sera. In addition, recombinant Sin a 1 possesses a high resistance to trypsin digestion, like the native mustard allergen.
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PMID:Expression in Escherichia coli of Sin a 1, the major allergen from mustard. 864 31

A cDNA encoding a precursor for a novel serine protease (neurosin) was cloned from a cDNA library prepared from a human colon adenocarcinoma cell line, COLO 201. The sequence consisted of 155 bp 5' non-coding region and a 732 bp open reading frame which was followed by a 551 bp 3' non-coding region. The predicted protein consists of 244 amino acids which is possibly processed to an active enzyme of 223 amino acids that shows some similarity (< 30%) to other members of serine protease family. As found in other trypsin-like proteases, the enzyme contains the catalytic triad which is characterized as the essential amino acid residues for the activity. Northern blot analyses of the mRNA showed the strongest expression in brain followed by a lower but significant one in spleen. A construct of cDNA encoding chimeric protein that carries pro-sequence of trypsin II and putative mature neurosin starting from Leu22 was transfected to COS-1 cells. Successful production of the active neurosin was shown after treating the supernatant of the culture of the transfectants with enterokinase.
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PMID:Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain. 900 50

This study examines the role of L-selectin in monocyte adhesion to arterial endothelium, a key pathogenic event of atherosclerosis. Using a nonstatic (rotation) adhesion assay, we observed that monocyte binding to bovine aortic endothelium at 4 degrees C increased four to nine times upon endothelium activation with tumor necrosis factor (TNF)-alpha. mAb-blocking experiments demonstrated that L-selectin mediates a major part (64 +/- 18%) of monocyte attachment. Videomicroscopy experiments performed under flow indicated that monocytes abruptly halted on 8-h TNF-alpha-activated aortic endothelium, approximately 80% of monocyte attachment being mediated by L-selectin. Flow cytometric studies with a L-selectin/IgM heavy chain chimeric protein showed calcium-dependent L-selectin binding to cytokine-activated and, unexpectedly, unactivated aortic cells. Soluble L-selectin binding was completely inhibited by anti-L-selectin mAb or by aortic cell exposure to trypsin. Experiments with cycloheximide, chlorate, or neuraminidase showed that protein synthesis and sulfate groups, but not sialic acid residues, were essential for L-selectin counterreceptor function. Moreover, heparin lyases partially inhibited soluble L-selectin binding to cytokine-activated aortic cells, whereas a stronger inhibition was seen with unstimulated endothelial cells, suggesting that cytokine activation could induce the expression of additional ligand(s) for L-selectin, distinct from heparan sulfate proteoglycans. Under flow, endothelial cell treatment with heparinase inhibited by approximately 80% monocyte attachment to TNF-alpha-activated aortic endothelium, indicating a major role for heparan sulfate proteoglycans in monocyte-endothelial interactions. Thus, L-selectin mediates monocyte attachment to activated aortic endothelium, and heparan sulfate proteoglycans serve as arterial ligands for monocyte L-selectin.
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PMID:Monocyte adhesion to activated aortic endothelium: role of L-selectin and heparan sulfate proteoglycans. 904 58

Human P-glycoprotein (P-gp) is a cell surface drug efflux pump that contains two nucleotide binding domains (NBDs). Mutations were made in each of the Walker B consensus motifs of the NBDs at positions D555N and D1200N, thought to be involved in Mg(2+) binding. Although the mutant and wild-type P-gps were expressed equivalently at the cell surface and bound the drug analogue [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) comparably, neither of the mutant proteins was able to transport fluorescent substrates nor had detectable basal nor drug-stimulated ATPase activities. The wild-type and D1200N P-gps were labeled comparably with [alpha-(32)P]-8-azido-ATP at a subsaturating concentration of 2.5 microM, whereas labeling of the D555N mutant was severely impaired. Mild trypsin digestion, to cleave the protein into two halves, demonstrated that the N-half of the wild-type and D1200N proteins was labeled preferentially with [alpha-(32)P]-8-azido-ATP. [alpha-(32)P]-8-Azido-ATP labeling at 4 degrees C was inhibited in a concentration-dependent manner by ATP with half-maximal inhibition at approximately 10-20 microM for the P-gp-D1200N mutant and wild-type P-gp. A chimeric protein containing two N-half NBDs was found to be functional for transport and was also asymmetric with respect to [alpha-(32)P]-8-azido-ATP labeling, suggesting that the context of the ATP site rather than its exact sequence is an important determinant for ATP binding. By use of [alpha-(32)P]-8-azido-ATP and vanadate trapping, it was determined that the C-half of wild-type P-gp was labeled preferentially under hydrolysis conditions; however, the N-half was still capable of being labeled with [alpha-(32)P]-8-azido-ATP. Neither mutant was labeled under vanadate trapping conditions, indicating loss of ATP hydrolysis activity in the mutants. In confirmation of the lack of ATP hydrolysis, no inhibition of [(125)I]IAAP labeling was observed in the mutants in the presence of vanadate. Taken together, these data suggest that the two NBDs are asymmetric and intimately linked and that a conformational change in the protein may occur upon ATP hydrolysis. Furthermore, these data are consistent with a model in which binding of ATP to one site affects ATP hydrolysis at the second site.
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PMID:Both ATP sites of human P-glycoprotein are essential but not symmetric. 1052 34

The design of chimeric proteins is a major field of interest in structural biology and biotechnology. The successful design of the chimeric protein composed by the minimized reactive site domain of the low-molecular-mass trypsin inhibitor from Brassica napus (var. oleifera) seed (Ser3-Lys35; mini-RTI-III) and murine dihydrofolate reductase (DHFR) is reported here. The DHFR-mini-RTI-III chimeric protein was expressed in Escherichia coli, purified by metal-chelate affinity chromatography and oxidatively refolded. The affinity of the purified and refolded DHFR-mini-RTI-III for bovine trypsin (K = 5.0 x 10(-10) M) was closely similar to that determined for native RTI-III (K = 2.9 x 10(-10) M), at pH 8.2 and 22.0 degrees C. DHFR-mini-RTI-III may be regarded as a tool in structure-function studies and for developing multifunctional and multidomain proteinase inhibitors.
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PMID:A chimeric mini-trypsin inhibitor derived from the oil rape proteinase inhibitor type III. 1097 4


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