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

---

Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Conditioned medium (CM) from muscle or fibroblast cultures dramatically increases the outgrowth of neurites from fetal rat spinal cord slices in vitro. The factor(s) in CM responsible for this enhanced outgrowth is chymotrypsin-sensitive, but neuraminidase-insensitive. At neutral pH, the factor(s) binds to a concanavalin A-agarose affinity column, a zinc metal chelate affinity column, a DE-52 anion exchange column, but not to a carboxymethyl-52 cation exchange column. These results suggest that the active CM factor(s) is a glycoprotein that is negatively charged at neutral pH. Following gel chromatography the major peak of outgrowth-promoting activity elutes at a molecular weight of approximately 50,000 daltons.
...
PMID:Characterization of neuritic outgrowth-promoting activity of conditioned medium on spinal cord explants. 712 51

Prostate-specific antigen (PSA) provides an excellent serum marker for prostate cancer, the most frequent form of cancer in American males. PSA is a 237-residue protease based on sequence homology to kallikrein-like enzymes. To predict the 3-dimensional structure of PSA, homology modeling studies were performed based on sequence and structural alignments with tonin, pancreatic kallikrein, chymotrypsin, and trypsin. The structurally conserved regions of the 4 reference X-ray proteins provided the core structure of PSA, whereas the loop structures were modeled on the loops of tonin and kallikrein. The unique "kallikrein loop" insert, between Ser 95b and Pro 95k of kallikrein, was constructed using molecular mechanics, dynamics, and electrostatics calculations. In the resulting PSA structure, the catalytic triad, involving residues His 57, Asp 102, and Ser 195, and hydrophobic and electrostatic interactions typical of serine proteases were extremely well conserved. Similarly, the 5-disulfide bonds of kallikrein were also conserved in PSA. These results, together with the fact that no major steric clashes arose during the modeling process, provide strong evidence for the validity of the PSA model. Calculation of the electrostatic potential contours of kallikrein and PSA was carried out using the finite difference Poisson-Boltzmann method. The calculations revealed matching areas of negative potential near the catalytic triad, but differences in the positive potential surrounding the active site. The PSA glycosylation site, Asn 61, is fully accessible to the solvent and is enclosed in a positive region of the isopotential map. The bottom of the substrate specificity pocket, residue S1, is a serine (Ser 189) as in chymotrypsin, rather than aspartate (Asp 189) as in tonin, kallikrein, and trypsin. This fact, plus other features of the S1 binding-pocket region, suggest that PSA would prefer substrates with hydrophobic residues at the P1 position. The location of a potential zinc ion binding site involving the side chain of histidines 91, 101, and 233 is also suggested. This PSA model should facilitate the understanding and prediction of structural and functional properties of this important cancer marker.
...
PMID:A structural model for the prostate disease marker, human prostate-specific antigen. 753 13

APO-1/Fas (CD95) ligand (APO-1L) induces apoptosis in sensitive target cells. Activation-induced T cell death and Ca2(+)-independent cytotoxicity in perforin knockout mice are mediated by APO-1L. To define whether APO-1L is expressed on the surface of activated T cells and to investigate the mechanisms leading to the release of a soluble form, we developed rabbit anti-APO-1L antibodies (Ab). The purified rabbit Ab detected the mature forms of the human and mouse APO-1L of approximately 42 and 40 kDa. In addition, the Ab recognized the non-glycosylated form of APO-1L of approximately 32-33 kDa. In activated human T cells, the soluble form of APO-1L was detectable with a molecular mass of 26 kDa. Immunofluorescence of three human T lymphoblastoid cell lines showed that activation of these cells by phorbol 12-myristate 13-acetate/ionomycin induced a significant increase in cell surface APO-1L only in the presence of metalloprotease inhibitors. Zn2+, but not Ca2+, prevented the increase in surface APO-1L observed in the presence of 1,10-phenanthroline. Blocking of other classes of proteases (serine- and acid-proteases, chymotrypsin) had no effect. Increased expression of surface APO-1L by metalloprotease inhibitors was not dependent on T cell activation, as the metalloprotease inhibitors also modulated the low level of constitutive APO-1L expression. These results suggest that cell surface expression of human APO-1L is regulated by Zn2(+)-dependent metalloproteases. Cleavage of surface APO-1L may act as a regulatory mechanism to prevent accumulation of the membrane-bound form and may cause systemic effects of the APO-1L.
...
PMID:Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases. 754 18

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.
...
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

Transcription factor IIIA (TFIIIA) employs an array of nine N-terminal zinc fingers to bind specifically to both 5S RNA and 5S DNA. The binding of TFIIIA to 5S RNA and 5S DNA was studied by using a protease footprinting technique. Brief treatment of free or bound TFIIA with trypsin or chymotrypsin generated fragments which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fragments retaining the N terminus of TFIIA were identified by immunoblotting with an antibody directed against the N terminus of TFIIIA. Proteolytic footprinting of TFIIIA complexed with 5S DNA derivatives reinforced other evidence that the three N-terminal zinc fingers of TFIIIA bind most tightly to 5S DNA. Proteolytic footprinting of TFIIIA in reconstituted 7S ribonucleoprotein particles revealed different patterns of trypsin sensitivity for TFIIIA bound to oocyte versus somatic 5S RNA. Trypsin cleaved TFIIIA between zinc fingers 3 and 4 more readily when the protein was bound to somatic 5S RNA than when it was bound to oocyte 5S RNA. A tryptic fragment of TFIIIA containing zinc fingers 4 through 7 remained tightly associated with somatic 5S RNA. Zinc fingers 4 through 7 may represent a tightly binding site for 5S RNA in the same sense that fingers 1 through 3 represent a tightly binding site for 5S DNA.
...
PMID:Proteolytic footprinting of transcription factor TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA. 768 46

A periplasmic insulin-cleaving proteinase (ICP), purified to its electrophoretic homogeneity in the SDS-PAGE from the Gram-negative bacterium Acinetobacter calcoaceticus, was examined and compared in its properties with the protease III (protease Pi, pitrilysin, EC 3.4.99.44) of Escherichia coli and the insulin-destroying proteinase (IDE, insulinase, EC 3.4.99.45) from eucaryotes. The enzyme was proven to be a metalloprotease like protease III and IDE, as was shown by the inhibitory effects exerted by EDTA and o-phenanthroline. Furthermore, dialysis against EDTA and o-phenanthroline led to a complete loss of activity, which could be restored by addition of Co2+, and, to a lesser extent, but at a lower metal ion concentration by Zn2+. Similar to protease III and IDE, ICP prefers the cleavage of small polypeptides (insulin, insulin B-chain, glucagon) to the cleavage of proteins (casein, human serum albumin, globin) and was inactive against synthetic amino acid derivates (esters, p-nitranilides, and furoylacroleyl substrates) of subtilisin, thermolysin, trypsin, and chymotrypsin. The peptide-bond-specificity of the ICP in the cleavage of the oxidized insulin B-chain was investigated and the results were compared to the specificity of protease III of E. coli, IDE, protease-24,11, and thermolysin. Cleavage sites in the oxidized insulin B-chain generated by ICP are Asn3-Gln4, His10-Leu11, Ala14-Leu15, Leu17-Val18, Gly23-Phe24, Phe24-Phe25, and Phe25-Tyr26. Principally, ICP cleaves between hydrophobic amino acids and amides. The ICP shares one of the only two cleavage sites with the protease III and four sites with the IDE.
...
PMID:A periplasmic insulin-cleaving proteinase (ICP) from Acinetobacter calcoaceticus sharing properties with protease III from Escherichia coli and IDE from eucaryotes. 773 84

Protease nexin-2 (PN-2) is the secreted isoform of the Alzheimer's Amyloid beta-Protein Precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. PN-2/A beta PP is a potent inhibitor of coagulation factor XIa (FXIa) and is secreted in large quantities by activated platelets suggesting a normal function in regulating this protease at sites of vascular injury. In the present study, the effect of Zn2+ on the protease inhibitory properties of PN-2/A beta PP was quantitatively investigated. Zn2+ (1 microM to 1 mM) had no effect on the inhibition of trypsin or chymotrypsin by PN-2/A beta PP. In contrast, Zn2+ at concentrations > 1 microM increased the inhibition of FXIa by PN-2/A beta PP. Enhancement of FXIa inhibition was virtually saturated at approximately 100 microM Zn2+ resulting in a final Ki approximately 6.0 x 10(-11) M. Zn2+ had no effect on the inhibition of FXIa by a purified, recombinant KPI domain of PN-2/A beta PP indicating that the native protein is required for the potentiation of FXIa inhibition. Heparin and Zn2+ were found to further augment each other's ability to stimulate the inhibition of FXIa by PN-2/A beta PP. Together, these findings suggest that the interaction of Zn2+ with PN-2/A beta PP may be important for optimal inhibition of FXIa.
...
PMID:Zinc (II) selectively enhances the inhibition of coagulation factor XIa by protease nexin-2/amyloid beta-protein precursor. 777 65

Latency of matrix metalloproteinase 3 (MMP-3) is regulated by the interaction of a free cysteine residue (Cys-75) in the conserved amino acid sequence Pro-Arg-Cys-Gly-Val-Pro-Asp located in the COOH-terminal portion of the propeptide with a chelated zinc atom in the active site of the catalytic domain. Proteolytic activation of full-length human pro-MMP-3 involves the removal of approximately 35 amino acids from the NH2-terminal portion of the propeptide, forming a 53-kDa unstable intermediate that undergoes intermolecular autocatalysis to form the 45-kDa mature active enzyme. In this study, we have evaluated the contribution of the NH2-terminal 35 amino acids to the maintenance of latency. Full-length human pro-MMP-3 was expressed in Escherichia coli and refolded to form latent pro-MMP-3 capable of activation by chymotrypsin or aminophenylmercuric acetate. Renaturation of pro-MMP-3 expressed in bacteria with 20 or more amino acids removed from the NH2-terminal region of the propeptide yielded only an active enzyme. COS-7 cells transiently transfected with pro-MMP-3 expression vectors containing the single amino acid substitutions Y20A, L21A, and C75S also secreted active forms of the enzyme. These data suggest that simultaneous interactions of the NH2- and COOH-terminal regions of the propeptide are required for maintenance of the latent form of the enzyme.
...
PMID:Multiple sites of the propeptide region of human stromelysin-1 are required for maintaining a latent form of the enzyme. 792 38

The relative effectiveness of two beta-cyclodextrin derivatives, i.e., dimethyl-beta-cyclodextrin (DM beta CD) and hydroxypropyl-beta-cyclodextrin (HP beta CD), in enhancing enteral absorption of insulin was evaluated in the lower jejunal/upper ileal segments of the rat by means of an in situ closed loop method. The incorporation of 10% (w/v) DM beta CD to a 0.5 mg/ml porcine-zinc insulin solution dramatically increased insulin bioavailability from a negligible value (approximately 0.06%) to 5.63%, when administered enterally at a dose of 20 U/kg. However, addition of 10% (w/v) HP beta CD did not improve enteral insulin uptake significantly with a bioavailability of only 0.07%. Similarly, the pharmacodynamic relative efficacy values obtained after the enteral administration of 20 U/kg insulin, 20 U/kg insulin with 10% HP beta CD, and 20 U/kg insulin with 10% DM beta CD were 0.24%, 0.26%, and 1.75%, respectively. Biodegradation studies of 0.5 mg/ml insulin hexamers by 0.5 microM alpha-chymotrypsin revealed no inhibitory effect on the enzymatic activity by the two cyclodextrins. On the contrary, the apparent first-order rate constant increased significantly in the presence of 10% DM beta CD, suggesting insulin oligomer dissociation by DM beta CD. Histopathological examination of the rat intestine was performed to detect tissue damage following enteral administration of the beta-cyclodextrin derivatives. Light microscopic inspection indicated no observable tissue damage, thereby arguing direct membrane fluidization as the primary mechanism for enhanced insulin uptake. This study indicates the feasibility of using cyclodextrins as mucosal absorption promoters of proteins and peptide drugs.
...
PMID:Cyclodextrins as mucosal absorption promoters of insulin. II. Effects of beta-cyclodextrin derivatives on alpha-chymotryptic degradation and enteral absorption of insulin in rats. 797 20

The factors that drive mineralization of matrix vesicles (MV) have proven difficult to elucidate; in the present studies, various detergent, chemical, and enzyme treatments were used to reveal the nature of the nucleational core. Incubation with detergents that permeabilized the membrane enhanced calcification of treated MV incubated in synthetic cartilage lymph. While detergents removed most of the membrane lipid, they left significant amounts of the MV annexins and nearly all of the Ca2+, Pi, and Zn2+. Extraction with 1 M NaCl removed much of the Ca2+ and Pi present in MV, markedly reducing Ca2+ accumulation; these effects could be prevented by low levels of Ca2+ and Pi in the NaCl extractant. Treatment with chymotrypsin appeared to damage proteins required for MV mineralization; further treatment with detergents to bypass the membrane reactivated MV mineralization. Treatment of MV with pH 6 citrate removed Ca2+ and Pi, destroying their ability to mineralize; subsequent treatment with detergents did not reactivate these MV. Incubation of the detergent-resistant core with o-phenanthroline complexed Zn2+ and stimulated mineralization; addition of Zn2+ to synthetic cartilage lymph blocked the ability of the core to mineralize. These studies show that once the nucleational core complex is formed, the membrane-enclosed domain is no longer essential for MV calcification. Our findings indicate that the MV core contains two main components as follows: a smaller membrane-associated complex of Ca2+, Pi, phosphatidylserine, and the annexins that nucleates crystalline mineral formation, and a larger pool of Ca2+ and Pi bound to lumenal proteins. These proteins appear to bind large amounts of mineral ions, stabilize the nucleational complex, and aid its transformation to the first crystalline phase. Once nucleated, the crystalline phase appears to feed on protein-bound mineral ions until external ions enter through the MV ion channels. Zn2+ appears to regulate gating of the ion channels and conversion of the nucleational complex to the crystalline state.
...
PMID:Characterization of the nucleational core complex responsible for mineral induction by growth plate cartilage matrix vesicles. 822 72


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---