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)

Amino and carboxyl termini of the bifunctional enzyme Fru 6-P, 2-kinase:Fru 2,6-bisphosphatase regulate the relative activities of the kinase/phosphatase. The N-terminus of the rat liver bifunctional enzyme is highly basic, containing a protein kinase A phosphorylation site that regulates these enzyme activities in a reciprocal manner. To determine the role of charged residues in the N-terminal peptide, mutant enzymes were constructed in which these residues were altered to residues carrying opposite charges, and the effect on the catalytic properties, thermal lability, and susceptibility to trypsin digestion and phosphorylation by protein kinase A was determined. Most of these mutations decreased k(cat)/K(ATP) and/or k(cat)/K(Fru) (6-P) of the kinase and increased k(cat)/K(Fru 2,6-P2) of the phosphatase. These mutant enzymes were more susceptible to trypsin digestion, phosphorylation by protein kinase A, and thermal inactivation. In general, the effect was greater with amino acid residues located more distant from the N-terminus. The resulting changes were not as large as observed with the phosphorylated enzyme. Mutation of Ser22 to Pro produced large changes in the kinetic properties comparable to those of phosphorylation, suggesting that the flexible region of the N-terminus containing five serines (Ser20 to S24) is essential for the enzyme activities. These results indicated that the charged residues as well as Ser20-Ser24 in the N-terminus of the liver Fru 6-P,2-kinase:Fru 2,6-Pase are essential in the allosteric regulation and probably involved in interactions with the catalytic domains that induce a conformation that has high Fru 6-P,2-kinase and low Fru 2,6-Pase activities. Any disruption of this N-terminal interaction results in inhibition of the kinase and activation of the phosphatase.
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PMID:Mutations in the charged residues of the amino terminus of rat liver fructose 6-phosphate,2-kinase:Fructose 2,6-bisphosphatase: effects on regulation. 1052 84

C-terminal tails of plant hexose/H(+)-symporters of the major facilitator superfamily contain a highly conserved motif of four amino acids: HWFW. A deletion of these four amino acids in the Chlorella HUP1 protein leads to a decrease in transport activity by a factor of 3-4. The mutated tail is highly sensitive to trypsin; it does not show alpha-helical conformation in contrast to the wild type C-terminal peptide with an alpha-helical content of at least 15%. The production of monoclonal antibody 416B8 recognizing an epitope within the central loop of HUP1 protein has been a prerequisite for the experiments described.
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PMID:The C-terminal tetrapeptide HWFW of the Chlorella HUP1 hexose/H(+)-symporter is essential for full activity and an alpha-helical structure of the C-terminus. 1069 91

The possible contribution of the mature portion of a mitochondrial precursor protein to its interaction with membrane lipids is unclear. To address this issue, we examined the interaction of the precursor to mitochondrial aspartate aminotransferase (pmAAT) and of a synthetic peptide corresponding to the 29-residue presequence peptide (mAAT-pp) with anionic phospholipid vesicles. The affinity of mAAT-pp and pmAAT for anionic vesicles is nearly identical. Results obtained by analyzing the effect of mAAT-pp or full-length pmAAT on either the permeability or microviscosity of the phospholipid vesicles are consistent with only a shallow insertion of the presequence peptide in the bilayer. Analysis of the quenching of Trp-17 fluorescence by brominated phospholipids reveals that this presequence residue inserts to a depth of approximately 9 A from the center of the bilayer. Furthermore, in membrane-bound pmAAT or mAAT-pp, both Arg-8 and Arg-28 are accessible to the solvent. These results suggest that the presequence segment lies close to the surface of the membrane and that the mature portion of the precursor protein has little effect on the affinity or mode of binding of the presequence to model membranes. In the presence of vesicles, mAAT-pp adopts considerable alpha-helical structure. Hydrolysis by trypsin after Arg-8 results in the dissociation of the remaining 21-residue C-terminal peptide fragment from the membrane bilayer, suggesting that the N-terminal portion of the presequence is essential for membrane binding. Based on these results, we propose that the presequence peptide may contain dual recognition elements for both the lipid and import receptor components of the mitochondrial membrane.
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PMID:Interaction of the precursor to mitochondrial aspartate aminotransferase and its presequence peptide with model membranes. 1093 77

Accurate definition of the carboxyl terminal of proteins is necessary for elucidating posttranslational processing at the C-terminal and more generally for characterizing protein primary structures. Here, we describe a strategy for isolating and characterizing the C-terminal peptide of a protein after proteolysis with endoprotease Lys-C. Isolation is achieved using anhydrotrypsin, a catalytically inert derivative of trypsin that binds peptides containing lysine or arginine residues at their C-termini without cleaving them. Rapid, accurate characterization of the isolated C-terminal peptide is achieved by mass spectrometry. Initial identification of the C-terminal peptide is obtained by comparing matrix-assisted laser desorption/ionization time-of-flight mass spectra of the digest prior to and after incubation with anhydrotrypsin. Characterization of the C-terminal sequence is achieved by capillary-HPLC electrospray ionization tandem mass spectrometry of the isolated peptide using a quadrupole ion trap mass spectrometer in the selective reaction monitoring mode. This strategy was successfully applied to the characterization of the C-terminal of proteins with molecular masses ranging up to 56 kDa.
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PMID:A method to define the carboxyl terminal of proteins. 1093 15

Insulin-like growth factor binding protein-6 (IGFBP-6) is an O-linked glycoprotein which specifically inhibits insulin-like growth factor (IGF)-II actions. The effects of O-glycosylation of IGFBP-6 on binding to glycosaminoglycans and proteolysis, both of which reduce the IGF binding affinity of other IGFBPs were studied. Binding of recombinant human nonglycosylated (n-g) IGFBP-6 to a range of glycosaminoglycans in vitro was approximately threefold greater than that of glycosylated (g) IGFBP-6. When bound to glycosaminoglycans, IGFBP-6 had approximately 10-fold reduced binding affinity for IGF-II. Exogenously added n-gIGFBP-6 but not gIGFBP-6 also bound to partially purified rat PC12 phaeochromocytoma membranes. Binding of n-gIGFBP-6 was inhibited by increasing salt concentrations, which is typical of glycosaminoglycan interactions. O-glycosylation also protected human IGFBP-6 from proteolysis by chymotrypsin and trypsin. Proteolysis decreased the binding affinity of IGFBP-6 for IGF-II, even with a relatively small reduction in apparent molecular mass as observed with chymotrypsin. Analysis by ESI-MS of IGFBP-6 following limited chymotryptic digestion showed that a 4.5-kDa C-terminal peptide was removed and peptide bonds involved in the putative high affinity IGF binding site were cleaved. The truncated, multiply cleaved IGFBP-6 remained held together by disulphide bonds. In contrast, trypsin cleaved IGFBP-6 in the mid-region of the molecule, resulting in a 16-kDa C-terminal peptide which did not bind IGF-II. These results indicate that O-glycosylation inhibits binding of IGFBP-6 to glycosaminoglycans and cell membranes and inhibits its proteolysis, thereby maintaining IGFBP-6 in a high-affinity, soluble form and so contributing to its inhibition of IGF-II actions.
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PMID:O-glycosylation of insulin-like growth factor (IGF) binding protein-6 maintains high IGF-II binding affinity by decreasing binding to glycosaminoglycans and susceptibility to proteolysis. 1095 Nov 95

Mouse SPACR cDNA was cloned by screening a mouse retina cDNA library using a PCR probe derived from human SPACR cDNA. Mouse SPACR cDNA comprises 3675 bp containing an open reading frame coding for 742 amino acids. Multitissue Northern blot analysis and in situ hybridization studies indicate that SPACR expression is restricted to retinal photoreceptors. The SPACR core protein was identified with Western blotting following SDS-PAGE with a SPACR C-terminal peptide polyclonal antibody and a chondroitin-6-sulfate Deltadisaccharide monoclonal antibody. The 150 kD immunopositive band was isolated, digested with trypsin and the peptides analysed by MALDI mass spectroscopy. Peptide mass mapping confirmed the identity of the 150 kD immunopositive band to be mouse SPACR core protein. Alignment comparisons of the deduced amino acid sequence of mouse and human SPACR show 64% homology. Like SPACR in the human interphotoreceptor matrix, the mouse orthologue contains a large central mucin-like domain flanked by consensus sites for N-linked oligosaccharide attachment, one EGF-like domain and four hyaluronan-binding motifs. Unlike human SPACR, which contains no conventional consensus sites for glycosaminoglycan attachment, mouse SPACR contains three. Recent biochemical studies of human and mouse SPACR protein indicate that this novel interphotoreceptor matrix molecule is a glycoprotein in human and a proteoglycan in the mouse. The presence of consensus sites for glycosaminoglycan attachment in the deduced sequence of mouse SPACR and the absence of these sites in human SPACR provide molecular verification of our biochemical results, suggesting that differences in post-translational modifications of SPACR may be important in SPACR function in foveate and non-foveate retinas.
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PMID:SPACR in the interphotoreceptor matrix of the mouse retina: molecular, biochemical and immunohistochemical characterization. 1099 55

A widely used method for assessing occupational and environmental exposure to 1,3-butadiene involves the detection of hemoglobin adducts formed by the reactive metabolite butadiene monoxide (BMO). This assay employs the N-alkyl Edman method, which was developed to determine adducts formed at the amine group of the N-terminal valine of hemoglobin. Disadvantages of this procedure include its limitation to detecting only one adduct per globin chain, despite the presence of numerous other, and potentially more reactive, nucleophilic amino acids in hemoglobin. The method is also not suitable for determining whether the reaction of BMO occurs at the N-terminal valine of alpha- or beta-globin. The primary goals of the current research are to determine the degree of modification of alpha- and beta-globin chains by BMO and to localize the reactive residues to specific regions of the globin polypeptides. The reaction products after in vitro incubation of C57Bl/6 mouse erythrocytes with BMO were isolated by acid extraction of heme and microprecipitation of globin, followed by the determination of the number and location of adducts by mass spectrometry. The modification degree was monitored by electrospray mass spectrometry, which was used to measure the time- and concentration-dependent formation of BMO-hemoglobin adducts (< or =10 adducts per globin). The results indicate that BMO reacts faster and to a higher degree with alpha-globin than with beta-globin. Structural analysis was performed by peptide mapping of globin peptides after trypsin digestion using liquid chromatography/mass spectrometry. These experiments allowed the localization of BMO-hemoglobin adducts to specific regions within alpha- and beta-globin, and also provided information about their relative reactivity. Interestingly, the initial site of adduct formation on alpha-globin is located near the N-terminal peptide, whereas the initial site on beta-globin is located at the C-terminal region. Collectively, the results establish differences in the reactivities of alpha- and beta-globin toward BMO, demonstrate the formation of multiple adducts at several alpha- and beta-globin sites, and show that the N-terminal valine residues are not the first to be modified by BMO.
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PMID:A comprehensive structural analysis of hemoglobin adducts formed after in vitro exposure of erythrocytes to butadiene monoxide. 1108 32

The most abundant albumin present in seeds of Theobroma cacao was purified to apparent homogeneity as judged by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH(2)-terminal sequence analysis. Tryptic peptide mass fingerprinting of the purified protein by HPLC/ESI-MS showed the presence of 16 masses that matched the expected tryptic peptides corresponding to 95% of the translated amino acid sequence from the cDNA of the 21 kDa cocoa albumin. Collision-induced dissociation MS/MS analysis of the C-terminal peptide isolated from the CNBr cleavage products provided unequivocal evidence that the mature cocoa albumin protein is nine amino acid residues shorter than expected from the reported cDNA of its corresponding gene. The experimentally determined M(r) value of 20234 was in excellent agreement with the truncated version of the amino acid sequence. The purified cocoa albumin inhibited the catalytic activities of bovine trypsin and chymotrypsin. The inhibition was stoichiometric with 1 mol of trypsin or chymotrypsin being inhibited by 1 mol of inhibitor with apparent dissociation constants (K(i)) of 9.5 x 10(-8) and 2. 3 x 10(-6) M, respectively, for inhibitor binding at pH 8.5 and 37 degrees C. No inhibition of the catalytic activities of subtilisin, papain, pepsin, and cocoa endoproteases was detected under their optimal reaction conditions.
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PMID:Primary structure of the abundant seed albumin of Theobroma cacao by mass spectrometry. 1108 24

Human brain carboxypeptidase B (HBCPB) is a novel brain protease that processes native brain beta-amyloid precursor protein (APP) in vitro. Immunoblot analysis of human serum and cerebrospinal fluid (CSF) using anti C14-module antibody, which recognizes the C-terminal peptide unique to HBCPB, detected the 30 and 40 kDa immunoreactive bands. Analysis of HBCPB prepared from both serum and CSF demonstrated proteolytic activities for brain APP. Protease inhibitor spectrum analysis also supports that these bands correspond to the mature form and and prepro form of HBCPB, respectively. As is the case in brain parenchyma, the prepro-form is dominant in CSF. In serum, however, the majority of HBCPB exists in the mature form, possibly due to an abundant trypsin-like proteolytic activity in serum. HBCPB expressed in serum and CSF, therefore, may have a significance as a peripheral marker of the brain protease, which participates in APP processing in human brain.
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PMID:Expression of human brain carboxypeptidase B, a possible cleaving enzyme for beta-amyloid precursor protein, in peripheral fluids. 1124 71

Pancreatitis-associated protein (PAP), a secretory acute-phase protein of the pancreatic acinar cell, is highly up-regulated early in acute pancreatitis. PAP expression returns to undetectable levels when the pancreas recovers. In the rat, three isoforms of PAP are known, all of which are upregulated during acute pancreatitis. Their functions remain obscure. Pancreatic stone protein (PSP/reg), which shows strong sequence homology to PAP, is secreted into pancreatic juice under physiologic and pathologic conditions. PSP/reg is highly susceptible to trypsin cleavage at its ARG11-ILE12 bond. Cleavage results in an N-terminal undecapeptide and a C-terminal peptide called pancreatic thread protein (PTP). PTP forms oligomeric fibrillar structures, which spontaneously sediment in vitro. PTP can be found in protein plugs or stones from patients with chronic pancreatitis. Rat PAP contains a trypsin cleavage site at the same position as PSP/reg. We hypothesize that PAP is susceptible to tryptic cleavage, and that the C-terminal cleavage product of PAP spontaneously precipitates at neutral pH. To test our hypothesis, we generated and purified recombinant PAP. Here we report the production of rat PAP I, II, and III in a yeast expression system using Pichia pastoris. We demonstrate in vitro the tryptic cleavage of rat PAP and the formation of a spontaneously precipitating peptide, which we call pancreatitis-associated thread protein (PATP). PATP displays pH-dependent solubility characteristics very similar to those of PTP.
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PMID:Conformational changes of pancreatitis-associated protein (PAP) activated by trypsin lead to insoluble protein aggregates. 1124 74


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