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)

Calcium sensitive actin severing protein, adseverin, with Mr 74,000, was cleaved into two fragments of Mr 42,000 and Mr 39,000 by V8 protease and trypsin, and both fragments were purified by high performance (pressure) liquid chromatography ion-exchange column chromatography. To understand how adseverin can sever actin filaments, we identified the actin-binding domains. The NH2 termini of native adseverin and the Mr 42,000 fragment were confirmed to be blocked by amino acid sequencing. Twelve amino acids of the Mr 39,000 fragment were sequenced from the NH2 terminus; the sequence of this part had a homology to the hinge region between segments 3 and 4 of gelsolin and villin. Thus, the Mr 42,000 fragment is the NH2-terminal half (N42), and the Mr 39,000 fragment is the COOH-terminal half (C39). Each fragment was examined for actin-severing, -nucleating, -capping, and phospholipid binding activities with and without calcium. N42 contained a calcium-dependent actin-severing activity regulated by phospholipid. C39 bound to G-actin in a calcium-dependent manner, but had no severing activity. The sequence homology and similar functional domain structure suggest a common structural basis for the calcium- and phospholipid-regulated actin-severing properties shared by adseverin, gelsolin, and villin.
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PMID:The Ca2(+)-dependent actin filament-severing activity of 74-kDa protein (adseverin) resides in its NH2-terminal half. 184 25

Acanthamoeba profilin was cross-linked to actin via a zero-length isopeptide bond using carbodiimide. The covalently linked 1:1 complex was purified and treated with cyanogen bromide. This cleaves actin into small cyanogen bromide (CNBr) peptides and leaves the profilin intact owing to its lack of methionine. Profilin with one covalently attached actin CNBr peptide was purified by gel filtration followed by gel electrophoresis and electroblotting on polybase-coated glass-fiber membranes. Since the NH2 terminus of profilin is blocked, Edman degradation gave only the sequence of the conjugated actin CNBr fragment beginning with Trp-356. The profilin-actin CNBr peptide conjugate was digested further with trypsin and the cross-linked peptide identified by comparison with the tryptic peptide pattern obtained from carbodiimide-treated profilin. Amino-acid sequence analysis of the cross-linked tryptic peptides produced two residues at each cycle. Their order corresponds to actin starting at Trp-356 and profilin starting at Ala-94. From the absence of the phenylthiohydantoin-amino acid residues in specific cycles, we conclude that actin Glu-364 is linked to Lys-115 in profilin. Experiments with the isoforms of profilin I and profilin II gave identical results. The cross-linked region in profilin is homologous with sequences in the larger actin filament capping proteins fragmin and gelsolin.
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PMID:Acanthamoeba actin and profilin can be cross-linked between glutamic acid 364 of actin and lysine 115 of profilin. 256 69

Serum gelsolin, a Ca2+-dependent protein regulating the length of actin filaments, undergoes conformational changes upon binding Ca2+. These were detected and analyzed by several approaches including ultraviolet difference spectroscopy, circular dichroism studies, analytical ultracentrifugation, thiol group titration, and limited proteolytic digestions. The effect of Ca2+ binding on the UV absorption difference spectrum and the near-UV circular dichroism spectrum was consistent with changes in the environments of tyrosine and phenylalanine residues. In the presence of Ca2+, the S0(20),w value decreased from 5.3 to 4.7. This latter result implies a transformation to a more asymmetric molecular shape. Gelsolin contained only two accessible thiol groups per mole of protein, one of which was titratable in the native protein; it was more accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the absence than in the presence of Ca2+. The limited digestion of gelsolin from serum and bovine aorta smooth muscle by two different proteases, chymotrypsin and trypsin, proceeded much faster in the presence of Ca2+ than in its absence with the production of three main fragments of about 40K, 32K, and 21K. This fragment mixture was found still able to shorten F-actin in a Ca2+-dependent manner; this severing activity was expressed by the isolated 40K peptide. Gelsolin was cross-linked to F- and G-actin by the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC), generating a covalent 130K binary complex (actin1-gelsolin1) followed by a covalent 180K ternary complex (actin2-gelsolin1).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the Ca2+-induced conformational changes in gelsolin and identification of interaction regions between actin and gelsolin. 301 7

A large body of biochemical evidence suggests that the F-actin filament can have internal cooperativity. We have observed large cooperative effects on the low-resolution structure of actin filaments under three very different conditions. First, when G-Ca(2+)-actin is polymerized by both Mg2+ and KCl, filaments may be found in two different populations, with two discrete positions seen for subdomain 2. When G-Ca2+ actin is polymerized by only Mg2+, a single F-Mg(2+)-actin population is seen. The structural data suggest that an entire filament exists with subdomain 2 in one state or the other when there is a heterogenous mixture of Mg2+ and Ca(2+)-actin. Second, when actin filaments are nucleated from gelsolin there is a conformational change that can be observed throughout the filament that is consistent with a large shift in the actin C terminus. There must be a large cooperative propagation of this effect throughout the filament from the nucleation point. Third, we have used phalloidin to stabilize F-actin in which two C-terminal residues have been proteolytically removed by trypsin. It has been shown biochemically that this stabilization occurs at substoichiometric amounts of phalloidin. Phalloidin, at either a 1:1 or a 1:20 molar ratio with actin, restores the connectivity between the long-pitch helical strands. F-actin's internal cooperativity will have large implications in vivo, particularly in muscle.
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PMID:Structural dynamics of F-actin: II. Cooperativity in structural transitions. 784 29

Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron "white" beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its "inactive" and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation.
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PMID:Structural analysis of gelsolin using synchrotron protein footprinting. 1296 45

The giant protein titin functions as a molecular spring in muscle and is responsible for most of the passive tension of myocardium. Because the titin spring is extended during diastolic stretch, it will recoil elastically during systole and potentially may influence the overall shortening behavior of cardiac muscle. Here, titin elastic recoil was quantified in single human heart myofibrils by using a high-speed charge-coupled device-line camera and a nanonewtonrange force sensor. Application of a slack-test protocol revealed that the passive shortening velocity (Vp) of nonactivated cardiomyofibrils depends on: (i) initial sarcomere length, (ii) release-step amplitude, and (iii) temperature. Selective digestion of titin, with low doses of trypsin, decelerated myofibrillar passive recoil and eventually stopped it. Selective extraction of actin filaments with a Ca2+-independent gelsolin fragment greatly reduced the dependency of Vp on release-step size and temperature. These results are explained by the presence of viscous forces opposing myofibrillar passive recoil that are caused mainly by weak actin-titin interactions. Thus, Vp is determined by two distinct factors: titin elastic recoil and internal viscous drag forces. The recoil could be modeled as that of a damped entropic spring consisting of independent worm-like chains. The functional importance of myofibrillar elastic recoil was addressed by comparing instantaneous Vp to unloaded shortening velocity, which was measured in demembranated, fully Ca2+-activated, human cardiac fibers. Titin-driven passive recoil was much faster than active unloaded shortening velocity in early phases of isotonic contraction. Damped myofibrillar elastic recoil could help accelerate active contraction speed of human myocardium during early systolic shortening.
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PMID:Damped elastic recoil of the titin spring in myofibrils of human myocardium. 1456 22

The basic mechanism for the nucleating effect of gelsolin on actin polymerization is the formation of a complex of gelsolin with two actin monomers. Probably due to changes in the C-terminal part of gelsolin, a stable ternary complex is only formed at [Ca(2+)] >10(-5) M [Khaitlina, S., and Hinssen, H. (2002) FEBS Lett. 521, 14-18]. Therefore, we have studied the binding of actin monomer to the isolated C-terminal half of gelsolin (segments 4-6) over a wide range of calcium ion concentrations to correlate the conformational changes to the complex formation. With increasing [Ca(2+)], the apparent size of the C-terminal half as determined by gel filtration was reduced, indicating a transition into a more compact conformation. Moreover, Ca(2+) inhibited the cleavage by trypsin at Lys 634 within the loop connecting segments 5 and 6. Though the inhibitory effect was observed already at [Ca(2+)] of 10(-7) M, it was enhanced with increasing [Ca(2+)], attaining saturation only at >10(-4) M Ca(2+). This indicates that the initial conformational changes are followed by additional molecular transitions in the range of 10(-5)-10(-4) M [Ca(2+)]. Consistently, preformed complexes of actin with the C-terminal part of gelsolin became unstable upon lowering the calcium ion concentrations. These data provide experimental support for the role of the type 2 Ca-binding sites in gelsolin segment 5 proposed by structural studies [Choe et al. (2002) J. Mol. Biol. 324, 691]. We assume that the observed structural transitions contribute to the stable binding of the second actin monomer in the ternary gelsolin-actin complex.
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PMID:Calcium-induced conformational changes in the C-terminal half of gelsolin stabilize its interaction with the actin monomer. 1546 56

The purpose of this study is to identify corneal proteins differentially expressed between keratoconus and normal epithelial samples. Proteins from the corneal epithelium were isolated from 6 keratoconus and 6 myopia patients (controls) and separated by 2D-gel electrophoresis. Six % and 12% SDS-PAGE gels were used to separate low and high molecular weight proteins. Gels were silver stained and protein spots were defined by Melanie II software. The proteins that were most altered in expression comparing keratoconus and controls were extracted, trypsin-digested, and identified by mass spectroscopy. Approximately 200-500 protein spots were detected on each gel. Nineteen spots were identified as differentially expressed between keratoconus and reference epithelium including cytokeratin 3 (< 7.8 fold), gelsolin (1.6 fold), S100A4 (1.9 fold), and enolase 1 (0.72 fold). Another identified protein found at very high levels was cytokeratin 12. Gelsolin, cytokeratin 3, and cytokeratin 12 have previously been described to be involved in other corneal diseases. Three proteins, gelsolin, alpha enolase, and S100A4 were identified to be differentially expressed in keratoconus compared to reference epithelium and thus may be involved in the pathogenesis.
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PMID:Proteome profiling of corneal epithelium and identification of marker proteins for keratoconus, a pilot study. 1608 75

Immunodepletion of albumin to improve the 2-D gel resolution of human plasma proteins has recently been described. With the importance of mouse models in many studies in which serum or plasma is often analyzed, we have adopted this approach to immunoprecipitate mouse albumin and evaluated its effectiveness for 2-D separation of mouse plasma proteins. Purified polyclonal antibodies against mouse albumin were effective depleting intact albumin as well as its numerous fragments from mouse plasma samples. Removal of albumin resulted in better resolution of mouse plasma proteins. Three proteins, alpha2-macroglobulin, coagulation factor XII, and hemopexin, that were previously either undetectable or poorly resolved, were identified from albumin-depleted 2-D gels by peptide mass fingerprinting. Albumin depletion also led to partial loss of several other proteins such as clusterin and gelsolin. This loss can be attributed to the interaction with albumin itself because the specificity of the antibody was demonstrated by Western blot. When applying this method to the 2-D separation of plasma from inflamed mouse induced by cutaneous burn injury with superimposed Pseudomonas aeruginosa infection, the upregulation of inter alpha-trypsin inhibitor heavy chain 4 (ITIH4) and hemopexin was unambiguously detected along with other mouse acute-phase proteins (APP), including haptoglobin and serum amyloid A. Based on the significant increase of ITIH4, we propose that this protein is a new member of mouse APP that are upregulated during the inflammatory response.
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PMID:Immunodepletion of albumin for two-dimensional gel detection of new mouse acute-phase protein and other plasma proteins. 1613 Jan 72

Osteonecrosis of the femoral head (ONFH) is a skeletal disorder characterized by ischemic deterioration, bone marrow edema and eventually femoral head collapse. The systemic regulation of ONFH in adult patients has not been examined. Serum proteomic is an innovative tool that potentially detects simultaneous expressions of serum proteins in pathological contexts. We compared the serum proteome profiles of 11 adult patients with ONFH (3 females and 8 males) and 11 healthy volunteers (3 females and 8 males). The proteins in the aliquots of sera were subjected to isoelectric focusing, two-dimensional gel electrophoresis and silver staining. The protein spots were matched and quantified using an imaging analysis system. The differentially expressed protein spots were subjected to in-gel trypsin digestion. The peptide mass fingerprints were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF) and a bioinformation search. We found that ONFH patients showed significantly higher abundances of kininogen 1 variant, complement factor C3 precursor, and complement factor H and lower levels of antithrombin III chain B, apolipoprotein A--IV precursor, and gelsolin isoform alpha precursor. These proteins of interest were reported to modulate thrombotic/fibrinolytic reactions, oxidative stress, vessel injury, tissue necrosis or cell apoptosis in several tissue types under pathological contexts. Taken together, the occurrence of ONFH was associated with various serum protein expressions. Our high--throughput serum proteomic findings indicated that multiple pathological reactions presumably occurred in ONFH.
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PMID:Comparative serum proteome expression of osteonecrosis of the femoral head in adults. 1857 10


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