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)

The 95,000 molecular weight protein (95K protein) of the false discharges of Limulus sperm, purified by means of preparative gel electrophoresis in the presence of sodium dodecyl sulfate, was compared with a 95K protein from Limulus muscle and chicken gizzard alpha-actinin. The results were as follows. 1) One-dimensional peptide mapping using four different proteases showed differences among these proteins. 2) Two-dimensional peptide mapping using trypsin showed that about 30% of the peptides in the digest of the sperm 95K protein were similar to those of chicken gizzard alpha-actinin and about 50% of the peptides were similar to those of the Limulus muscle 95K protein. 3) The sperm 95K protein contained relatively large amounts of Gly, Pro, and Ser and relatively small amounts of Glu and Leu compared to the muscle proteins. 4) Antibodies against the sperm 95K protein did not cross-react with the Limulus muscle 95K protein or chicken gizzard alpha-actinin. These results suggest that the 95K protein of sperm is different from alpha-actinin in primary structure.
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PMID:Comparison of the 95,000 molecular weight protein from Limulus sperm with muscle alpha-actinin. 11 67

A calcium-activated factor (CaAF) has been isolated and partially purified from the post-myofibrillar supernatant fraction of rabbit skeletal muscle. The 200-fold purified CaAF hydrolyzed denatured casein, [3-H]acetyl hemoglobin, and N-ethyl[3-H]maleimide-labeled alpha-actinin. The proteolytic activity has a pH optimum at 6.9 and is dependent on the presence of Ca2+ (optimum concentration, 10 mM). Digestion of isolated myofibrils with CaAF results in removal of Z-lines and in a parallel loss of a 90, 000-dalton protein that has a mobility identical with that of alpha-actinin as determined by polyacrylamide gel electrophoresis. A protein with the properties of alpha-actinin (identical electrophoretic mobility, and ability to accelerate the Mg2+-activated ATPase of reconstituted actomyosin) was isolated from the supernatant of CaAF-treated myofibrils. The release of alpha-actinin from myofibrils by the calcium-activated neutral protease occurs in the absence of detectable change in the electrophoretic profiles of the other myofibrillar proteins, or in the ethylene glycol bis(beta-aminoethyl ether)-N, N' tetraacetic acid (EGTA) sensitivity of Mg2+-activated ATPase. In contrast to the specific removal of Z-lines and of alpha-actinin by CaAF, trypsin treatment of myofibrils results in extensive degradation of myosin heavy chains and of the inhibitory component of troponin (TN-I), and in loss of EGTA sensitivity of myofibrillar ATPase. The degradation of TN-I and loss of EGTA sensitivity occur before the Z-line disappearance.
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PMID:Removal of Z-lines and alpha-actinin from isolated myofibrils by a calcium-activated neutral protease. 80 38

5 min of tryptic digestion of purified rabbit skeletal alpha-actinin decreases by approximately 75% the ability of alpha-actinin to cross-link F-actin filaments as measured viscometrically at 27 degrees C, but has little effect on the sedimentation coefficient of alpha actinin at 20 degrees C or an alpha-actinin's ability to increase the Mg2+-modified ATPase activity and rate of turbidity increase of reconstituted actomyosin suspensions. Twenty to sixty min of trypsin treatment reduces the sedimentation coefficient of alpha-actinin and destroys much of alpha-actinin's ability to increase the MG2+-modified ATPase and rate of turbidity increase of reconstituted actomyosin suspensions. Therefore, the ability of alpha-actinin to increase the rate of in vitro measures of muscle contraction may not result directly from alpha-actinin's ability to cross-link F-actin filaments. Trypsin does not split alpha-actinin into large fragments as it does myosin. Previous studies have shown that 35 to 65% of total tryptic-susceptible peptide bonds in alpha-actinin are split after 60 min of incubation with trypsin and that 30% of these bonds split in 60 min are cleaved during the first 5 min in a rapid reaction. That splitting of this group of peptide bonds has little effect on the sedimentation coefficient of alpha-actinin indicates that these bonds are located in a region of the alpha-actinin molecule where noncovalent forces are strong enough to maintain conformation of the native alpha-actinin molecule even after these bonds have been split. This ostensible segregation of alpha-actinin's ability to cross-link F-actin filaments from its ability to increase rate of in vitro assays of contraction by tryptic digestion may suggest that alpha-actinin could have at least two different physiological roles: (1) to bind actin filaments to each other or to basal structures, and (2) to enhance the effectiveness of actin in supporting movement.
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PMID:Effect of trypsin on rabbit skeletal muscle alpha-actinin. 99 Feb 86

A multicatalytic proteinase (MCP) purified from lobster claw and abdominal muscles degrades a variety of peptide and protein substrates. The enzyme is activated by low concentrations (0.03%) of sodium dodecyl sulfate (SDS) and brief (1 min) heating at 60 degrees C. The lobster MCP can assume three stable and functionally distinct states in vitro; these are classified as the basal, heat-activated, and SDS-activated forms. The basal MCP possessed high trypsin-like peptidase activity and low chymotrypsin-like peptidase, peptidylglutamyl-peptide hydrolase, and caseinolytic activities; incubation of the basal form with SDS stimulated the peptidylglutamyl-hydrolase activity about 30-fold and inhibited the other three activities 80% to 100%. Heating the basal form stimulated caseinolytic activity about 6-fold with little effect on the peptidase activities. The heat-activated enzyme also degraded myosin, tropomyosin, troponin, and actin depolymerizing factor; alpha-actinin was resistant to proteolysis. Incubation of the heat-activated MCP with SDS inhibited the trypsin-like, chymotrypsin-like, and proteinase activities 95 to 100% and stimulated the peptidylglutamyl-hydrolase activity about 16-fold. Incubation of myosin with either the basal or the heat-activated forms in the presence of SDS generated identical proteolytic fragments of the myosin heavy chain, suggesting that SDS induced a third form that can be produced from either the basal or the heat-activated forms. The heat-activated form produced proteolytic fragments of myosin heavy chain different from those generated by either basal or heat-activated enzymes in the presence of SDS. Furthermore, 100 mM KCl stimulated the caseinolytic activity of the heat-activated form 24% and inhibited the trypsin-like and peptidylglutamyl-hydrolase activities 56 and 20%, respectively. These results, though indirect, suggest that heating induced a proteinase activity that was distinct from the three peptidase activities. Activation of the basal form with SDS was reversible, since precipitation of dodecyl sulfate with 100 mM KCl restored trypsin-like activity and inhibited peptidylglutamyl-hydrolase activity. In contrast, removal of dodecyl sulfate from the SDS-activated form that was derived from the heat-activated MCP induced its conversion to the basal form. Thus, although heat-activation was irreversible, the heat-activated form was converted back to the basal form via the SDS-activated form.
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PMID:Sodium dodecyl sulfate and heat induce two distinct forms of lobster muscle multicatalytic proteinase: the heat-activated form degrades myofibrillar proteins. 189 47

Actobindin is a protein from Acanthamoeba castellanii with bivalent affinity for monomeric actin. Because it can bind two molecules of actin, actobindin is a substantially more potent inhibitor of the early phase of actin polymerization than of F-actin elongation. The complete amino acid sequence of 88 residues has been deduced from the determined sequences of overlapping peptides obtained by cleavage with trypsin, Staphylococcus V8 protease, endoproteinase Asp-N, and CNBr. Actobindin contains 2 trimethyllysine residues and an acetylated NH2 terminus. About 76% of the actobindin molecule consists of two nearly identical repeated segments of approximately 33 residues each. This could explain actobindin's bivalent affinity for actin. The circular dichroism spectrum of actobindin is consistent with 15% alpha-helix and 22% beta-sheet structure. A hexapeptide with sequence LKHAET, which occurs at the beginning of each of the repeated segments of actobindin, is very similar to sequences found in tropomyosin, muscle myosin heavy chain, paramyosin, and Dictyostelium alpha-actinin. A longer stretch in each repeated segment is similar to sequences in mammalian and amoeba profilins. Interestingly, the sequences around the trimethyllysine residues in each of the repeats are similar to the sequences flanking the trimethyllysine residue of rabbit reticulocyte elongation factor 1 alpha, but not to the sequences around the trimethyllysine residues in Acanthamoeba actin and Acanthamoeba profilins I and II.
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PMID:The covalent structure of Acanthamoeba actobindin. 237 77

Limited trypsinolysis of native alpha-actinin by trypsin was carried out. This procedure resulted in the formation of two large fragments with Mr of 30 and 70 kD which cover almost the whole subunit of alpha-actinin. Using the sedimentation equilibrium method, it was demonstrated that the bisubunit structure of alpha-actinin is provided for by C-terminal fragments of the subunits. Data from circular dichroism analysis suggest that the fragments formed are independent structural units, i.e., domains.
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PMID:[Domain organization of alpha-actinin from the rabbit skeletal muscle]. 317 51

Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmission electron microscopy revealed that the cells had fine structural features typical of trophoblasts. In contrast to syncytial trophoblasts of intact term placentae, these cells did not stain for hCG, human placental lactogen, pregnancy-specific beta 1-glycoprotein or low mol wt cytokeratins by immunoperoxidase methods. Endothelial cells, fibroblasts, or macrophages did not contaminate the purified cytotrophoblasts, as evidenced by the lack of immunoperoxidase staining with antibodies against vimentin or alpha 1-antichymotrypsin. The cells produced progesterone (1 ng/10(6) cells . 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 micrograms/ml). They also produced estrogens (1360 pg/10(6) cells . 4 h) when supplied with androstenedione (1 ng/ml) as a precursor. When placed in culture, the cytotrophoblasts consistently formed aggregates, which subsequently transformed into syncytia within 24-48 h after plating. Time lapse cinematography revealed that this process occurred by cell fusion. The presumptive syncytial groups were proven to be true syncytia by microinjection of fluorescently labeled alpha-actinin, which diffused completely throughout the syncytial cytoplasm within 30 min. Immunoperoxidase staining of cultured trophoblasts between 3.5 and 72 h after plating revealed a progressive increase in cytoplasmic pregnancy-specific beta 1-glycoprotein, hCG, and human placental lactogen concomitant with increasing numbers of aggregates and syncytia. At all time points examined, occasional single cells positive for these markers were identified. RIA of the spent culture media for hCG revealed a significant increase in secreted hCG, paralleling the increase in hCG-positive cells and syncytia identified by immunoperoxidase methods. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.
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PMID:Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. 351 58

Myofibrils from bovine longissimus muscle were obtained at 2 h postmortem and incubated in .10 to .35 M ionic strength buffers under various conditions in vitro. Increasing ionic strength or increasing the incubation time from 1 to 72 h decreased the turbidity of suspensions of myofibrils and increased myofibrillar solubilization (P less than .01 for both measures). The use of KCl or NaCl to elevate ionic strength gave essentially identical results, but lactate generally was ineffective in changing either the percentage myofibrillar solubilization or the turbidity of suspensions of myofibrils. Gel electrophoresis under denaturing conditions indicated that KCl was more effective than NaCl in causing the release of C-protein from myofibrils, and both salts were quite effective in dissociating M-protein, actin, troponin-T, tropomyosin, myosin light chain-3 and a 30,000-dalton molecular weight protein from myofilaments. Small increases in alpha-actinin also were observed, especially in samples incubated for 72 h. Substantially more myosin light chain-3, tropomyosin (or paratropomyosin) and troponin-T, and less actin and the 30,000-dalton protein, were released in samples incubated at pH 5.5 than at pH 7.0 (P less than .05). Electron micrographs indicated loss of thick filament ultrastructure after incubation for 24 h in either .1 or .3 M ionic strength, but the Z-lines were largely unaffected. In samples that had first been incubated with trypsin for 10 min, the Z-lines were virtually indistinguishable at .1 M ionic strength, and absolutely no myofibrillar structures could be discerned in samples incubated in .3 M ionic strength buffer.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ionic strength and myofibrillar protein solubilization. 362 3

Incubation of alpha-actinin with trypsin leads to the formation of several fragments with molecular weight of 55 000, 38 000, 30 000 and 15 000 which are rather resistant against further proteinase action. Two from five exposed cysteine residues modified by N-ethylmaleimide in the polypeptide chain of the subunit are located in the 55 000 fragment, one in the 30 000 fragment, and the remaining two appear to belong to those parts of the polypeptide chain that are subject to degradation to small peptides under the action of trypsin. Masked SH-groups are localized to the 30 000 fragment.
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PMID:[Limited trypsinolysis of alpha-actin and the distribution of sulfhydryl groups in the molecular fragments]. 400 53

Incubation of native alpha-actinin with trypsin leads to the formation of intermediate fragments that are quite resistant to further enzyme action (M 98, 85, 80, 70, 64, 55, 38, 30, 15 kDa). Analysis of these fragments made it possible to elucidate the course of trypsinolysis and to localize the fragments in the polypeptide chain of alpha-actinin subunit.
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PMID:[Fragments of limited trypsinolysis of native alpha-actinin]. 409 62


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