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 skeletal muscle ryanodine receptor of malignant hyperthermia-susceptible (MHS) pigs contains a mutation at residue 615 that is highly correlated with various abnormalities in the regulation of sarcoplasmic reticulum (SR) Ca2+ channel activity. In isolated SR membranes the Arg615 to Cys615 ryanodine receptor mutation is now shown to be directly responsible for an altered tryptic peptide map, due to the elimination of the Arg615 cleavage site. Furthermore, trypsin treatment released 86-99 kDa ryanodine receptor fragments encompassing residue 615 from the SR membranes. We conclude that the 86-99 kDa domain containing residue 615 is near the cytoplasmic surface of the ryanodine receptor and likely near important Ca2+ channel regulatory sites.
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PMID:Structural and functional correlates of a mutation in the malignant hyperthermia-susceptible pig ryanodine receptor. 133 12

The effect of trypsin digestion on the (i) fragmentation pattern, (ii) activity, (iii) [3H]ryanodine binding, and (iv) sedimentation behavior of the skeletal sarcoplasmic reticulum (SR) ryanodine receptor-Ca2+ release channel complex has been examined. Mild tryptic digestion of heavy, junctional-derived SR vesicles resulted in the rapid disappearance of the high molecular weight (Mr approximately 400,000) Ca2+ release channel protein on sodium dodecyl sulfate gels and appearance of bands of lower Mr upon immunoblot analysis, without an appreciable effect on [3H]ryanodine binding or the apparent S value (30 S) of the 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized channel complex. Further degradation to bands of Mr greater than 70,000 on immunoblots correlated with a reduction of channel size from 30 S to 10-15 S and loss of high affinity [3H]ryanodine binding to the trypsinized receptor, while low affinity [3H]ryanodine binding and [3H]ryanodine bound prior to digestion were retained. Parallel 45Ca2+ efflux measurements also indicated retention of the Ca2+, Mg2+, and ATP regulatory sites, although Ca2+-induced 45Ca2+ release rates were changed. In planar lipid bilayer-single channel measurements, addition of trypsin to the cytoplasmic side of the high conductance (100 pS in 50 mM Ca2+), Ca2+-activated SR Ca2+ channel initially increased the fraction of channel open time and was followed by a complete and irreversible loss of channel activity. Trypsin did not change the unitary conductance, and was without effect on single channel activity when added to the lumenal side of the channel.
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PMID:Structural and functional correlation of the trypsin-digested Ca2+ release channel of skeletal muscle sarcoplasmic reticulum. 253 70

The subunit structure of the rabbit skeletal muscle ryanodine receptor-Ca2+ release channel complex was examined following solubilization of heavy sarcoplasmic reticulum membranes in two zwitterionic detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Zwittergent 3-14. High and low affinity [3H]ryanodine binding was retained upon solubilization of the complex in Chaps but was lost in Zwittergent 3-14. The purified complex migrated as a single peak with an apparent sedimentation coefficient of approximately 30 and approximately 9 S upon density gradient centrifugation and with isoelectric points of 3.7 and 3.9 upon two-dimensional gel electrophoresis in Chaps and Zwittergent 3-14, respectively. Electron microscopy of negatively stained samples indicated that the distinct four-leaf clover structure of the ryanodine receptor observed in Chaps disappeared following Zwittergent treatment of the 30 S complex and instead showed smaller, round particles. Ferguson plot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partial and fully cross-linked and incompletely denatured complexes suggested a stoichiometry of four Mr approximately 400,000 peptides/30 S ryanodine receptor oligomer. [3H]Ryanodine binding to the membrane-bound receptor in 50 microM--1 mM free Ca2+ revealed the presence of both high affinity (KD = 8 nM, Hill coefficient (nH) = 0.9) and low affinity (nH approximately 0.45) sites with a ratio of 1:3. Reduction in free Ca2+ to less than or equal to 0.1 microM or trypsin digestion of the membranes resulted in loss of high affinity but not low affinity ryanodine binding (Hill KD = 5,000 nM, nH = 0.9). Addition of 20 mM caffeine to the nanomolar Ca2+ medium decreased the Hill KD to 1,000 nM without changing the Hill coefficient. Occupation of the low affinity sites altered the rate of [3H]ryanodine dissociation from the high affinity sites. Single channel recordings of the purified ryanodine receptor channel incorporated into planar lipid bilayers also indicated the existence of high and low affinity sites for ryanodine, occupation of which resulted in formation of a subconducting and completely closed state of the channel, respectively. These results are compatible with a subunit structural model of the 30 S ryanodine receptor-Ca2+ release channel complex which comprises a homotetramer of negatively charged and allosterically coupled polypeptides of Mr approximately 400,000.
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PMID:The ryanodine receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum. Evidence for a cooperatively coupled, negatively charged homotetramer. 255 Apr 60

A putative constituent of the junctional processes, connecting the terminal cisternae of sarcoplasmic reticulum and the transverse tubules of skeletal muscle fibers, is a greater than or equal to 350,000-dalton (Da) protein that displays ryanodine binding and Ca2+ channel properties. Ryanodine modulation of Ca2+ fluxes suggests that the ryanodine receptor and calcium channel are integral parts of one functional unit corresponding to the greater than or equal to 350,000-Da protein [Inui, M., Saito, E., & Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747; Campbell, K. P., Knudson, C. M., Imagawa, T., Leung, A. L., Sutko, J. L., Kahl, S. D., Raab, C. R., & Madson, L. (1987) J. Biol. Chem. 262, 6460-6463]. We subjected vesicular fragments of junctional-cisternal membrane to stepwise trypsin digestion. The greater than or equal to 350,000-Da protein is selectively cleaved in the early stage of digestion, with consequent disappearance of the corresponding band in electrophoretic gels. The Ca2+-ATPase is cleaved at a later stage, while calsequestrin is not digested under the same experimental conditions. While the Ca2+-ATPase yields two complementary fragments that are relatively resistant to further digestion, the greater than or equal to 350,000-Da protein yields fragments that are rapidly broken down to small peptides. Under conditions producing extensive digestion of the greater than or equal to 350,000-Da protein, the junctional processes are still visualized by electron microscopy, with no discernible alterations of their ultrastructure. The functional properties of the Ca2+ release channel are also maintained following trypsin digestion, including blockage by Mg2+ and ruthenium red and activation by Ca2+ and nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Trypsin digestion of junctional sarcoplasmic reticulum vesicles. 296 48

Tryptic digestion of the junctional sarcoplasmic reticulum membranes in sucrose but not NaCl buffer leads to complete loss of ryanodine binding capacity. The presence of MgCl2 in the sucrose buffer prevents the loss of ryanodine binding by the trypsin treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the treated membranes reveal that the 400-kDa protein band disappeared under all the different digestion conditions. However, the presence of 135-kDa tryptic fragment is observed only when ryanodine binding is retained. Quantitative analysis of the gels shows that the loss of ryanodine binding is well correlated with the cleavage of the 135-kDa tryptic fragment. This correlation is obtained when the cleavage was controlled either by the digestion time or by NaCl or MgCl2 concentrations. The same concentrations of MgCl2 and NaCl affect the ryanodine binding activity, the cleavage of the 135-kDa tryptic fragment, and the solubility and stability of the [3H]ryanodine-receptor complex in a detergent-containing medium. Tryptic digestion of the ryanodine receptor/junctional Ca2+ release channel, which leads to complete loss of ryanodine binding capacity, has no effect or slightly stimulates the Ca2+ accumulation activity of these membranes.
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PMID:Trypsin destruction of the high affinity ryanodine binding sites of the junctional sarcoplasmic reticulum. 318 12

Two isoforms of the ryanodine receptor (termed alpha and beta) are coexpressed in avian fast twitch skeletal muscle, whereas a single isoform is expressed in avian cardiac muscle. We have investigated the relationship between these three proteins, comparing several different properties. First, the three receptor isoform subunits have different mobilities on SDS-polyacrylamide gels. Second, monoclonal antibodies against the chicken skeletal muscle receptor isoforms recognize shared and unique epitopes in each receptor protein, indicating there is not a simple antigenic relationship between the isoforms. Third, the three receptor isoforms exhibit different susceptibilities to proteolysis by trypsin, and limited tryptic digestion yields a different peptide map for each isoform. Fourth, in native sarcoplasmic reticulum membranes, the chicken muscle receptor isoforms are phosphorylated to different extents by the multifunctional calcium/calmodulin-dependent protein kinase II (beta > cardiac > alpha). Fifth, the sites phosphorylated by the calcium/calmodulin-dependent protein kinase in the chicken cardiac and skeletal receptor isoforms are not equivalent. A polyclonal serum, produced against a synthetic peptide containing the site phosphorylated by this kinase in the mammalian cardiac muscle receptor, by immunoprecipitation showed markedly different avidities for the receptor isoforms, and recognized only the cardiac receptor isoform on Western blots. Sixth, the chicken ryanodine receptor isoforms differ in the extent to which they bind azido[125I]calmodulin (alpha > beta > cardiac). These results indicate that three distinct ryanodine receptor proteins are expressed in chicken striated muscles.
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PMID:Three ryanodine receptor isoforms exist in avian striated muscles. 768 27

At least three distinct ryanodine receptor genes appear to be expressed in mammalian brain. We have used biochemical and immunological methods to characterize the major form of ryanodine binding protein purified from brain. [3H]Ryanodine binding to the purified brain receptor is stimulated by Ca2+, ATP, KCl, and phosphorylation and is inhibited by calmodulin, Mg2+, and ruthenium red. Immunoblot and immunoprecipitation analysis using a panel of monoclonal and polyclonal antibodies against skeletal and cardiac muscle ryanodine receptors, and two novel polyclonal antibodies against the brain ryanodine receptor, reveals that the major form of ryanodine receptor expressed in brain is immunologically similar to the cardiac ryanodine receptor, but is distinct from the skeletal muscle receptor. Digestion of cardiac and brain ryanodine receptors with trypsin or alpha-chymotrypsin generates similar proteolytic patterns as detected by immunoblot analysis or by autoradiography after labeling with a hydrophobic probe, suggesting that the two proteins are similar in both their large cytoplasmic and hydrophobic transmembrane domains. Taken together, these data indicate that the cardiac ryanodine receptor/Ca2+ release channel is the major form of ryanodine receptor expressed in brain, and that it likely functions in releasing Ca2+ from caffeine-sensitive intracellular Ca2+ stores in neurons by a mechanism of regulated Ca(2+)-induced Ca2+ release.
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PMID:Characterization of the major brain form of the ryanodine receptor/Ca2+ release channel. 769 41

A polypeptide of high molecular mass has been detected in mammalian brain by a monoclonal antibody, 5C3, raised against skeletal muscle ryanodine receptor. 5C3 does not crossreact with the cardiac ryanodine receptor, the isoform which is believed to be located in many regions of the brain. Endogenous proteases in brain formed a prominent immunogenic fragment of 116 kDa whereas five immunostaining polypeptides greater than 200 kDa were observed in skeletal muscle. Mild trypsin digestion of brain microsomes resulted in fragments of approximately 400 and approximately 280 kDa, of similar mass to two peptides formed from the skeletal muscle ryanodine receptor. However a peptide of 28 kDa, resistant to trypsin, was observed in muscle but not in brain. The brain polypeptide recognised by 5C3 is therefore not identical to the skeletal muscle ryanodine receptor.
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PMID:Monoclonal antibody to skeletal muscle ryanodine receptor detects a polypeptide in rat brain: comparison of immunogenic fragments after limited proteolysis. 801 88

The 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized 30 S ryanodine receptor (RyR)/Ca2+ release channel complex from rabbit skeletal muscles, purified by density gradient centrifugation, was reconstituted with an excess of phospholipid into proteoliposomes by removal of the detergent by dialysis. Reconstituted proteoliposomes were concentrated by centrifugation, frozen and thawed, and sonicated. [3H]Ryanodine binding measurements indicated close to 50% recovery of calculated binding activity following reconstitution of the purified RyR and dynamic light scattering measurements a mean vesicle diameter of approximately 150 nm. Using these values, a functional RyR was estimated to be present in only a small fraction (< 15%) of the reconstituted vesicles. SDS-polyacrylamide gel electrophoresis of trypsin-treated proteoliposomes revealed that about four-fifths of the reconstituted 30 S complex was readily accessible to proteolytic attack. Vesicle-45Ca2+ flux and fusion of proteoliposomes with planar lipid bilayers showed that the reconstituted channel complex could be activated by Ca2+ and ATP, inhibited by Mg2+ and ruthenium red, and modified by ryanodine, similarly as observed for native sarcoplasmic reticulum vesicles. These results suggest that the method described here results in the reconstitution of a functional channel and thus provides the opportunity to study the structure and function of the sarcoplasmic reticulum Ca2+ release channel under well defined conditions.
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PMID:Reconstitution of the skeletal muscle ryanodine receptor-Ca2+ release channel protein complex into proteoliposomes. 817 60

Site-specific antibodies against different regions of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor) were developed and used as probes for immunoblotting of the major tryptic fragments resulting from partial digestion of the ryanodine receptor in sarcoplasmic reticulum membranes. Five major tryptic fragments, some of which migrated as doublets, with apparent masses of 150/140, 110/100, 55, 170/160, and 76 kDa were ordered so that they covered the bulk of the protein from the NH2 to the COOH terminus. Tryptic subfragments of 53, 63, and 115/95 kDa were also derived from the 150/140-, 110/100-, and 170/160-kDa fragments, respectively. All of these fragments and subfragments were detected only in the insoluble membrane fraction of the trypsinized sarcoplasmic reticulum. Upon Na2CO3 extraction, the 150/140-, 110/100-, and 55-kDa fragments could be solubilized, suggesting their origin in the cytoplasmic domain of the ryanodine receptor. The 170/160- and 76-kDa fragments and the 115/95-kDa subfragment remained insoluble, suggesting their origin in the transmembrane region of the ryanodine receptor. The 150/140-, 110/100-, 170/160-, and 76-kDa fragments and the 115/95 subfragment co-migrated near the bottom of a sucrose density gradient after CHAPS solubilization, suggesting that they were associated in an oligomeric complex. By contrast, the 53- and 63-kDa subfragments and the 55-kDa fragment were detected near the top of the sucrose gradient after CHAPS solubilization, suggesting that they were not involved in the formation of the core of the oligomeric complex. These studies identify 7 sites that are exposed to trypsin in the ryanodine receptor in sarcoplasmic reticulum, 3 of which are novel and 4 of which are in the same location as proteolytic cleavage sites identified previously (Marks, A. R., Fleischer, S., and Tempst, P. (1990) J. Biol. Chem. 265, 13143-13149).
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PMID:Positioning of major tryptic fragments in the Ca2+ release channel (ryanodine receptor) resulting from partial digestion of rabbit skeletal muscle sarcoplasmic reticulum. 822 72


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