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)

Exercised mdx mice were used to evaluate the efficacy of two pharmacologic entities, cromolyn and compound 48/80. Beginning at 2 weeks of age, mdx mice were treated with either cromolyn (50 mg/kg/day), prednisone (2mg/kg/day), compound 48/80 (1mg/kg/day), or diluent vehicle. At 4 weeks of age, treated mice were subjected to twice weekly, forced treadmill running which has previously been shown to cause expressed weakness in mdx mice (Hudecki, Pollina et al., 1993). Strength was evaluated weekly through 6 weeks of age using a previously described "pull-test" procedure (Hudecki, Pollina et al., 1993). Serum creatine kinase (CK) and mast cell tryptase activities were evaluated from 6 week blood samples. There was a significant increase in strength in mdx mice treated with cromolyn (p < or = 0.05), while no significant increase in strength was found in mice treated with compound 48/80, or prednisone compared to vehicle controls. While no significant change in tryptase activity was found between treatments, CK activity was significantly increased in the cromolyn group compared to vehicle controls. However, when tryptase and CK were expressed as a combined factor (Tryp x CK), the cromolyn treated group was significantly different from all other groups. The results of this study suggest a possible use for cromolyn-like compounds in the treatment of Duchenne muscular dystrophy.
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PMID:Cromolyn increases strength in exercised mdx mice. 882 68

Very recent experimental data, obtained by using the permeabilized cell technique or tissue homogenates for investigation of the mechanisms of regulation of respiration in the cells in vivo, are shortly summarized. In these studies, surprisingly high values of apparent Km for ADP, exceeding that for isolated mitochondria in vitro by more than order of magnitude, were recorded for heart, slow twitch skeletal muscle, hepatocytes, brain tissue homogenates but not for fast twitch skeletal muscle. Mitochondrial swelling in the hypo-osmotic medium resulted in the sharp decrease of the value of Km for ADP in correlation with the degree of rupture of mitochondrial outer membrane, as determined by the cytochrome c test. Very similar effect was observed when trypsin was used for treatment of skinned fibers, permeabilized cells or homogenates. It is concluded that, in many but not all types of cells, the permeability of the mitochondria outer membrane for ADP is controlled by some cytoplasmic protein factor(s). Since colchicine and taxol were not found to change high values of the apparent Km for ADP, the participation of microtubular system seems to be excluded in this kind of control or respiration but studies of the roles of other cytoskeletal structures seem to be of high interest. In acute ischemia we observed rapid increase of the permeability of the mitochondrial outer membrane for ADP due to mitochondrial swelling and concomitant loss of creatine control of respiration as a result of dissociation of creatine kinase from the inner mitochondrial membrane. The extent of these damages was decreased by use of proper procedures of myocardial protection showing that outer mitochondrial membrane permeability and creatine control of respiration are valuable indices of myocardial preservation. In contrast to acute ischemia, chronic hypoxia seems to improve the cardiac cell energetics as seen from better postischemic recovery of phosphocreatine, and phosphocreatine overshoot after inotropic stimulation. In general, adaptational possibilities and pathophysiological changes in the mitochondrial outer membrane system point to the central role such a system may play in regulation of cellular energetics in vivo.
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PMID:On the regulation of cellular energetics in health and disease. 890 74

Protease digestion experiments have been used to characterize the structure of an equilibrium intermediate in the unfolding of creatine kinase (CK) by low concentrations (0.625 M) of guanidine hydrochloride (GdnHCl). Eighteen of the major products of digestion by trypsin, chymotrypsin and endoproteinase Glu-C have been identified by microsequencing after separation by SDS/PAGE and electroblotting on poly(vinylidene difluoride) membranes. The C-terminal portion (Gly215 to Lys380) was much more resistant to digestion than the N-terminal portion (Pro1 to Gly133), although the area most sensitive to proteolysis was in the middle of the CK sequence (Arg134 to Arg214). These experiments are consistent with the two-domain model for the CK monomer. The structure of the intermediate is proposed to consist of a folded C-terminal domain and a partly folded N-terminal domain separated by an unfolded central linker. Protease susceptibility is clustered within two N-terminal regions and one central region. These regions are evidently exposed as a result of the partial unfolding and/or separation of the N-terminal domain. Further evidence for the structure of this intermediate comes from gel filtration studies. Treatment of CK with 0.625 M GdnHCl resulted in slow aggregation at 37 degrees C, but not at 12 degrees C, a phenomenon previously reported for phosphoglycerate kinase. The aggregation did not occur at higher GdnHCl concentrations and was unaffected by a reducing agent. It is proposed that aggregation is a consequence of non-specific interactions between hydrophobic regions, possibly domain/domain interfaces, which become exposed in the intermediate.
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PMID:Protease digestion studies of an equilibrium intermediate in the unfolding of creatine kinase. 900 4

Though some mechanisms of photoreception have been well characterized, others remain obscure. Presumably, most, if not all, of the major players in photoreceptor-specific functions are present in large amounts in the photoreceptor layer, and a catalog of these proteins will prove a useful tool for vision researchers. As a first step toward a complete catalog of photoreceptor cells, we have developed a novel method for isolating the photoreceptor cell monolayer from bovine retina. Electron microscopic studies of both the photoreceptor layer and the residual retina from which the photoreceptor layer had been removed, indicate that the preparation contains the photoreceptor outer segments and the majority of the inner segments. Proteins were extracted from the isolated photoreceptor cell layer as well as the rest of the retina with isoelectric focusing lysis buffer, and the protein components were separated by two-dimensional gel electrophoresis. The obtained protein maps reveal several classes of proteins that appear to be expressed more abundantly or specifically in the photoreceptor layer than in the rest of the retina. Four of these protein spots were excised and in-gel digested with trypsin, and the digests were extracted with solvent. The mixture of peptides digested from each protein was analyzed by high performance liquid chromatography interfaced with electrospray ionization tandem quadrupole mass spectrometry or by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Some of the peptides were isolated and their sequences were determined by gas phase Edman degradation. RNA transcripts extracted from the photoreceptor layer or the whole retina were subjected to Northern blot analysis as well as to reverse transcriptase-polymerase chain reaction amplification of probes for the successful selection of cDNA clones. These data permit both the identification of virtually any protein detectable on a two-dimensional gel, and also enable the corresponding cDNA clone to be selected. We have validated this approach by identifying aspartate aminotransferase and creatine kinase from the populations of abundant photoreceptor layer proteins. Both aspartate aminotransferase and creatine kinase are of mitochondrial origin and are thought to play crucial roles in photoreceptor functions by producing glutamate and ATP, respectively. We also identified two photoreceptor layer specific proteins: an acidic and high molecular weight protein, interphotoreceptor retinoid-binding protein, and an acidic and small molecular weight protein, recoverin.The technique presented here will allow vision researchers to discover and identify the proteins that are expressed specifically or abundantly in the photoreceptor cell as well as the proteins that undergo post-translational modification or modulation in expression under a defined biological condition. With the use of this technology, we anticipate that a researcher who knows only the 2-D gel position of a protein of interest can identify the protein, isolate a cDNA clone, and move into molecular genetic studies. Moreover, this streamlined technology will enable one to assemble a catalog of photoreceptor proteins using a minute amount of materials in a short period of time. We believe that such a catalog will serve as a valuable resource for vision investigators and will accelerate the rate of research progress.
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PMID:Initiating ocular proteomics for cataloging bovine retinal proteins: microanalytical techniques permit the identification of proteins derived from a novel photoreceptor preparation. 1043 56

Protein S-thiolation, the formation of mixed disulphides of cysteine residues in proteins with low-molecular-mass thiols, occurs under conditions associated with oxidative stress and can lead to modification of protein function. In the present study, we examined the site of S-thiolation of the enzyme creatine kinase (CK), an important source of ATP in myocytes. Inactivation of this enzyme is thought to play a critical role in cardiac injury during oxidative stress, such as during reperfusion injury. Reaction of rabbit CK M isoenzyme with GSSG, used to model protein S-thiolation, was found to result in enzyme inactivation that could be reversed by GSH or dithiothreitol. Measurement of GSH that is released during the thiolation reaction indicated that the maximum extent of CK thiolation was approx. 1 mol of GSH/mol of protein, suggesting thiolation on one reactive cysteine residue. Accordingly, matrix-assisted laser-desorption ionization MS confirmed that the molecular mass of CK was increased, consistent with addition of one GSH molecule/molecule of CK. Using trypsin digestion, HPLC and MS analysis, the active-site cysteine residue (Cys(283)) was identified as the site of thiolation. Reversal of thiolation was shown to be rapid when GSH is abundant, rendering dethiolation of CK thermodynamically favoured within the cell. We conclude that S-glutathionylation of CK could be one mechanism to explain temporary reversible loss in activity of CK during ischaemic injury. The maintainance of GSH levels represents an important mechanism for regeneration of active CK from S-glutathionylated CK.
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PMID:Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue. 1076 88

Comparative studies of conformation and activity changes of a number of enzymes during denaturation have shown that inactivation generally precedes detectable global conformational changes. Kinetically, the inactivation rates of enzymes during denaturation are much faster than the rates of global conformational changes under identical conditions. It is suggested that the conformation of enzyme active sites are held together by weaker forces and consequently more flexible compared to the molecule as a whole. Conformational changes at the active sites were demonstrated directly by fluorescent and spin probes introduced at the active site of creatine kinase, glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A. In addition, the susceptibility of ribonuclease A to proteolysis is markedly increased in dilute GuHCl. By separation and N-terminal sequence analysis of the peptide fragments liberated by hydrolysis with trypsin or proteinase K, the cleavage points can be identified to show that without gross unfolding of the RNase molecule as a whole, loosening up of active site conformation has indeed occurred during inactivation in dilute GuHCl. For the role of active site flexibility in enzyme catalysis, it is possible that each intermediate step of the whole cycle of catalysis requires the enzyme molecule to be in a different conformation state. Active site flexibility would therefore be essential for the full expression of enzyme activity. It has recently been demonstrated that conformational change, especially that at the active site, accompanies enzyme catalysis and the activation of a number of enzymes involves the loosening up of the active site structure.
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PMID:[Conformational flexibility of enzyme active sites]. 1254 69

Heterogeneity of ADP diffusion and regulation of respiration were studied in permeabilized cardiomyocytes and cardiac fibers in situ and in silico. Regular arrangement of mitochondria in cells was altered by short-time treatment with trypsin and visualized by confocal microscopy. Manipulation of matrix volumes by changing K(+) and sucrose concentrations did not affect the affinity for ADP either in isolated heart mitochondria or in skinned fibers. Pyruvate kinase (PK)-phosphoenolpyruvate (PEP) were used to trap ADP generated in Ca,MgATPase reactions. Inhibition of respiration by PK-PEP increased 2-3 times after disorganization of regular mitochondrial arrangement in cells. ADP produced locally in the mitochondrial creatine kinase reaction was not accessible to PK-PEP in intact permeabilized fibers, but some part of it was released from mitochondria after short proteolysis due to increased permeability of outer mitochondrial membrane. In in silico studies we show by mathematical modeling that these results can be explained by heterogeneity of ADP diffusion due to its restrictions at the outer mitochondrial membrane and in close areas, which is changed after proteolysis. Localized restrictions and heterogeneity of ADP diffusion demonstrate the importance of mitochondrial functional complexes with sarcoplasmic reticulum and myofibrillar structures and creatine kinase in regulation of oxidative phosphorylation.
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PMID:Heterogeneity of ADP diffusion and regulation of respiration in cardiac cells. 1271 70

The time course and dose-response to proteolysis of three dimeric isozymes of creatine kinase, CK-MM (muscle), CK-BB (brain), and CK-MB (heart) and the homologous monomer, arginine kinase were compared. Chymotrypsin and trypsin cause a rapid and significant loss of intact CK-BB, but limited hydrolysis of CK-MM. After 1h of hydrolysis by chymotrypsin, 80% of CK-MM is intact as judged by quantification of monomers after electrophoresis in sodium dodecyl sulfate. While 50% of the intact monomers of CK-MB remain under these conditions, no CK-BB monomers are detected. These results indicate that treatment with chymotrypsin leads to a CK-MB devoid of the B-subunit. When treated with trypsin for 1h, CK-MM is totally resistant to hydrolysis and all CK-BB is highly degraded. However, CK-MB exhibits approximately 90% intact monomers, indicating survival of intact B-subunit in CK-MB. This suggests that heterodimerization of a B-subunit with an M-subunit may have a protective effect against hydrolysis by trypsin. In view of the considerably larger number of potentially tryptic sensitive sites on the muscle isozyme, the resistance of CK-MM and susceptibility of CK-BB dimers to trypsin implies that differences in subunit tertiary structure are a factor in proteolysis of the homodimeric isozymes. Arginine kinase is rapidly degraded by trypsin, but is minimally affected by chymotrypsin. The finding that both a monomeric (arginine kinase) and dimeric (CK-BB) phosphagen kinase are highly susceptible to proteolysis by trypsin indicates that quaternary structure is not, in and of itself, an advantage in resistance to proteolysis. Since both arginine kinase and muscle creatine kinase are resistant to chymotryptic hydrolysis, it seems unlikely that in general, the increased packing density, which may result from dimerization can account for the stability of CK-MM towards trypsin.
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PMID:Proteolytic susceptibility of creatine kinase isozymes and arginine kinase. 1282 Nov 44

Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and beta actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.
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PMID:Identification of mouse brain proteins associated with isoform 3 of metallothionein. 1580 40

Abnormalities of the anterior cingulate cortex have previously been described in schizophrenia, major depressive disorder and bipolar disorder. In this study 2-DE was performed followed by mass spectrometric sequencing to identify disease-specific protein changes within the anterior cingulate cortex in these psychiatric disorders. The 2-DE system comprised IPGs 4-7 and 6-9 in the first, IEF dimension and SDS-PAGE in the second dimension. Resultant protein spots were compared between control and disease groups. Statistical analysis indicated that 35 spots were differentially expressed in one or more groups. Proteins comprising 26 of these spots were identified by mass spectroscopy. These represented 19 distinct proteins; aconitate hydratase, malate dehydrogenase, fructose bisphosphate aldolase A, ATP synthase, succinyl CoA ketoacid transferase, carbonic anhydrase, alpha- and beta-tubulin, dihydropyrimidinase-related protein-1 and -2, neuronal protein 25, trypsin precursor, glutamate dehydrogenase, glutamine synthetase, sorcin, vacuolar ATPase, creatine kinase, albumin and guanine nucleotide binding protein beta subunit. All but three of these proteins have previously been associated with the major psychiatric disorders. These findings provide support for the view that cytoskeletal and mitochondrial dysfunction are important components of the neuropathology of the major psychiatric disorders.
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PMID:Proteomic analysis of the anterior cingulate cortex in the major psychiatric disorders: Evidence for disease-associated changes. 1663 10


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