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 activity of cytosolic calcium-dependent neutral protease (calpain) is commonly measured using casein as a substrate. A novel, modified caseinolysis assay is now developed. It involves incubation of calpain with substrate casein, followed by removal of an aliquot to which Coomassie brilliant blue G-250 dye reagent is added. The assay is based on the observation that the dye interacts only with protein but not the proteolytic products (small peptides and amino acids). Unlike the existing caseinolysis assay, this novel assay does not require separation of substrate from products and measurement is done in the visible range (595 nm). It is also more sensitive than existing colorimetric assays which use dye-conjugated protein substrates. The assay can also be used to measure the activity of other proteases such as trypsin and papain.
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PMID:A Coomassie brilliant blue G-250-based colorimetric assay for measuring activity of calpain and other proteases. 845 37

Adducin is a 200-kDa heterodimeric protein of the cortical cytoskeleton of mammalian erythrocytes. Analogs are also abundant in brain and several other tissues. In vitro, adducin bundles F-actin and enhances the binding of spectrin to actin. Previous studies have established that the beta subunit of adducin binds calmodulin (CaM) in a Ca(2+)-dependent fashion with intermediate affinity (approximately 200 nM) and that this activity is destroyed by proteolysis. We have confirmed the trypsin sensitivity of CaM binding by beta-adducin and the existence of a 38- to 39-kDa protease-resistant core. Calpain I digestion generates a larger core fragment (49 kDa) that is also devoid of CaM-binding activity. Use of recombinant beta-adducin peptides generated from partial cDNA clones identified strong CaM-binding activity within the protease-sensitive domain in residues 425-461: KQQKEKTRWLNTPNTYLRVNVADEVQRNMGSPRPKTT in single-letter amino acid codes. This region of the molecule is highly conserved between mouse, rat, and human and shares structural features with CaM-binding sequences in other proteins. Multiple flanking PEST sequences (sequences rich in proline, glutamic acid, serine, and threonine residues that enhance proteolytic sensitivity) may contribute to the protease sensitivity of this region. Consensus sequences for phosphorylation by cAMP-dependent kinases and by protein kinase C (or CaM-dependent kinase) are also found within or near this CaM-binding domain. Collectively, these data suggest a structural basis for the regulation of adducin by Ca(2+)-dependent CaM binding and possibly by covalent phosphorylation and calpain I-mediated proteolysis as well.
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PMID:Calmodulin-binding domain of recombinant erythrocyte beta-adducin. 847 88

The antitumor drug aclacinomycin A was previously shown to inhibit the degradation of ubiquitinated proteins in rabbit reticulocyte lysates with an IC50 of 52 microM (Isoe, T., Naito, M., Shirai, A., Hirai, R., and Tsuruo, T.(1992) Biochim. Biophys. Acta 1117, 131-135). We report here that from all the catalytic activities of the 20 S proteasome tested, the chymotrypsin-like activity was the only one affected by the antitumor drug. An important requirement for inhibition of the chymotrypsin-like activity seemed to be the presence of hydrophobic nonpolar residues in positions P1 to P3. Degradation of Z-E(OtBu)AL-pNA and Z-LLL-AMC at pH 7.5 was dramatically (87-98%) inhibited by 50 microM of the drug, while that of Z-GGL-pNA (containing uncharged polar residues in positions P2 and P3) and succinyl-LLVY-AMC (containing an uncharged polar residue in the P1 position) was inhibited only 11 and 24%, respectively. Aclacinomycin A had no effect on cathepsin B, stimulated trypsin, and inhibited chymotrypsin and, to a lesser extent, calpain. The aglycone and sugar moieties of the cytotoxic drug are essential for inhibition. The results presented here support a major role for the chymotrypsin-like activity in the degradation of ubiquitinated proteins. Aclacinomycin A is the first described non-peptidic inhibitor showing discrete selectivity for the chymotrypsin-like activity of the 20 S proteasome.
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PMID:The antitumor drug aclacinomycin A, which inhibits the degradation of ubiquitinated proteins, shows selectivity for the chymotrypsin-like activity of the bovine pituitary 20 S proteasome. 866 10

Active Ca2+ transport was measured in microsomal vesicles prepared from bovine retinae and was compared with that in disk membranes of the photoreceptor cells of the same retina. The 45Ca uptake was dependent on the presence of Mg(2+)-ATP and was inhibited by vanadate or when GTP substituted for ATP. The dependence of calcium uptake on the external free Ca2+ concentration gave a KM = 13 microM or a KM = 0.1 microM for disks and microsomal vesicles, respectively. A phosphorylated intermediate (E-P) of Ca(2+)-ATPase of about 100 kDa was isolated in microsomal vesicles. The E-P formation was strongly inhibited by thapsigargin and partially by 2,5-di-(-butyl)benzohydroquinone. Digestion of disks or microsomes with calpain had no effect on the phosphorylated intermediate, while digestion with trypsin produced two fragments of approximately 55 kDa and 35 kDa. These results suggest that bovine retinal microsomes contain a calcium pump belonging to the SERCA family.
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PMID:Endoplasmic reticulum Ca(2+)-ATPase in microsomal vesicles isolated from bovine retinae. 874 9

We previously cloned a cDNA encoding a protein tyrosine phosphatase (PTP) containing sequence homology to protein 4.1, designated PTPMEG. Recombinant protein and amino- and carboxyl-terminal peptides were used to obtain polyclonal antibodies against PTPMEG to identify endogenous PTPMEG in A172 cells and to show that the enzyme is primarily localized to the membrane and cytoskeletal fractions of these cells. We prepared recombinant protein in Sf9 and COS-7 cells to further characterize it. The protein was phosphorylated in both cell types on serine and threonine residues. The multiple sites of phosphorylation were all within the intermediate domain of the protein between amino acids 386 and 503. This region also contains two PEST sequences and two proline-rich motifs that may confer binding to Src homology 3 domains. The recombinant protein was cleaved by trypsin and calpain in this region and thereby activated 4-8-fold as assayed using Raytide as substrate. We immunoprecipitated the protein from human platelets with both amino- and carboxyl-terminal antipeptide antibodies to assess the state of the enzyme in these cells. The full-length molecule was found in extracts from unstimulated platelets, whereas extracts from both calcium ionophore- and thrombin-treated platelets contained proteolyzed and activated forms of the enzyme, indicating that proteolysis by calpain is evoked in response to thrombin. Prior incubation of platelets with calpeptin, an inhibitor of calpain, blocked the agonist-induced proteolysis.
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PMID:The properties of the protein tyrosine phosphatase PTPMEG. 891 Mar 69

Failure of inactivation is the typical response of voltage-gated Na+ channels to the cytosolic presence of proteolytic enzymes, protein reagents such as N-bromoacetamide (NBA) or iodate, and antibodies directed against the linker between domains III and IV of the alpha-subunit. The present patch clamp experiments with cardiac Na+ channels aimed to test the hypothesis that these interventions may provoke the occurrence of non-inactivating Na+ channels with distinct kinetic properties. A site-directed polyclonal antibody (anti-SLP2, target sequence 1481-1496 of the cardiac Na+ channel alpha-subunit) eliminated fast Na+ inactivation to induce burst activity which was accompanied by the occurrence of two open states. A deactivation process terminated channel activity during membrane depolarization proceeding with time constants of close to 40 ms (at -40 mV). NBA-modified and iodatemodified Na+ channels were kinetically indistinguishable from the anti-SLP2-modified type since they likewise deactivate and, thus, attain an only moderate Po of close to 20%. This is fundamentally different from the behaviour of enzymatically-modified Na+ channels: after cytosolic proteolysis with alpha-chymotrypsin, trypsin or pronase, mean Po during membrane depolarization amounted to approximately 40% because deactivation operated extremely slowly and less efficiently (time constants 100-200 ms at -40 mV, as a minimum) or was virtually non-operating. Invitro cleavage of the synthetic linker sequence 1481-1496 confirmed that this part of the alpha-subunit provides a substrate for these peptidases or reactants for NBA but cannot be chemically modified by iodate. This iodate resistance indicates that iodate-modified Na+ channels are based on a structural alteration of still another region which is also involved in Na+ inactivation, besides the linker between domains III and IV of the alpha-subunit. Endogenous peptidases such as calpain did not affect Na+ inactivation. This stresses the stochastic nature of a kinetic peculiarity of cardiac Na+ channels, mode-switching to a non-inactivating mode.
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PMID:Two types of modified cardiac Na+ channels after cytosolic interventions at the alpha-subunit capable of removing Na+ inactivation. 903 54

Two forms of Ca(2+)-pump were identified in bovine brain synaptic membranes as aspartylphosphate intermediates and were characterized. The 140 kDa and 97 kDa phosphoproteins were digested by calpain, producing two phosphorylated fragments, of M.W. 124 and 80 kDa respectively, not inhibited by thapsigargin, and displayed a trypsin digestion pattern with the formation of one phosphorylatable fragment of about 80 kDa. These results suggest that both pumps belong to the Plasma Membrane-type of Ca2+ ATPases, differing from the Sarco- or Endoplasmic Reticulum kind. A plasma membrane Ca(2+)-ATPase proteinaceous inhibitor with molecular weight between 6,000 and 10,000 Da was resolved from synaptic terminal cytosol, where it is enriched by fourfold with respect to frontal cortex brain cytosol. Such enrichment is already evident in the correspondent crude fractions. The presence of calcium pump and its proteinaceous inhibitor inside the synaptic terminals from bovine brain is discussed in terms of free calcium level regulation in neuron synaptoplasm.
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PMID:Ca(2+)-ATPase pump forms and an endogenous inhibitor in bovine brain synaptosomes. 905 65

The precursor of the non-A beta component of Alzheimer's disease amyloid (NACP) is a presynaptic protein whose function has been suspected to be tightly involved in neuronal biogenesis including synaptic regulations. NACP was suggested to seed the neuritic plaque formation in the presence of A beta during the development of Alzheimer's disease (AD). Recombinant NACP purified through heat treatment, DEAE-Sephacel anion-exchange, Sephacryl S-200 size-exclusion, and S-Sepharose cation-exchange chromatography steps appeared as a single band on SDS-PAGE with Mr of 19 kDa. Its N-terminal amino acid sequence clearly confirmed that the protein was NACP. Interestingly, however, the protein was split into a doublet on a nondenaturing (ND)-PAGE with equal intensities. The doublet was located slightly above a 45-kDa marker protein on a 12.5% ND-PAGE. In addition, the size of NACP was more carefully estimated as 53 kDa with high-performance gel-permeation chromatography using a TSK G3000sw size-exclusion column. Recently, Lansbury and his colleagues (Biochemistry 35, 13709-13715) have reported that NACP exists as an elongated "natively unfolded" structure which would make the protein more actively involved in protein-protein interactions and Kim (Mol. Cells 7, 78-83) has also shown that the natively unfolded protein is extremely sensitive to proteases. Here, we report that the structure of NACP could be altered by certain environmental factors. Aluminum, a suspected risk factor for AD, converged the doublet of NACP into a singlet with slightly lower mobility on ND-PAGE. Spectroscopic analysis employing uv absorption, intrinsic fluorescence, and circular dichroism indicated that NACP experienced the structural alterations in the presence of aluminum such as the secondary structure transition to generate about 33% alpha-helix. This altered structure of NACP became resistant to proteases such as trypsin, alpha-chymotrypsin, and calpain. Therefore, it is suggested that aluminum, which influences two pathologically critical processes in AD such as the protein turnover and the protein aggregation via the structural modifications, could participate in the disease.
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PMID:Aluminum-induced structural alterations of the precursor of the non-A beta component of Alzheimer's disease amyloid. 926 46

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situ transglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly less in situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTG in situ.
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PMID:Modulation of the in situ activity of tissue transglutaminase by calcium and GTP. 944 73

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the transamidation of specific polypeptide-bound glutamine residues, a reaction that is inhibited by GTP. There is also preliminary evidence that, in situ, calpain and GTP may regulate tTG indirectly by modulating its turnover by the calcium-activated protease calpain. In the present study, the in vitro and in situ proteolysis of tTG by calpain, and modulation of this process by GTP, was examined. tTG is an excellent substrate for calpain and is rapidly degraded. Previously it has been demonstrated that GTP binding protects tTG from degradation by trypsin. In a similar manner, guanosine-5'-O-(3-thiotriphosphate) protects tTG against proteolysis by calpain. Treatment of SH-SY5Y cells with 1 nM maitotoxin, which increases intracellular calcium levels, resulted in a significant increase in in situ TG activity, with only a slight decrease in tTG protein levels. In contrast, when GTP levels were depleted by pretreating the cells with tiazofurin, maitotoxin treatment resulted in an approximately 50% decrease in tTG protein levels, and a significant decrease in TG activity, compared with maitotoxin treatment alone. Addition of calpain inhibitors inhibited the degradation of tTG in response to the combined treatment of maitotoxin and tiazofurin and resulted in a significant increase in in situ TG activity. These studies indicate that tTG is an endogenous substrate of calpain and that GTP selectively inhibits the degradation of tTG by calpain.
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PMID:Tissue transglutaminase is an in situ substrate of calpain: regulation of activity. 964 71


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