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 glycoprotein thrombospondin is distributed between the extracellular matrix and the platelet-sequestered pool in the resting state and it undergoes redistribution upon platelet stimulation. It is believed to play a role in matrix structure and in coagulation. We have studied the structural domains of endothelial cell (EC) thrombospondin by use of the serine proteases thrombin, trypsin and chymotrypsin and have characterized the heparin-binding domains of this molecule. For this purpose we used purified thrombospondin synthesized and secreted by bovine aortic endothelial cells grown in the presence of radiolabeled methionine. We find that the susceptibility of EC thrombospondin to proteolysis is five-fold smaller than that of platelet thrombospondin. In the presence of 2 mM Ca ions the molecule is cleaved by 20 U/ml thrombin at a single locus, to yield fragments of 160 kDa and 35 kDa. Trypsin digestion for 5 min at room temperature at an enzyme-to-substrate ratio of 1:20 produces a stable fragment of 140 kDa but not the 30-kDa fragment observed in platelet thrombospondin. Chymotrypsin, under identical conditions to those used for trypsin, cleaves EC thrombospondin into four stable fragments of 160 kDa, 140 kDa, 27 kDa and 18 kDa. Chelation of Ca by EDTA increases susceptibility of the molecule to proteolysis. Under the conditions used a cryptic thrombin-cleavage site, not hitherto observed in platelet thrombospondin, was observed in EC thrombospondin. The location of this site is near a chymotrypsin-susceptible site, which has been observed in the long connecting arm, which is particularly Ca-stabilized. Heparin-binding capacity of EC thrombospondin was observed in at least two separate loci. Both thrombin and chymotrypsin produced small fragments (35 kDa and 27 kDa respectively) which bound to heparin with high affinity, and large fragments (160 kDa for thrombin and 140 kDa for chymotrypsin) which had low affinity. Chelation of Ca substantially decreased the low-affinity binding of the large fragments but not the high-affinity binding of the small fragments. Two-dimensional gel electrophoresis of the chymotryptic heparin-binding fragments shows that each molecule gave rise to a heterogeneous array of fragments of high molecular mass bound by disulfide bonds, indicating that there is a difference in the rate of cleavage between the three subunits of EC thrombospondin. Trypsin, despite its limited degradation, completely eliminated the heparin-binding capacity of both high and low-affinity loci, in contrast to platelet thrombospondin where the high affinity remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The structure of endothelial cell thrombospondin. Characterization of the heparin-binding domains. 282 10

The (K+ + H+)-ATPase from gastric mucosa has been treated by limited proteolytic digestion with trypsin to study the conformational states of the enzyme. The existence of a K+- and an ATP-form of the enzyme follows from the kinetics of inactivation and from the specific cleavage products. In the presence of K+ the 95 kDa chain is cleaved into two fragments of 56 and 42 kDa, whereas in the presence of ATP fragments of 67 and 35 kDa are formed. When Mg2+ is present during tryptic digestion cleavage products which are specific for both the ATP- and the K+-form of the enzyme are yielded. In analogy to ATP, Mg2+ is able to convert the enzyme from a K+-conformation to a more protected form. Moreover Mg2+ supports the protecting effect of ATP against tryptic inactivation. The K0.5 for ATP is lowered from 1.6 mM (no Mg2+) to 0.2 mM in the presence of 10 mM Mg2+. Mg2+, which in previous studies has been shown to induce a specific conformation, apparently induces a conformation different from the K+-form of the enzyme and has ATP-like effects on the enzyme. In addition it has been found that in the initial rapid phase of the digestion process the K+-ATPase activity is interrupted at a step which is very likely the interconversion of the phosphoenzyme forms E1P and E2P, since neither the K+-stimulated p-nitrophenylphosphatase activity nor the phosphorylation of the enzyme are inhibited in this phase. During the tryptic digestion in the presence of K+ there is a good correlation between the residual ATPase activity and the amount of the catalytic subunit left, suggesting that the latter is homogeneous. After tryptic digestion in the presence of K+, phosphorylation only occurs in the 42 kDa and not in the 56 kDa band. The same experiments in the presence of ATP yield only phosphorylation in the 67 kDa band and not in the 35 kDa band. A provisional model for the structure of the catalytic subunit is given.
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PMID:Conformational states of (K+ + H+)-ATPase studied using tryptic digestion as a tool. 282 83

Three polycation-stimulated (PCSH-, PCSM- and PCSL-) protein phosphatases are characterized by distinct specificities and regulatory properties. The properties of the catalytic subunits obtained from the 3 basic types of PCS phosphatases are apparently identical. The 35 kDa catalytic subunits are insensitive to inhibitor-1 and modulator protein and in contrast with the holoenzymes are less sensitive to stimulation by protamine, displaying a similar degree of stimulation and an identical concentration optimum; preincubation with polycations also results in a time-dependent deactivation. The phosphorylase phosphatase activity of the three catalytic subunits is stimulated to a similar extent by low but comparable concentrations of detergents, but not by metal ions. Upon limited proteolysis by trypsin the basal, but to a lesser extent the polycation-stimulated activity of the holoenzymes and the catalytic subunits is decreased.
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PMID:Characterization of the catalytic subunits of the different types of polycation-stimulated protein phosphatases. 282 2

The low molecular weight polypeptide required for energy-dependent proteolysis, ubiquitin, is rapidly inactivated by 100,000 X g supernatants of rabbit liver extracts. Ubiquitin inactivation results from limited proteolysis by an endogenous contaminating lysosomal thiol protease having trypsin-like specificity. Evidence for this includes a pH optimum of 5.0 for the first order constant of ubiquitin inactivation and observation that inactivation is inhibited by EDTA, o-phenanthroline, iodoacetamide, p-chloromercuribenzoic acid, phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethyl ketone, leupeptin, soybean trypsin inhibitor, and aprotinin. Metals stimulate but are not required for ubiquitin inactivation with the effect apparently mediated by a low molecular weight heat-labile component of crude extracts. When this heat-labile component is removed by gel exclusion chromatography a number of metals inhibit ubiquitin inactivation. In the presence of excess dithiothreitol, inhibition is relatively specific for Zn(II). Inhibition by Zn(II) is specifically overcome competitively by Cd(II) or by a concentration of ubiquitin in excess of Zn(II). The responsible cathepsin possesses a molecular mass of 35 kDa by gel exclusion chromatography and shows marked thermal lability at neutral pH but stability at acid pH. Proteolytic inactivation of ubiquitin results from limited cleavage of the carboxyl-terminal glycine dipeptide required for isopeptide bond formation and is supported by data on isoelectric point changes on subsequent digestion with carboxypeptidase B and by direct amino acid analysis. When the responsible cathepsin is inactivated, liver extracts display ATP,ubiquitin-dependent proteolysis that cannot be ascribed to contaminating erythrocytes. Thus the previous inability to demonstrate energy-dependent proteolysis in liver extracts is accounted for by the artifactual inactivation of ubiquitin.
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PMID:The inactivation of ubiquitin accounts for the inability to demonstrate ATP, ubiquitin-dependent proteolysis in liver extracts. 298 63

Glycogen synthase (labelled in sites-3) and glycogen phosphorylase from rabbit skeletal muscle were used as substrates to investigate the nature of the protein phosphatases that act on these proteins in the glycogen and microsomal fractions of rat liver. Under the assay conditions employed, glycogen synthase phosphatase and phosphorylase phosphatase activities in both subcellular fractions could be inhibited 80-90% by inhibitor-1 or inhibitor-2, and the concentrations required for half-maximal inhibition were similar. Glycogen synthase phosphatase and phosphorylase phosphatase activities coeluted from Sephadex G-100 as broad peaks, stretching from the void volume to an apparent molecular mass of about 50 kDa. Incubation with trypsin decreased the apparent molecular mass of both activities to about 35 kDa, and decreased their I50 for inhibitors-1 and -2 in an identical manner. After tryptic digestion, the I50 values for inhibitors-1 and -2 were very similar to those of the catalytic subunit of protein phosphatase-1 from rabbit skeletal muscle. The glycogen and microsomal fractions of rat liver dephosphorylated the beta-subunit of phosphorylase kinase much faster than the alpha-subunit and dephosphorylation of the beta-subunit was prevented by the same concentrations of inhibitor-1 and inhibitor-2 that were required to inhibit the dephosphorylation of phosphorylase. The same experiments performed with the glycogen plus microsomal fraction from rabbit skeletal muscle revealed that the properties of glycogen synthase phosphatase and phosphorylase phosphatase were very similar to the corresponding activities in the hepatic glycogen fraction, except that the two activities coeluted as sharp peaks near the void volume of Sephadex G-100 (before tryptic digestion). Tryptic digestion of the hepatic glycogen and microsomal fractions increased phosphorylase phosphatase about threefold, but decreased glycogen synthase phosphatase activity. Similar results were obtained with the glycogen plus microsomal fraction from rabbit skeletal muscle or the glycogen-bound form of protein phosphatase-1 purified to homogeneity from the same tissue. Therefore the divergent effects of trypsin on glycogen synthase phosphatase and phosphorylase phosphatase activities are an intrinsic property of protein phosphatase-1. It is concluded that the major protein phosphatase in both the glycogen and microsomal fractions of rat liver is a form of protein phosphatase-1, and that this enzyme accounts for virtually all the glycogen synthase phosphatase and phosphorylase phosphatase activity associated with these subcellular fractions.
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PMID:The protein phosphatases involved in cellular regulation. Evidence that dephosphorylation of glycogen phosphorylase and glycogen synthase in the glycogen and microsomal fractions of rat liver are catalysed by the same enzyme: protein phosphatase-1. 300 40

Using flow dialysis, we found two classes of calcium-binding sites on tubulin: high-affinity binding sites (1.56 +/- 0.38 per tubulin dimer) with a dissociation constant of (4.86 +/- 0.12).10(-6) M and low-affinity binding sites (5.82 +/- 0.50 per tubulin dimer) with a dissociation constant of (6.4 +/- 0.4).10(-5) M. In the presence of 6.10(-5) M MgSO4, we found 0.64 +/- 0.18 calcium-binding sites per tubulin dimer with a dissociation constant of (4.7 +/- 0.5).10(-6) M and 1.2 +/- 0.2 sites per dimer with a dissociation constant of (3.5 +/- 0.4).10(-5) M. Under controlled conditions, trypsin and chymotrypsin selectively cleaved alpha- and beta-subunits, respectively, forming major fragments of 35 kDa and 20 kDa from the alpha-subunit, and major fragments of 31 kDa and 22 kDa from the beta-subunit. The high-affinity calcium-binding sites were detected in the carboxyl-terminal region of each tubulin subunit. Computer analysis of the subunit amino-acid sequences suggested possible locations of the putative calcium-binding sites.
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PMID:Calcium binding to tubulin. 320 16

We report here that, in culture, the expression of glial fibrillary acidic protein (GFAP) by astrocytes, as well as their shape (flat-polygonal vs. stellate) can be regulated by 4 serum antagonistic factors. Three of these factors are stimulatory, while the fourth exerts an inhibitory effect upon these astrocytic properties. As suggested by temperature and trypsin treatments, the inhibitory factor is a polypeptide or a protein of 15-35 kDa. The stimulatory factors are smaller: two of them have a mol. wt. between 0.2 and 5 kDa; the third is smaller than 0.2 kDa. Treatments with chloroform/methanol, ammonium sulfate, neuraminidase, and papain, indicate that at least one glycolipid and one glycoprotein are involved. We speculate that, during development, cells from the astrocytic line could be susceptible selectively to one or another of these factors, which would explain their great plasticity.
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PMID:Expression of glial fibrillary acidic protein by differentiated astrocytes is regulated by serum antagonistic factors. 321 58

Trypsin and chymotrypsin were used as probes of structure-divalent cation relationships in G-actin molecule. The pattern of fragments produced has been analyzed by sodium dodecyl sulfate gel electrophoresis. The tryptic product of G-actin, 33 kDa is a protease-resistant fragment in the presence of divalent cations. However, once divalent cations are eliminated from the solution during the digestion, the 33 kDa fragment starts to degrade into smaller peptides via a 30 kDa fragment. On the other hand the chymotryptic product of G-actin, 35 kDa (precursor of 33 kDa) is rather stable even in the absence of divalent cations. In addition it is observed that the presence of divalent cation is necessary for the degradation of G-actin to the 33 kDa fragment by trypsin. The ultra violet and intrinsic tryptophan fluorescence spectra of G-actin are changed after the elimination of divalent cations. These results suggest that the structure of G-actin molecule depends on the presence or absence of divalent cations, and that the divalent cation-dependency of G-actin structure is still conserved even after the tryptic digestion.
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PMID:Effect of divalent cation on the structure of skeletal muscle G-actin molecule. 335 76

The 'native' Mg-ATP-dependent protein phosphatase was isolated from rabbit skeletal muscle by a procedure that avoided the use of organic solvents or heating at 90-100 degrees C. The purified enzyme was composed of two major proteins (molecular mass 37 kDa and 31 kDa) that were present in a 1:1 molar ratio, and accounted for 70-80% of the material. The 37-kDa component comigrated with the catalytic subunit of protein phosphatase-1, and its identity with this protein was established by peptide mapping, and by its cleavage to the characteristic 34-kDa and 33-kDa fragments following incubation with chymotrypsin. The 31-kDa protein comigrated with inhibitor-2, and its identity with this protein was established by its heat stability, ability to inhibit protein phosphatase-1 at nanomolar concentrations, and its phosphorylation on a threonine residue by glycogen synthase kinase 3. It is therefore concluded that the 'native' Mg-ATP-dependent protein phosphatase is composed of the catalytic subunit of protein phosphatase-1 (37 kDa) and inhibitor-2 (31 kDa) in a 1:1 molar ratio. The 'native' Mg-ATP-dependent protein phosphatase had virtually identical properties to the enzyme reconstituted from inhibitor-2 and the 37-kDa catalytic subunit of protein phosphatase-1. Each preparation had a similar specific activity and was inhibited by identical concentrations of inhibitor-1. Both enzymes could be activated by incubation with glycogen synthase kinase-3 and Mg-ATP, or by Mn2+ and trypsin (or chymotrypsin). However, Mn2+ alone, or proteinase digestion in the absence of Mn2+, failed to activate either preparation. Incubation with glycogen synthase kinase-3 and Mg-ATP did not dissociate the 'native' or 'reconstituted' enzymes, whereas treatment with Mn2+ and trypsin decreased their apparent molecular masses from 70 kDa to 35 kDa. Incubation with chymotrypsin converted the 'native' and 'reconstituted' enzymes to forms that required preincubation with glycogen synthase kinase-3, Mg-ATP and inhibitor-2, in order to exhibit catalytic activity. The Mg-ATP-dependent protein phosphatase reconstituted from the 'nicked' 33-kDa catalytic subunit dissociated upon activation, in contrast to the enzyme reconstituted from the undegraded 37-kDa catalytic subunit. The results suggest that a 3-4-kDa fragment at one end of the polypeptide is involved in strengthening interaction between the undegraded 37-kDa catalytic subunit and the phosphorylated form of inhibitor-2.
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PMID:The protein phosphatases involved in cellular regulation. Comparison of native and reconstituted Mg-ATP-dependent protein phosphatases from rabbit skeletal muscle. 609 83

Flufenamate, non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte (I50 = 6 . 10(-7) M). The concentration dependence of the binding to ghosts reveals two saturable components. [14C]Flufenamate binds with high affinity (Kd1 = 1.2 . 10(-7) M) to 8.5 . 10(5) sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity (Kd2 = 10(-4) M) to a second set of sites (4.6 . 10(7) per cell). Pretreatment of cells with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [14C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport of [14C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [14C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [14C]flufenamate. Thus it appears that 35 kDa peptide is necessary is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.
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PMID:Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds. I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein. 704 2


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