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)

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.
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PMID:Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum. 250 29

Acylgalactosylceramide (AGC) synthesis was measured in vivo, and in a cell free system. 24 hours post-injection of [3H] palmitic acid into rat brain, more than 60% of the AGC radioactivity was associated with an ester linkage. Isolated rat myelin was incubated in the presence of [14C] palmitic acid, 2mM ATP, 50 microM CoA and 10 mM MgCl2 and acylation of myelin cerebrosides occurred at a linear rate for at least 60 min. Incubation of isolated myelin under standard conditions with [3H] cerebrosides and [14C] palmitic acid produced double labeled AGC. Labeling of AGC was maximum at pH 7.5 and 37 degrees C and appeared to be enzyme mediated inasmuch as it was reduced by myelin incubation with trypsin and drastically reduced by preheating the myelin for 5 min at 80 degrees C. Omission of ATP, CoA, MgCl2 or all three did not reduce fatty acid incorporation into AGC when compared to the values in the complete system. Addition of Triton X100 or Sodium Dodecyl Sulfate had little or no effect on the acylation of cerebrosides. Pulse chase experiments indicated that the reaction involved the net addition of fatty acid to the cerebrosides, rather than a rapid fatty acid exchange.
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PMID:Intramyelinic conversion of cerebrosides into acylgalactosylceramides. 262 89

An orbivirus of the Palyam serogroup was isolated from Culicoides oxystoma collected in a cowshed in Kagoshima, Southern Kyushu Island, Japan. This is the first isolation of an orbivirus of the Palyam serogroup in Japan. The virus was a spherical non-enveloped RNA virus, approximately 60 nm in diameter. The virus was resistant to ethyl ether, sodium deoxycholate and freezing-thawing, but readily inactivated by trypsin. The virus was not stabilized by 1 M MgCl2, was labile at pH 3.0 and was not precipitated by protamine sulfate. Indirect immunofluorescent staining of infected Vero cells indicated the virus to be antigenically related to D'Aguilar and Bunyip Creek viruses of the Palyam serogroup. Neutralization tests showed the virus to have no relationship with D'Aguilar virus, but to have a one-way cross-reaction with Bunyip Creek virus. The virus was tentatively designated as Kagoshima virus. A serological survey indicated dissemination of the virus in cattle populations in Kagoshima Prefecture.
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PMID:Isolation and preliminary characterization of an orbivirus of the Palyam serogroup from biting midge Culicoides oxystoma in Japan. 264 23

Inflamed gingiva contain a serine proteinase which could not previously be identified on the basis of its substrate specificity and inhibitor response. Using the substrate ZAlaArgArgAFC at alkaline pH, the enzyme was shown to be extracted more efficiently in high salt buffer. Inclusion of NaCl in assays, however, caused progressive reduction of activity. There was also inhibition by CaCl2, MgCl2 and 2 mM TosLysCH2Cl but not by 2 mM TosPheCH2Cl. Heparin produced significant activation. In gel filtrations with 1.0 M NaCl, activity appeared in fractions corresponding to a molecular weight of about 135,000. These properties are all consistent with tryptase from human mast cells. The enzyme may participate in both the connective tissue destruction and the inflammatory and immunological processes of gingivitis and periodontitis.
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PMID:Identification of a tryptase-like enzyme in extracts of inflamed human gingiva by effector and gel-filtration studies. 268 10

The labeling of chloroplast coupling factor 1 by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) was studied. When the enzyme was incubated with approximately 10 microM BzATP and 6 mM MgCl2 at pH 7.9 for approximately 20 min and passed through two Sephadex G-50 centrifuge columns, three BzATP molecules were bound per coupling factor molecule. Photolysis of radioactive enzyme-bound BzATP followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the BzATP bound primarily to the beta-polypeptide. If unbound BzATP was not removed by centrifuge columns prior to photolysis, significant labeling of the alpha-polypeptide also occurred. After photolysis, the BzATP-labeled enzyme was treated with trypsin, and two radioactive peptides were isolated by high-performance liquid chromatography on a C18 column. The two peptides were sequenced and found to correspond to amino acids 360-378 and 393-397 of the beta-polypeptide. For the sequence 360-378, two specific amino acids were found to be radioactive (Tyr-362 and Asp-369). This region of the polypeptide is highly conserved in several different species and probably corresponds to part of the nucleotide binding region of the catalytic site. In the case of amino acids 393-397, a very low level of radioactivity was found for all amino acids. The significance of this peptide for the binding of nucleotides to coupling factor 1 could not be established.
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PMID:Amino acid sequence of the nucleotide binding region of chloroplast coupling factor 1. 288 50

Digestion of the F1-ATPase of Escherichia coli with trypsin stimulated ATP hydrolytic activity and removed the delta and epsilon subunits of the enzyme. A species represented by the formula alpha 1(3) beta 1(3) gamma 1, where alpha 1, beta 1 and gamma 1 are forms of the native alpha, beta and gamma subunits which have been attacked by trypsin, was formed by trypsin digestion in the presence of ATP. In the presence of ATP and MgCl2, conversion of gamma to gamma 1 was retarded and the enzyme retained the epsilon subunit. These results imply that binding of ATP to the beta subunits alters the conformation of ECF1 to increase the accessibility of the gamma subunit to trypsin. The likely trypsin cleavage sites in the alpha, beta and gamma subunits are discussed. ECF1 from the alpha subunit-defective mutant uncA401, or after treatment with N,N'-dicyclohexylcarbodiimide or 4-chloro-7-nitrobenzofurazan, was present in a conformation in which the gamma subunit was readily accessible to trypsin and could not be protected by the presence of ATP and MgCl2. In a similar manner to native E. coli F1-ATPase, the hydrolytic activity of the trypsin-digested enzyme was stimulated by the detergent lauryldimethylamine N-oxide. Since the digested enzyme lacked the epsilon subunit, a putative inhibitor of hydrolytic activity, a mechanism for the stimulation which involves loss or movement of this subunit is untenable.
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PMID:Ligand-induced conformational changes in the Escherichia coli F1 adenosine triphosphatase probed by trypsin digestion. 289 Mar 77

A platelet membrane glycoprotein, 61 kDa, has been identified, which binds specifically to insoluble collagen. The detection of this protein was accomplished by incubating radiolabeled Triton-solubilized platelet supernatant with insoluble collagen, and, after washing the collagen pellet, extracting the 61-kDa glycoprotein from the pellet with sodium dodecyl sulfate buffer. The optimal conditions for specific binding were incubation of 120 micrograms of total platelet supernatant protein with 2 mg of collagen at 4 degrees C for 0.5 h in 0.5 ml of the incubating buffer (20 mM Tris, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, and 0.2% Triton, pH 7.4). The 61-kDa glycoprotein is cleaved by trypsin into a major peptide (44 kDa) and a smaller peptide(s) linked together by disulfide bonds in a molecule which still binds to collagen. When intact platelets are treated first with trypsin and then with dithiothreitol, the 44-kDa peptide is released and was shown to bind to collagen. We conclude that the 61-kDa glycoprotein is a platelet membrane protein which specifically interacts through its extracellular domain with insoluble collagen, and, thus, must be considered as a possible component of the initial platelet-matrix adhesion process which leads to platelet aggregation in vivo.
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PMID:Specific adsorption of a platelet membrane glycoprotein by human insoluble collagen. 294 18

The actin-dependent ATPase activity of myosin is retained in the separated heads (S1) which contain the NH2-terminal 95-kDa heavy chain fragment and one or two light chains. The S1 heavy chain can be degraded further by limited trypsin treatment into characteristic 25-, 50-, and 20-kDa peptides, in this order from the NH2-terminal end. The 20-kDa peptide contains an actin-binding site and SH1 and SH2, two thiols whose modification dramatically affects ATPase activity. By treating myosin filaments with trypsin at 4 degrees C in the presence of 2 mM MgCl2, we have now obtained preferential cleavage at the 50-20-kDa heavy chain site without any cleavage at the head-rod junction and hinge region in the rod. Incubation of these trypsinized filaments at 37 degrees C in the presence of MgATP released a new S1 fraction which lacked the COOH-terminal 20-kDa heavy chain peptide region. This fraction, termed S1'(75K), has more than 50% of the actin-activated Mg2+-ATPase activity of S1 and the characteristic Ca2+-ATPase and K+-EDTA ATPase activities of myosin. These results show that SH1 and SH2 are not essential for ATPase activity and that binding of actin to the 20-kDa region is not essential for the enhancement of the Mg2+-ATPase activity.
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PMID:A new, smaller actin-activatable myosin subfragment 1 which lacks the 20-kDa, SH1 and SH2 peptide. 295 48

The prevailing conformations of partially purified pig kidney (Na+ + K+)-ATPase interacting with ligands related to its phosphatase activity were determined following time-dependent trypsin digestion and inactivation as well as the amounts of Rb+ or Ca2+ bound to the enzyme after passage through cation-exchange resin columns. In the presence of 150 mM choline chloride, alone or with 3 mM MgCl2, 3 mM MnCl2 or 1 mM CaCl2, the major enzyme conformation was E1. Similar forms were seen with 5 mM p-nitrophenyl phosphate with and without 3 mM MgCl2. KCl, at 0.5 mM or 150 mM, produced an E2 enzyme state; the effects of 0.5 mM KCl were completely counteracted by 5 mM p-nitrophenyl phosphate. Under optimal conditions for phosphatase activity (3 mM MgCL2/5 mM p-nitrophenyl phosphate/10 mM KCl) the (Na+ + K+)-ATPase was in the E2 state. At low ionic strength and 20 degrees C and under 85% of maximal RbCl-stimulated phosphatase turnover (1 mM RbCl/3 mM MgCl2/5 mM p-nitrophenyl phosphate) no Rb+ occlusion could be detected. Ca2+, at low ionic strength and in the presence of 3 mM MgCl2, stimulated an ouabain-sensitive phosphatase activity. The rates of hydrolysis obtained wit 1 mM CaCl2 were similar to those seen with 0.5 mM KCl; under both conditions, similar patterns of trypsin digestion and inactivation of the enzyme were obtained. On the other hand, Ca2+ could not mimic Rb+ in its ability to induce an E2-occluding state. These results suggest that during phosphatase activity of (Na+ + K+)-ATPase, the most abundant form is a non-occluding E2 and that at least one of the mechanisms of potassium stimulation of that activity it to take the enzyme into the E2 state.
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PMID:Phosphatase activity of (Na+ + K+)-ATPase. Ligand interactions and related enzyme forms. 299 63

The hydrolysis of ATP catalyzed by purified (Na,K)-ATPase from pig kidney was more sensitive to Mg2+ inhibition when measured in the presence of saturating Na+ and K+ concentrations [(Na,K)-ATPase] than in the presence of Na+ alone, either at saturating [(Na,Na)-ATPase] or limiting [(Na,0)-ATPase] Na+ concentrations. This was observed at two extreme concentrations of ATP (3 mM where the low-affinity site is involved and 3 microM where only the catalytic site is relevant), although Mg2+ inhibition was higher at low ATP concentration. In the case of (Na,Na)-ATPase activity, inhibition was barely observed even at 10 mM free Mg2+ when ATP was 3 mM. When (Na,K)-ATPase activity was measured at different fixed K+ concentrations the apparent Ki for Mg2+ inhibition was lower at higher monovalent cation concentration. When K+ was replaced by its congeners (Rb+, NH+4, Li+), Mg2+ inhibition was more pronounced in those cases in which the dephosphorylating cation forms a tighter enzyme-cation complex after dephosphorylation. This effect was independent of the ATP concentration, although inhibition was more marked at lower ATP for all the dephosphorylating cations. The K0.5 for ATP activation at its low-affinity site, when measured in the presence of different dephosphorylating cations, increased following the sequence Rb+ greater than K+ greater than NH+4 greater than Li+ greater than none. The K0.5 values were lower with 0.05 mM than with 10 mM free Mg2+ but the order was not modified. The trypsin inactivation pattern of (Na,K)-ATPase indicated that Mg2+ kept the enzyme in an E1 state. Addition of K+ changed the inactivation into that observed with the E2 enzyme form. On the other hand, K+ kept the enzyme in an E2 state and addition of Mg2+ changed it to an E1 form. The K0.5 for KCl-induced E1-to-E2 transformation (observed by trypsin inactivation profile) in the presence of 3 mM MgCl2 was about 0.9 mM. These results concur with two mechanisms for free Mg2+ inhibition of (Na,K)-ATPase: "product" and dead-end. The first would result from Mg2+ interaction with the enzyme in the E2(K) occluded state whereas the second would be brought about by a Mg2+-enzyme complex with the enzyme in an E1 state.
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PMID:Effects of ATP and monovalent cations on Mg2+ inhibition of (Na,K)-ATPase. 300 46


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