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)

We have recently described a cell-free system that reconstitutes the vesicular transport of 300-kD mannose 6-phosphate receptors from late endosomes to the trans-Golgi network (TGN). We report here that the endosome----TGN transport reaction was significantly inhibited by low concentrations of the alkylating agent, N-ethylmaleimide (NEM). Addition of fresh cytosol to NEM-inactivated reaction mixtures restored transport to at least 80% of control levels. Restorative activity was only present in cytosol fractions, and was sensitive to trypsin treatment or incubation at 100 degrees C. A variety of criteria demonstrated that the restorative activity was distinct from NSF, an NEM-sensitive protein that facilitates the transport of proteins from the ER to the Golgi complex and between Golgi cisternae. Cytosol fractions immunodepleted of greater than or equal to 90% of NSF protein, or heated to 37 degrees C to inactivate greater than or equal to 93% of NSF activity, were fully able to restore transport to NEM-treated reaction mixtures. The majority of restorative activity sedimented as a uniform species of 50-100 kD upon glycerol gradient centrifugation. We have termed this activity ETF-1, for endosome----TGN transport factor-1. Kinetic experiments showed that ETF-1 acts at a very early stage in vesicular transport, which may reflect a role for this factor in the formation of nascent transport vesicles. GTP hydrolysis appears to be required throughout the transport reaction. The ability of GTP gamma S to inhibit endosome----TGN transport required the presence of donor, endosome membranes, and cytosol, which may reflect a role for guanine nucleotides in vesicle budding. Finally, ETF-1 appears to act before a step that is blocked by GTP gamma S, during the process by which proteins are transported from endosomes to the TGN in vitro.
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PMID:Identification of a novel, N-ethylmaleimide-sensitive cytosolic factor required for vesicular transport from endosomes to the trans-Golgi network in vitro. 199 60

High-resolution human chromosomes were obtained from lymphocytes after thymidine synchronization. The block was released either with thymidine to produce GTG (G-bands by trypsin using Giemsa) and RHG (R-bands by heating using Giemsa) banding or with BrdU (5-bromo-2'-deoxyuridine) for RBG (R-bands by BrdU using Giemsa) banding. RHG and RBG band patterns are only 75 to 85% congruent. The dissimilarities increase with the band number per genome and vary from one chromosome region to another. After high-resolution RBG banding, the BrdU-substituted bands show an unequal condensation delay, which can be, according to the bands involved, very important, minimal, or even absent. The bands showing the highest degree of condensation delay are the bands replicating the latest. The GTG- and RHG-band patterns show complementary matching for about 90% of the bands. It was found that two third of the chromosome surface appears positively stained after R-banding. This suggests that more DNA is replicated during early S-phase than during late S-phase. To obtain a fully developed RBG-band pattern in 90 to 95% of harvested mitoses, a period of 4.5 hours after the removal of the blocking agent is optimal. Such a brief release period also implies that late S-phase is much shorter than early S-phase.
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PMID:High-resolution R-banding at the 1250-band level. III. Comparative analysis of morphologic and dynamic R-band patterns (RHG and RBG). 207 51

Limited trypsinolysis was used to study conformational changes in elongation factors Tu and G. The trypsin cleavage rates of the factors differed and depended on both their interaction with ligands and the presence or absence of ribosomes. When the factors were bound to ribosomes, changes in their sensitivity to trypsin were observed depending on whether GDP or GTP was present in the complex, i.e. on the hydrolysis state of the guanine nucleotide ligand. The possible significance of factor structural changes for their functioning is discussed.
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PMID:Elongation factors Tu and G change their conformation on interaction with ribosomes. 210 75

Incubation of adenylosuccinate synthetase from Escherichia coli with low concentrations of pyridoxal 5'-phosphate (PLP) resulted in a rapid loss of activity (92%), concomitant with the formation of a Schiff base. The inactivation of the enzyme by PLP is apparently first order with respect to PLP. The pseudo-first order rate constant, Kapp, showed a hyperbolic dependence on the concentration of PLP, indicating that a kinetically significant PLP.enzyme intermediate is formed during the inactivation process. Stoichiometry and peptide isolation studies showed that 2 lysine residues were modified during reaction of the enzyme with PLP. The three substrates of adenylosuccinate synthetase (GTP, IMP, and aspartate) showed different effects in their ability to protect the enzyme against PLP inactivation. Complete protection of the enzyme against inactivation can be observed only in the presence of high concentrations of GTP. One lysine residue was protected under these conditions. In contrast to GTP, addition of the other two substrates either alone or together to reaction mixtures did not render protection. Peptide mapping by digesting the enzyme with trypsin revealed that the lysine shielded by GTP is Lys140. Replacing the Lys140 with Ile140 by site-directed mutagenesis resulted in total loss of the activity. These results suggest that Lys140 may play an important role in enzymatic activity.
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PMID:Chemical modification of adenylosuccinate synthetase from Escherichia coli by pyridoxal 5'-phosphate. Identification of an active site lysyl residue. 210 56

The biological activity of proteins encoded by the ras family of oncogenes is dependent on whether they are bound to GTP or GDP: the type of nucleotide bound is dependent on the rate of GTP hydrolysis (promoted by the GTPase-activating protein, GAP) and the rate of nucleotide exchange with cytosolic pools. A protein that stimulates the rate of exchange of guanine nucleotide on p21ras has been identified and characterized in cytoplasmic extracts of human placenta. The exchange-promoting protein runs on a gel filtration column with an apparent relative molecular weight of about 60,000. It is sensitive to heat and to trypsin. The exchange-promoting protein acts reversibly and does not cause degradation of p21ras. It is inactive towards the alpha subunit of a heterotrimeric GTP-binding protein (Go alpha) but acts on a large number of different mutant ras proteins, including transforming and effector mutants that are insensitive to the action of GAP. This protein, which we have termed REP (ras exchange-promoting), has the characteristics expected of a physiological activator of p21ras in cellular growth-signal-transduction pathways.
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PMID:Identification of a nucleotide exchange-promoting activity for p21ras. 211 14

A 23 kDa GTP-binding protein was purified from pig heart sarcolemma. This protein was not ADP-ribosylated by cholera, pertussis and botulinum C3 toxins. In pig heart sarcolemma pertussis toxin ADP-ribosylated 40 kDa subunit of Gi-protein, cholera toxin--45 kDa subunit of Gs-protein, botulinum C3 toxin ADP-ribosylated a group of proteins with Mr 22, 26 and 29 kDa. Antiserum generated against the peptide common for all alpha-subunits of G-proteins did not react with purified 23 kDa protein. Trypsin cleaved the 23 kDa protein in the presence of guanyl nucleotides into a 22 kDa fragment. Proteolysis of the 39 kDa alpha 0-subunit from bovine brain plasma membranes and ADP-ribosylated 40 kDa alpha i-subunit from pig heart sarcolemma in the presence of GTP gamma S yielded the 37 and 38 kDa fragments, respectively. In the presence of GTP and GDP the proteolysis of alpha 0 yielded the 24 and 15 kDa fragments, while the proteolysis of ADP-ribosylated alpha i-subunit yielded a labelled 16 kDa peptide. Irrespective of nucleotides trypsin cleaved the ADP-ribosylated 26 kDa substrate of botulinum C3 toxin into two labelled peptides with Mr 24 and 17 kDa. The data obtained indicate the existence in pig heart sarcolemma of a new 23 kDa GTP-binding protein with partial homology to the alpha-subunits of "classical" G-proteins.
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PMID:[Identification and purification of GTP-binding regulatory proteins from plasma membranes of swine heart]. 211 90

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu have been derived from the atomic coordinates of the trypsin-modified form of EF-Tu-GDP and by comparison with the ras p21 structures. The significance of the differences in the guanine nucleotide binding sites of EF-Tu and ras p21 are discussed. Crystallization of the EF-Tu-GMPPNP complex is reported.
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PMID:Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu. 211 11

We previously demonstrated that the chick oviduct estrogen receptor exists in three interconvertible forms. Two of these forms bind estradiol with high but distinct affinities. A third form exists as a non-estrogen binding recyclable form, Rnb, which upon treatment with ATP/Mg2+ is quantitatively converted to the lower affinity estradiol binding form. We now describe the isolation from chick oviduct cytosol of a factor involved in this conversion and its 1100-fold purification by ammonium sulfate fractionation, DEAE ion-exchange chromatography, and size-exclusion HPLC. The factor elutes from the size-exclusion column with an apparent molecular weight of 40,000. This highly purified factor potentiates estradiol binding in a dose-dependent manner in the presence of ATP/Mg2+. Its activity is destroyed by heating or by trypsin treatment but is relatively stable to freezing and thawing and is inert to treatment with reducing agents. ATP is an essential nucleotide substrate; GTP and cyclic nucleotides are inactive. Studies of cation dependence demonstrate that Mg2+ is also essential; Ca2+ alone is completely ineffective in catalyzing receptor potentiation and does not synergize with Mg2+. In the presence of excess ATP/Mg2+ and a fixed concentration of Fy, the Km for potentiation of estradiol binding is approximately 0.4 nM.
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PMID:Receptor interconversion model of hormone action. 1. Purification of a factor involved in conferring estradiol binding properties to the estrogen receptor. 214 Jun 95

The cyclic GMP phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is kept inactive in the dark by its gamma subunits and is activated following illumination by the GTP form of the alpha subunit of transducin (T alpha-GTP). Recent studies have shown that the stoichiometry of the inhibited holoenzyme is alpha beta gamma 2. T alpha-GTP and gamma act reciprocally. We have investigated the activation mechanism using fluorescein-labeled gamma subunit (gamma F) as a probe. gamma F containing a single covalently attached fluorescein was prepared by reaction of PDE with 5-(iodoacetamido)fluorescein and purification by reversed-phase high-pressure liquid chromatography (HPLC). gamma F, like native gamma, inhibits the catalytic activity of trypsin-activated PDE and transducin-activated PDE. Inhibition by gamma F was overcome by further addition of T alpha-GTP. gamma F binds very weakly to ROS membranes stripped of PDE and other peripheral membrane proteins. gamma F added to ROS membranes became incorporated into a component that could be extracted with a low ionic strength buffer. HPLC gel filtration showed that gamma F became part of the PDE holoenzyme. Incorporation occurred in less than 1 min in the presence of light and GTP, but much more slowly (t1/2 approximately 500 s) in the absence of GTP. This result indicates that transducin activates PDE by binding to the holoenzyme and accelerating the dissociation of gamma from the inhibitory sites. The binding of gamma F to trypsin-activated PDE alpha beta was monitored by steady-state emission anisotropy measurements and compared with PDE activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Activation mechanism of retinal rod cyclic GMP phosphodiesterase probed by fluorescein-labeled inhibitory subunit. 215 46

GTP-binding proteins were studied in synaptic vesicles prepared from bovine brain by differential centrifugation and separated further from plasma membranes using gel permeation chromatography. Following separation by SDS-PAGE of proteins from the different fractions, and transfer to nitrocellulose sheets, the presence and localization of low-molecular-mass GTP-binding proteins were assessed by [alpha-32 P]GTP binding. The vesicle-membrane fraction (SV) was enriched in synaptophysin (p38, a synaptic vesicle marker) and contained low-molecular-mass GTP-binding proteins; these consisted of a major 27 kDa protein and minor components (Mr 26 and 24 kDa) which were trypsin-sensitive and immunologically distinguishable from ras p21 protein. GTP-binding proteins of low molecular mass, but displaying less sensitivity to trypsin, were also found in the plasma membrane fraction (PM; enriched in Na+/K(+)-ATPase). In addition, the PM fraction contained GTP-binding proteins with higher Mr (Gi alpha and G0 alpha), together with another GTP-binding protein, ras p21. Putative function(s) of these GTP-binding proteins with low mass are discussed.
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PMID:Identification and localization of low-molecular-mass GTP-binding proteins associated with synaptic vesicles and other membranes. 216 11


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