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 myelin/oligodendrocyte glycoprotein (MOG) is found exclusively in the CNS, where it is localized on the surface of myelin and oligodendrocyte cytoplasmic membranes. The monoclonal antibody 8-18C5 identifies MOG. Several studies have shown that anti-MOG antibodies can induce demyelination, thus inferring an important role in myelin stability. In this study, we demonstrate that MOG consists of two polypeptides, with molecular masses of 26 and 28 kDa. This doublet becomes a single 25-kDa band after deglycosylation with trifluoromethanesulfonic acid or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that there are no or few O-linked sugars and that the doublet band represents differential glycosylation. Partial trypsin cleavage, which also gave a doublet band of lower molecular weight, confirmed this idea. MOG was purified by polyacrylamide gel electrophoresis, followed by electroelution. Three N-terminal sequences of eight to 26 amino acids were obtained. By western blot analysis, no binding was found between MOG and cerebellar soluble lectin. MOG does not seem to belong to the signal-transducing GTP-binding proteins. Reduced MOG concentrations were observed in jimpy and quaking dysmyelinating mutant mice, giving further support to its localization in compact myelin of the CNS.
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PMID:Purification and partial structural and functional characterization of mouse myelin/oligodendrocyte glycoprotein. 137 75

We have developed a reconstituted model system to study the interaction of the Golgi membranes isolated from rabbit liver with taxol-stabilized bovine-brain microtubules without microtubule-associated proteins (MAPs). The Golgi membranes are associated with microtubules. The sheets of vesicles and the membranous tubules are observed along microtubules by direct visualization using differential-interference-contrast, dark field, or fluorescence microscopy. The monoclonal antibody against Golgi membranes suggests that the Golgi membranes, but not the contaminating vesicles, are interacting with microtubules. The degree of association is assayed quantitatively using rhodamine-labeled microtubules after separation of the complex from unbound microtubules by centrifugation upon sucrose gradient. The association is inhibited by crude MAPs, purified MAP2, or 1.0 mM ATP. However, the association neither requires the cytosol from rat liver or bovine brain nor N-ethylmaleimide, brefeldin A, or GTP-gamma-S. The association is mediated by trypsin-sensitive peripheral protein(s) on the Golgi membranes.
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PMID:Interaction of the Golgi membranes isolated from rabbit liver with microtubules in vitro. 139 49

The nucleotide binding site of the uncoupling protein (UCP) from brown adipose tissue was mapped by photoaffinity labeling with 2-azidoadenosine 5'-triphosphate (2-azido-ATP) and by affinity labeling with 3'-O-(5-fluoro-2,4-dinitrophenyl)adenosine 5'-triphosphate (FDNP-ATP). Both analogs bind with high affinity and specificity to the UCP in intact mitochondria, as well as to the isolated solubilized protein. Reversible binding at 4 degrees C in the dark is competitively blocked by GTP. Like the natural ligands ATP and GTP, both analogs are capable of inhibiting the H+/OH- conductance of the UCP as measured in proteoliposomes with reconstituted UCP. 2-azido-ATP was incorporated into UCP in mitochondria in the presence of carboxyatractylate, while FDNP-ATP was inserted into isolated UCP by prolonged incubation at room temperature under pH variation. Both reactions can be blocked by GTP. The incorporation of 2-azido-ATP could be localized between residues 258 and 283 by cleavage with CNBr. Solid-phase sequencing of the homoserine-linked radioactive peptide indicated that the 2-azido-ATP was linked to threonine-263. The incorporation of FDNP-ATP could be assigned by cleavage with CNBr and alternatively with trypsin at a locus of covalent attachment between residues 238 and 255. On the basis of published data that no tyrosine participates in nucleotide binding of the UCP, the probable residue reacting with FDNP-ATP is cysteine-253.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Labeling of two different regions of the nucleotide binding site of the uncoupling protein from brown adipose tissue mitochondria with two ATP analogs. 142 Jan 70

Heterotrimeric Go bound to the membranes of bovine brain, but Go alpha remained bound to the membranes even after activation with GTP gamma S. Furthermore, Go alpha bound to a Triton X-100-insoluble fraction of the membranes in a saturable manner. However, the 37-kDa Go alpha eliminated by trypsin at the amino-terminus could not bind to the fraction. Using a blot overlay approach of the Triton-insoluble fraction, only a 20-kDa protein was identified that interacts with Go alpha. These results indicate that Go alpha binds to a 20-kDa Triton-insoluble protein in the bovine brain membranes.
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PMID:Interaction of alpha subunit of GTP-binding protein Go with a 20-kDa Triton-insoluble membrane protein in bovine brain. 145 1

Highly purified peroxisomal membranes stripped from their peripheral membrane proteins and only minimally contaminated with other membranes, contained three GTP-binding proteins of 29, 27 and 25 kDa, respectively. Bound radioactive GTP was displaced by unlabelled GTP, GTP analogs and GDP but not by GMP or other nucleotides. GTP binding was markedly decreased by trypsin treatment of intact purified peroxisomes; it increased 2-3-fold after pretreatment of the animals with a peroxisome proliferator. We conclude that the peroxisomal membrane contains small GTP-binding proteins that are exposed to the cytosol and that are firmly anchored in the membrane. We speculate that these proteins are involved in peroxisome multiplication by fission or budding during peroxisome biogenesis and proliferation.
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PMID:Presence of small GTP-binding proteins in the peroxisomal membrane. 150 80

Cell-substrate adhesion is crucial at various stages of development and for the maintenance of normal tissues. Little is known about the regulation of these adhesive interactions. To investigate the role of GTPases in the control of cell morphology and cell-substrate adhesion we have injected guanine nucleotide analogs into Xenopus XTC fibroblasts. Injection of GTP gamma S inhibited ruffling and increased spreading, suggesting an increase in adhesion. To further investigate this, we made use of GRGDSP, a peptide which inhibits binding of integrins to vitronectin and fibronectin. XTC fibroblasts injected with non-hydrolyzable analogs of GTP took much more time to round up than mock-injected cells in response to treatment with GRGDSP, while GDP beta S-injected cells rounded up in less time than controls. Injection with GTP gamma S did not inhibit cell rounding induced by trypsin however, showing that cell contractility is not significantly affected by the activation of GTPases. These data provide evidence for the existence of a GTPase which can control cell-substrate adhesion from the cytoplasm. Treatment of XTC fibroblasts with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate reduced cell spreading and accelerated cell rounding in response to GRGDSP, which is essentially opposite to the effect exerted by non-hydrolyzable GTP analogs. These results suggest the existence of at least two distinct pathways controlling cell-substrate adhesion in XTC fibroblasts, one depending on a GTPase and another one involving protein kinase C.
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PMID:A GTPase controls cell-substrate adhesion in Xenopus XTC fibroblasts. 151 94

The integrin VLA-4 (alpha 4: beta 1; CD49d/CD29) is involved in both cellular adhesion to extracellular matrix and cell-cell interactions. We have determined that the human gene coding for VLA-alpha 4 is located on the long arm of chromosome 2 by using fluorescence in situ hybridization. The VLA-alpha 4 gene has been more precisely mapped to the 2q31-q32 region after GTG banding (G-bands by trypsin using Giemsa). These data suggest that the VLA-4 gene belongs to the COL3A1-(ELN-FN)-COL6A3 linkage group and establishes a potential genetic relationship between the alpha 4 and alpha v integrin subunits.
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PMID:Mapping of the human VLA-alpha 4 gene to chromosome 2q31-q32. 153 88

Bovine liver glutamate dehydrogenase reacts with the bifunctional affinity label 5'-(p-(fluorosulfonyl)benzoyl)-8-azidoadenosine (5'-FSBAzA) in a two-step process: a dark reaction yielding about 0.5 mol of -SBAzA/mol of subunit by reaction through the fluorosulfonyl moiety, followed by photoactivation of the azido group whereby covalently bound -SBAzA becomes cross-linked to the enzyme [Dombrowski, K. E., & Colman, R. F. (1989) Arch. Biochem. Biophys. 275, 302-308]. We now report that the rate constant for the dark reaction is not reduced by ADP or GTP, but it is decreased 7-fold by 2 mM NADH and 40-fold by 2 mM NADH + 0.2 mM GTP, suggesting that 5'-FSBAzA reacts at the GTP-dependent NADH inhibitory site. The amino acid residues modified in each phase of the reaction have been identified. Modified enzyme was isolated after each reaction phase, carboxymethylated, and digested with trypsin, chymotrypsin, or thermolysin. The digests were fractionated by chromatography on a phenylboronate agarose column followed by HPLC. Gas-phase sequencing of the labeled peptides identified Tyr190 as the major amino acid which reacts with the fluorosulfonyl group; Lys143 was also modified but to a lesser extent. The predominant cross-link formed during photolysis is between modified Tyr190 and the peptide Leu475-Asp476-Leu477-Arg478, which is located near the C-terminus of the enzyme. Thus, 5'-FSBAzA is effective in identifying critical residues distant in the linear sequence, but close within the regulatory nucleotide site of glutamate dehydrogenase.
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PMID:Identification of amino acids modified by the bifunctional affinity label 5'-(p-(fluorosulfonyl)benzoyl)-8-azidoadenosine in the reduced coenzyme regulatory site of bovine liver glutamate dehydrogenase. 156 33

Eel liver glutamate dehydrogenase (GDH) [EC 1.4.1.3] was eightfold activated by trypsin and the molecular weight of the subunit of the native GDH decreased from 54,000 to 50,000. The C-terminal amino acid of both subunits was Thr. One peptide was released after proteolysis of the native GDH by trypsin and purified by anhydrotrypsin agarose and reversed-phase HPLC. The isolated peptide consisted of 39 amino acids and its amino acid sequence was as follows: H2NS-E-A-V-E-K-E-D-D-P-N-F-F-K-M-V-E-G-F-F-D-K-G-A-A-I- V-E-N-K-L-V-E-E-D-L-K-T-R-COOH. The peptide contained the N-terminal of the native GDH and its molecular weight was calculated to be 4,413. We concluded that the trypsin-catalyzed activation was caused by release of this peptide from the native GDH. p-Chloromercuribenzoic acid inhibited the activity of the trypsin-treated GDH, but stimulated that of the native GDH. The response of trypsin-treated GDH to ADP and GTP was decreased compared with that of the native GDH.
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PMID:The trypsin-catalyzed activation of glutamate dehydrogenase purified from eel liver. 163 63

Atrial natriuretic factor (ANF-R1) receptor is a 130-kDa protein that contains a cytoplasmic guanylate cyclase domain. We report that ATP interacts in an allosteric manner with the ANF-R1 receptor, resulting in reduced ANF binding and enhanced ANF-stimulated guanylate cyclase activity. The modulatory properties of various nucleotides indicate a preference for the adenine family with a rank order of potency of ATP greater than App(NH)p greater than or equal to ADP greater than or equal to AMP while cyclic and guanine nucleotides except GTP are inactive. The negative modulation by ATP of ANF binding is specific for the ANF-R1 receptor subtype since the amount of ANF bound by the guanylate cyclase uncoupled ANF-R2 subtype is increased in the presence of ATP. Furthermore, the effects of ATP on ANF-R1 receptor binding function are still observed with the affinity-purified ANF-R1 receptor, suggesting an allosteric binding site for ATP on the ANF-R1 receptor. In intact membranes, limited proteolysis of the ANF-R1 receptor with trypsin dose-dependently prevents the ATP-induced decrease in ANF binding concomitantly with the formation of a membrane-associated ANF-binding fragment of 70 kDa. These results confirm the direct modulatory role of ATP on hormone binding activity of ANF-R1 receptor and suggest that the nucleotide regulatory binding site is located in the intracellular domain vicinal to the protease-sensitive region.
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PMID:Allosteric modulation by ATP of the bovine adrenal natriuretic factor R1 receptor functions. 165 83


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