Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have recently developed a kinetically controlled, step-wise affinity cross-linking technique for specific, high-yield, covalent linkage of murine epidermal growth factor (mEGF) via its N terminus to the EGF receptor. EGF receptor from A431 cells was cross-linked to radiolabeled mEGF (125I-mEGF) by this technique and the 125I-mEGF-receptor complex was purified and denatured. Tryptic digestion of this preparation gave rise to a unique radiolabeled peptide that did not comigrate with trypsin-treated 125I-mEGF in SDS/Tricine gels but that could be immunoprecipitated with antibodies to mEGF. The immunoprecipitated peptide was isolated by electrophoresis in SDS/Tricine gels, eluted, and sequenced. The sequence was found to correspond to that of a tryptic peptide of the EGF receptor beginning with Gly-85, which is in domain I, a region N terminal to the first cysteine-rich region of the receptor. Selective loss of signal in the 17th sequencing cycle suggests that the point of attachment of N-terminally modified 125I-mEGF to the receptor is Tyr-101. The data presented here provide identification by direct protein microsequencing of a site of interaction of EGF and the EGF receptor.
 ...
PMID:Direct identification of residues of the epidermal growth factor receptor in close proximity to the amino terminus of bound epidermal growth factor. 138 Jan 67

Two antigenic sites recognized by neutralizing monoclonal antibodies (MAbs) directed against the fusion (F) glycoprotein of human respiratory syncytial virus were mapped on the primary structure of the protein by (i) the identification of amino acid substitutions selected in antibody-escape mutants and (ii) the reactivity of synthetic peptides with MAbs. The first site contained several overlapping epitopes which were located within the trypsin-resistant amino-terminal third of the large F1 subunit. Only one of these epitopes was faithfully reproduced by a short synthetic peptide; the others might require specific local conformations to react with MAbs. The second antigenic site was located in a trypsin-sensitive domain of the F1 subunit towards the carboxy-terminal end of the cysteine-rich region. One of these epitopes was reproduced by synthetic peptides. In addition, mutagenized F protein with a substitution of serine for arginine at position 429 did not bind MAbs to the second site. These results are discussed in terms of F protein structure and the mechanisms of virus neutralization.
 ...
PMID:Characterization of two antigenic sites recognized by neutralizing monoclonal antibodies directed against the fusion glycoprotein of human respiratory syncytial virus. 138 4

The topographic location of the region comprising amino acids 359-440 of the Neurospora crassa plasma membrane H(+)-ATPase has been elucidated using reconstituted proteoliposomes and protein chemical techniques. Proteoliposomes containing H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were cleaved with trypsin and the resulting digest was subjected to centrifugation on a glycerol step gradient to separate the released and liposome-bound peptides. The released peptides were recovered in the upper regions of the step gradient, whereas the liposome-bound peptides were recovered near the 40% glycerol interface. The released peptides present in the upper fractions were reduced, 14C-carboxy-methylated, and then separated by high performance liquid chromatography. Two radioactive cysteine-containing peptides with retention times of about 162 and 182 min were identified as H(+)-ATPase peptides comprising residues Leu363-Lys379 and Leu388-Arg414, respectively, by comparison to standards prepared from the purified ATPase. This information thus establishes a cytoplasmic location for residues 359-418 in the H(+)-ATPase polypeptide chain. It also infers a cytoplasmic location for residues 419-440, since this stretch of amino acids is too short to cross the membrane and return between regions known to be cytoplasmically located. These results and the results of other recent experiments establish the topographical location of nearly all of the 919 residues in the H(+)-ATPase molecule.
 ...
PMID:Cytoplasmic location of amino acids 359-440 of the Neurospora crassa plasma membrane H(+)-ATPase. 138 55

Isopentenyl-diphosphate:dimethylallyl-diphosphate isomerase (EC 5.3.3.2) catalyzes the 1,3-allylic rearrangement of the homoallylic substrate isopentenyl diphosphate (IPP) to its allylic isomer, dimethylallyl diphosphate (DMAPP). Incubation of yeast IPP isomerase with 3-methyl-3,4-epoxybutyl diphosphate (EIPP) resulted in a time-dependent first-order loss of activity characteristic of an active-site-directed irreversible process, where k2 = 0.63 +/- 0.10 min-1 and KI = 0.37 +/- 0.11 microM. A 1:1 covalent E-I complex was formed upon incubation with [1-14C]EIPP. The inhibited enzyme was treated with trypsin to give two radioactive fragments, which were purified by reversed-phase HPLC on a C18 column. The modified amino acid in each fragment was identified as C139 by sequencing the radiolabeled peptides. Incubation of IPP isomerase with [2,4,5-13C3]EIPP gave a 13C-labeled E-I complex. A 1H-13C heteronuclear multiquantum correlation spectrum had strong cross-peaks at 1.2/28 and 2.9/48 ppm, which we assigned to the labeled methyl group and C(4) methylene, respectively, of the inhibitor. In addition, a weak signal at 2.17/42 ppm may be from the C(2) methylene. Comparison of these chemical shifts with those of a synthetic adduct isolated from treatment of EIPP with cysteine indicates C139 attacks C(4) of EIPP to generate a thioether linkage between the enzyme and the inhibitor.
 ...
PMID:Isopentenyl-diphosphate isomerase: irreversible inhibition by 3-methyl-3,4-epoxybutyl diphosphate. 139 Jul 79

Caltrins, small basic proteins that inhibit calcium uptake by epididymal spermatozoa, have been purified from seminal vesicle content of the mouse and rat. Mouse caltrin (M(r) 8,476) contains 75 amino acid residues, 14 basic, 5 acidic, and 7 cysteines while rat caltrin (M(r) 6,217) has 56 residues, 10 basic, 5 acidic, and 6 cysteines; their pI values are 10.2 and 9.3, respectively. The proteins did not react with Ellman's reagent unless the cystine residues were previously reduced. The primary structures were determined by sequencing fragments generated by trypsin, clostripain, and endoproteinase Lys-C digestion. The sequences were ordered to give the total structural formula. The two molecules have no sequence similarity and are different from those of the bull and guinea pig previously reported. Only rat caltrin has a sequence of 13 residues nearly identical to that in guinea pig caltrin I. Both rat and mouse caltrin react with antibodies against bovine and guinea pig caltrins. Reduction and alkylation of cysteine residues suppressed the immunologic response of mouse caltrin; however, modified rat caltrin retained partially its immunoreactivity with the antiserum against guinea pig caltrin I. The same treatment abolished the calcium transport inhibitory activity of mouse caltrin and greatly reduced that of rat caltrin. It is likely that rat and mouse caltrins have the same physiological function as proposed for bovine caltrin; namely, to regulate the development of the Ca(2+)-dependent processes that "capacitate" sperm for fertilization.
 ...
PMID:Purification, structure, and characterization of caltrin proteins from seminal vesicle of the rat and mouse. 140 Apr 6

Heat-stable enterotoxins (STa) are small, cysteine-rich peptides secreted by Escherichia coli that are able to induce diarrhea through the stimulation of an intestine-specific receptor-guanylyl cyclase known as STaR. A 15-amino acid peptide, guanylin, was recently purified from rat jejunum and proposed to be a potential endogenous activator of this receptor. We describe here the cloning and characterization of human and mouse cDNAs encoding precursor proteins of 115 and 116 amino acids, respectively, having guanylin present at their C termini. Expression of the human cDNA in mammalian cells leads to the secretion of proguanylin, an inactive 94-amino acid protein. Guanylin generated by either trypsin or acid treatment of proguanylin was purified and found to bind to, and activate, STaR. Northern blot and in situ hybridization show high-level expression of guanylin mRNA restricted to the intestine, with localization to Paneth cells at the base of the small intestinal crypts. These results demonstrate that guanylin is an endogenous activator of STaR.
 ...
PMID:Precursor structure, expression, and tissue distribution of human guanylin. 140 6

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)
 ...
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

Microsomes from rat liver were used to investigate the mechanisms by which thiol compounds protect cellular membranes against damage from oxidants. Glutathione (GSH), dihydrolipoate and dithioerythritol, but not cysteine, ameliorated the loss of thiol groups of microsomal proteins attacked by Fe/ADP/NADPH or Fe/ADP/ascorbate prooxidant systems. The protection by GSH, but not dihydrolipoate or dithioerythritol, appeared to be enzymic since it was lost after microsomes were heated or treated with trypsin. The blocking of microsomal protein thiols with N-ethylmaleimide also diminished the protective effect of GSH. Lipid peroxidation, as assessed by chemiluminescence and vitamin-E loss, was inhibited in parallel with the protection of protein thiols. In microsomes lacking vitamin E, the protection of protein thiols by exogenous thiols was diminished. However, the GSH-dependent protection of vitamin E showed no preference for alpha-tocopherol over other tocopherol homologs. It is suggested that a GSH-dependent enzyme maintains protein thiols in the face of oxidative damage during microsomal peroxidation. A maintenance of protein thiols might not only protect important metabolic functions, but may also afford an antioxidant capacity to membranes, and account for one facet of the GSH-dependent inhibition of lipid peroxidation.
 ...
PMID:Protection by glutathione and other thiol compounds against the loss of protein thiols and tocopherol homologs during microsomal lipid peroxidation. 144 67

Cellular extracts of Tetrahymena thermophila were found to contain substantial levels of proteolytic activity. Protein digestion occurred over broad ranges of pH, ionic strength, and temperature and was stimulated by treatment with thiol reductants, EDTA and sodium dodecyl sulfate. Incubation at temperatures > or = 60 degrees C or with high concentrations of chaotropic reagents such as 10 M urea or 6 M guanidine-HCl caused an apparent irreversible loss of activity. Activity was also strongly diminished by increasing concentrations of divalent cations. Several peptide aldehydes, p-hydroxymercuribenzoate, and alkylating reagents such as iodoacetate, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, N-methylmaleimide, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane were potent inhibitors of proteolytic activity. Aprotinin diminished activity by approximately 40% while benzamidine, 3,4-dichlorosocoumarin, and trypsin inhibitors from soy bean, lima bean, and chicken egg caused relatively modest inhibition of proteolytic activity. Phenylmethanesulfonyl fluoride had no apparent effect. Electrophoretic separation of proteins on SDS-polyacrylamide gels copolymerized with gelatin substrate revealed that at least eight active proteolytic enzymes were present in cell extracts ranging in apparent molecular weight from 45,000 to 110,000. Five of these apparent proteases were detected in 70% ammonium sulfate precipitates. Gelatinase activity was not detectable when extracts were pretreated with iodoacetate or E-64, indicating that all of the enzymes observed in activity gels were sensitive to thiol alkylation. Cellular extracts of T. thermophila appeared to contain multiple forms of proteolytic enzymes which were stimulated by thiol reductants and inhibited by thiol modifying reagents. Accordingly, the proteolytic enzymes present in cell extracts appear to be predominantly cysteine proteinases.
 ...
PMID:An assessment of proteolytic enzymes in Tetrahymena thermophila. 145 53

The structure of the Neurospora crassa plasma membrane H(+)-ATPase has been investigated using a variety of chemical and physiochemical techniques. The transmembrane topography of the H(+)-ATPase has been elucidated by a direct, protein chemical approach. Reconstituted proteoliposomes containing purified H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin, and the numerous peptides released were purified by HPLC and subjected to amino acid sequence analysis. In this way, seventeen released peptides were unequivocally identified as located on the cytoplasmic side of the membrane, and numerous intervening segments could be inferred to be cytoplasmically located by virtue of the fact that they are too short to cross the membrane and return between sequences established to be cytoplasmically located. Additionally, three large membrane-embedded segments of the H(+)-ATPase were isolated using our recently developed methods for purifying hydrophobic peptides, and identified by amino acid sequence analysis. This information established the topographical location of virtually all of the 919 residues in the H(+)-ATPase molecule, allowing the formulation of a reasonably detailed model for the transmembrane topography of the H(+)-ATPase polypeptide chain. Separate studies of the cysteine chemistry of the H(+)-ATPase have demonstrated the existence of a single disulfide bridge in the molecule, linking the NH2- and COOH-terminal membrane-embedded domains. And, analyses of the circular dichroism and infrared spectra of the purified H(+)-ATPase have elucidated the secondary structure composition of the molecule. A first-generation model for the tertiary structure of the H(+)-ATPase based on this information and other considerations is presented.
 ...
PMID:Probing the structure of the Neurospora crassa plasma membrane H(+)-ATPase. 146 Dec 58


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>