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)

A high-affinity, low-capacity estradiol-binding molecule (RE) has been demonstrated in the basal zone trophoblast (BZT) of the pregnant rat. On day 11 of pregnancy (day 0 = first sperm-positive day) RE is present in BZT cytosol, where it has a ka of 1.2 X 10(6)M-1 sec-1, t1/2 = 12.7 min, at 20 C. The Kd, under similar conditions, consists of 2 components, 1.3 X 10(-4) sec-1, t1/2 = 90 min, and 5.9 X 10(-5) sec-1, t1/2 = 196 min. When one uses the faster component, the equilibrium constant, Kd, obtained from kd/ka is 1.1 X 10(-10)M, in close agreement with that obtained from Scatchard analysis of specific estradiol (E2) binding at 20 C. On day 11 there were approximately 12,000 sites/cell in BZT cytosol. Scatchard analysis of nuclear RE on day 11 indicated a Kd of 1.85 X 10(-10)M and approximately 21,000 sites/nucleus, but, in day 15 BZT, nuclear RE was undetectable. Neither cytosol nor nuclei prepared from placental labyrinthine zone (LZT) tissue (fetal placenta) showed evidence of high-affinity, low-capacity E2 binding. Sucrose density gradient analysis on 5-20% linear gradients showed the cytosol RE to be approximately 4S whether in high or low-salt conditions. When measured against binding by 3H-labeled estradiol (*E2), the cytosol BTZ RE was competed for strongly (80-90%) by estrone, estriol, diethylstilbestrol, and estradiol-17alpha at 200 times excess. Nafoxidine-HCl, also at 200X excess, competed to approximately 50%. Corticosterone, progesterone, testosterone, dehydroepiandrosterone, and pregnenolone did not compete. The hormone specificity of nuclear BZT RE was similar to that of the comparable cytosol RE with the exception that nafoxidine did not compete. This was probably due to differences in kinetics, nafoxidine requiring a longer time to reach equilibrium than the other estrogens. The size of the nuclear RE by sucrose density gradient analysis was approximately 2S by KCl extraction (which was inefficient) or 4S by trypsin extraction. We conclude that the BZT of the day-11 rat placenta contains an estrogen-binding molecule with many of the attributes of a true receptor.
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PMID:A high-affinity estrogen-binding protein in rat placental trophoblast. 18 65

Cultured thyroid cells accumulate the lipophilic cation triphenylmethylphosphonium, indicating that there is an electrical potential (interior negative) across the plasma membrane. Thyrotropin stimulates the uptake of the lipophilic cation 3-fold, and the proton conductor carbonylcyanide-m-chlorophenylhydrazone causes efflux of triphenylmethylphosphonium accumulated in the presence or absence of thyrotropin. The stimulatory effect of thyrotropin on triphenylmethylphosphonium accumulation is not mimicked by human chorionic gonadotropin, a glycoprotein hormone with a similar structure whose target organ is not the thyroid, and the effect is abolished if the thyrotropin-receptor activity of the cells is destroyed by treatment with trypsin. Analogous effects are observed with thyroid plasma membrane vesicles which are essentially devoid of mitochondrial and soluble enzyme activities. Triphenylmethylphosphonium uptake and stimulation by thyrotropin occurs when NaCl, KCl, or Tris.HCl concentration gradients are artifically imposed across the vesicle membrane ([salt](out) > [salt](in)). It seems likely, therefore, that triphenylmethylphosphonium uptake is driven by a chloride diffusion potential (interior negative) and that thyrotropin either increases the permeability of the membrane to anions or decreases its permeability to cations. Thyrotropin-stimulated triphenylmethylphosphonium uptake in the vesicle preparations reaches a quasi steady-state within 3 min; in contrast, thyrotropin-stimulated adenylate cyclase activity is negligible during this period of time, becomes measurable after about 4 min, and is optimal after 12-15 min. Thus, a primary mode of action of thyrotropin on the thyroid cell may be an alteration in the electrical potential across the plasma membrane. The relevance of this observation to the mechanism of action of other glycoprotein hormones, certain bacterial toxins, and interferon is discussed.
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PMID:Effects of thyrotropin on the thyroid cell membrane: hyperpolarization induced by hormone-receptor interaction. 19 88

Sequential extraction of bovine corneal homogenates with aqueous 0.154M NaCl, 0.5M NaCl and 3M guanidine HCl revealed the presence, in the two sodium chloride extracts, of trypsin inhibitory factors. Upon gel-filtration chromatography of the o.5M NaCl soluble corneal material on Sephadex G-75, two peaks with trypsin inhibitory activity were resolved. One peak was eluted in the void volume, whereas a second peak had mobility corresponding to a molecular weight fraction much lower than, and therefore distinct from, alpha 1-antitrypsin inhibitor. The possible implication of this inhibitory factor in the pathogenesis of corneal ulceration is briefly discussed.
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PMID:Demonstration of a protease inhibitor in the cornea. 22 6

Streptomyces griseus trypsin has been isolated from Pronase by ion-exchange chromatography on CM-Sephadex and SE-Sephadex. The isolated enzyme was homogeneous by the criteria tested except for a low degree of contamination by an enzyme with nontryptic activity. The latter could be partially resolved by chromatography on Bio-Rex 70. The molar absorbancy at 280 nm was found to be 3.96 times 10-4 M-1/cm and the E1cm1% was found to be 17.3. The molecular weight was 22,800 plus or minus 800. The enzyme was found to be stable at 0 degrees from pH 2 to 10. At 30 degrees the enzyme was maximally stable at pH 3-4 and significantly stabilized in the neutral and alkaline range by 15 mM Ca2+. Some evidence was obtained for a reversible denaturation of the enzyme at pH 12.0 and 2.0. The K-m for N-alpha-benzoyl-L-arginine ethyl ester at pH 8.0 in 20 mM CaCl2-0.1 M KCl-10 mM Tris-HCl buffer at 30 degrees was found to be 7.7 plus or minus 1.9 times 10-6 M and the esterase activity was observed to be dependent on an ionizing group with pK-a equals 5.85. In 2H2O this pKa was increased to 6.35 and the rate of hydrolysis dicreased threefold. The rate of hydrolysis was independent of pH between 8 and 10. The inhibition of the enzyme with L-1-chloro-3-tosylamido-4-phenyl-2-butanone was shown to be associated with the alkylation of its single histidine residue. This residue is present in a homologous amino acid sequence as the active-site histidine in trypsin and chymotrypsin. Optical rotatory dispersion and circular dichroism measurements over the pH range 5.3-10.5 indicated no significant conformational change until the pH was increased above 10.1. The observation that, under the conditions tested, acetylation and carbamylation of the NH2-terminal valine were incomplete is consistent with the view that this group is buried as an ion pair and only becomes available for deprotonation and reaction upon denaturation of the enzyme at pH values greater than 10.0.
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PMID:Enzymic and physicochemical properties of Streptomyces griseus trypsin. 23 80

Kunitz bovine trypsin inhibitor gave with alpha-chymotrypsin a stoichiometric complex stable at neutral pH. The complex has been characteristized by amino acid composition, molecular sieving and zone electrophoresis. Complete dissociation occurred at pH 4.0 as shown by gel filtration, alpha-Chymotrypsin was displaced from the complex by trypsin either in solution or by affinity chromatography on trypsin-Sepharos: alpha-chymotrypsin was recovered in the filtrate (yield about 100%) and the inhibitor was eluted from trypsin-Sepharose with 0.1 M HCl (yield: 83%). Lysine-15 of the inhibitor was shown to be involved in the interaction between alpha-chymotrypsin and the inhibitor. When the complex was maleylated, the maleylated chymotrypsin-bound inhibitor was displaced by affinity chromatography on trypsin-Sepharose. Teh recovered derivative was oxidized, subjected to tryptic hydrolysis and the products separated by peptide mapping and analyzed. The peptides were compared with those obtained with non-maleylated inhibitor and fully maleylated free inhibitor. In the fully maleylated inhibitor, the four lysyl residues of the molecule were blocked but in the maleylated chymotrypsin-bound inhibitor, Lys-15 was unmodified in contrast to Lys-26, Lys-41 and Lys-46; therefore Lys-15 is shielded by chymotrypsin in the complex. On the other hand, when inhibitor with a selectively reduced carboxamidomethylated Cys-14-Cys-38 dislufide bridge was allowed to react with chymotrypsin, cleavage occurred not only at Tyr-21, Tyr-35 and Phe-45 but also at Lys-15, cleavage not observed in the case of the fully oxidized inhibitor. This result shows that under particular conditions the bond Lys-15-Ala-16 can be the substrate for chymotrypsin and the side chain of Lys-15 can be inserted in the chymotrypsin specificity pocket. Apparently the contact area of inhibitor with chymotrypsin seems to be similar to that with trypsin [J. Chauvet and R. Acher (1967) J. Biol. Chem. 242, 4274-4275].
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PMID:The reactive sites of Kunitz bovine-trypsin inhibitor. Role of lysine-15 in the interaction with chymotrypsin. 23 47

A purified preparation of bovine thrombokinase (activated Factor X) loses the ability to hydrolyze TAME (p-toluenesulfonyl-L-arginine methyl ester) when it is incubated at 37 degrees in 0.25 M Tris. HCl buffer, pH 7.4 with lauroxypropyl biguanide, N1, N5-dimethyl, N1-lauroxypropyl biguanide, N1-p-chlorophenethyl, N5-phenethyl biguanide, or N1-methyl, N1-p-chlorobenzyl, N5-o,p-dichlorobenzyl biguanide. Activity is lost much more slowly when 0.15 M NaCl is also present. Lauroxypropyl biguanide is the most potent of the compounds tested, 0.22 mM causing thrombokinase to lose almost all of its activity in about 30 minutes at 37 degrees in pH 7.4 buffered saline. Topical bovine thrombin also loses activity when incubated with either of the lauroxypropyl biguanides but not with the diphenethyl or the dibenzyl compound. Instead, the latter biguanides accelerate thrombin's hydrolysis of TAME. The percent acceleration is not affected or only slightly decreased by the presence of 0.15 M NaCl or KCl, and it is also unaffected by incubating the enzyme with the compounds in buffered saline for 4 to 120 minutes. Purified bovine trypsin is stabilized by both lauroxypropyl and the diphenethyl biguanide when incubated at 37 degrees in pH 7.4 buffered saline for the 60 minute test period but neither compounds has any effect on its rate of hydrolysis of TAME. It is postulated that the enzymes first react rapidly and reversibly with all of the test biguanides and, depending upon the enzyme and the substrate, the rate of hydrolysis of the substrate is unaffected, accelerated or inhibited. The lauroxypropyl biguanides also undergo a second, slower reaction with both thrombokinase and thrombin that produces loss of enzymatic activity. The dibenzyl and diphenethyl biguanides also undergo this second slow reaction with thrombokinase but not with thrombin, and none of the biguanides undergo this second reaction with trypsin.
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PMID:The effects of biguanides on thrombokinase, thrombin and trypsin. 24 90

Although the hypothesis that vasopressin and its associated neurophysin are synthesized together in one macromolecular common precursor was put forward more than a decade ago, direct conformation of this hypothesis has been lacking. A [35S]cysteine-labeled putative precursor for vasopressin-related neurophysin (Mr 20,000, pI 6.1) has been isolated from the supraoptic nuclei of rats. This precursor was subjected to limited proteolysis with trypsin which produced a Mr 10,000 protein and peptide products. The former was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and its behavior in isoelectric focusing systems (pI 4.6-4.8). The tryptic peptides proved to be vasopressin-like because they: (i) were rich in cysteine, (ii) comigrated with vasopressin on gel filtration columns in 6 M guanidine HCl, (iii) bound to a neurophysin-Sepharose affinity column at pH 5.7, and (iv) were recognized by antibodies against vasopressin. These data on the Mr 20,000, pI 6.1 protein represent direct experimental evidence for a candidate for the common precursor of vasopressin and neurophysin. We propose that this common precursor be called "propressophysin."
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PMID:Trypsin liberates an arginine vasopressin-like peptide and neurophysin from a Mr 20,000 putative common precursor. 29 5

lac repressor can be dissected by trypsin into a homogenous tetrameric core (accounting for residues 60 to 347), carrying inducer binding activity, and the monomeric amino-terminal peptides ("headpieces") accounting for residues 1 to 59 and 1 to 51, respectively. This restriction of the action of trypsin on lac repressor is obtained in 1 M Tris-HCl (pH 7.5)-30% in glycerol at 25 degrees C since only the peptide bonds at lysine-59 and to a lesser extent after at arginine-51 are cleaved under these conditions. The headpieces can be purified by gel filtration. They have ordered secondary structure as revealed by circular dichroism studies. The monomeric headpieces show the relatively weak binding to nonoperator DNA but not the highly specific and strong binding to operator DNA typical for tetrameric lac repressor.
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PMID:Isolation of amino-terminal fragment of lactose repressor necessary for DNA binding. 32 Oct 12

Strains of types II and III group B streptococci do not appear to be uniformly susceptible to opsonization by antibody-containing human sera, as studied using both a chemiluminescence and a radiolabeled bacterial uptake technique. We could not demonstrate a correlation of serum-sensitive or resistant strains with capsular antigen quantities, although serum absorption studies with whole organisms and HCl, trichloroacetic acid, and saline extracts indicated that the antibody to type-specific capsular polysaccharide is important in opsonizing both serum-resistant and serum-sensitive strains. Since trypsin treatment produced significantly enhanced opsonization of serum-resistant and serum-sensitive strains, proteins present on some group B streptococci may be important antiphagocytic factors.
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PMID:Strain specificity of opsonins for group B streptococci types II and III. 37 17

Homogeneous fragments of exosporium were isolated and purified from mature spores of a highly sporogenic mutant derived from Clostridium botulinum type A strain 190L. The exosporium was composed of three lamellae and showed a hexagonal array when negatively stained. The hexagonal array of isolated exosporium was resistant to sodium dodecyl sulfate, urea, dithiothreitol, and proteolytic enzymes such as trypsin, pronase, and nagarse, except for pepsin. The hexagonal array was partially disintegrated with 5 M guanidine-HCl and almost completely disrupted with 8 M urea in combination with 1% mercaptoethanol under alkaline conditions. The purified exosporium fraction was composed mainly of protein (69.1%) and lipids (13.8%). A small amount of amino sugars (2.5%) was present, but neutral sugars could not be detected. The exosporium protein had a predominantly acidic amino acid composition accompanied by low levels of cystine, methionine, and histidine.
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PMID:Isolation and partial characterization of exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A. 38 51


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