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 binding of triiodothyronine by Rana catesbeiana tadpole tail fin, tail muscle, kidney, and liver cytosol was studied using dextran-coated charcoal to separate bound and free hormone. A metal ion dependency was suggested by the fact that EDTA decreased the binding of triiodothyronine 80 to 90% in tail fin and tail muscle cytosol. Inhibition of binding in kidney or liver was less, 40 to 50%. This inhibition could be restored by adding an excess of divalent cations with an order of potency of Mn2+ greater than Ca2+ congruent to Co2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. Other chelators, e.g. o-phenanthroline, 8-hydroxyquinoline, and ethylene glycol bis(beta-aminoethylether)-N,N'-tetraacetate also decreased the binding of triiodothyronine, whereas citrate, oxalate, imidazole, and glycine had no effect. The triiodothyronine binding capacity of tail fin cytosol was reduced by EDTA treatment and dialysis against buffer. Ca2+ in the 1 to 10 mM range and Mn2+ at 1 mM could restore the binding to normal levels. Higher Mn2+ increased binding 70% above normal or to Ca2+-restored levels. The triiodothyronine cytosol binding activity was nondialyzable, heat-labile. pH-dependent, pronase-digestible, but unaffected by incubation with trypsin, RNase, and DNase, suggesting that the cytosol binding sites are acidic proteins. Scatchard analysis of triiodothyronine binding by the cytosol of different tissues, revealed Kassoc of 7.1 x 10(6) M(-1), 11.6 x 10(6) M(-1), 3.6 X 10(6) M(-1), and 68.0 x 10(6) M(-1) for tail fin, tail muscle, kidney, and liver cytosol, respectively. The corresponding maximal binding capacities in picomoles per mg of crude cytosol protein in these four tissues were 10.4, 0.86, 1.3, and 0.04, respectively.
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PMID:Metal ion dependence of the binding of triiodothyronine by cytosol proteins of bullfrog tadpole tissues. 0 Mar 82

1. Guanylate cyclase of washed particles and plasma membranes showed S-shaped progress curves when titrated with either GTP or Mn2+ ions; similar results were obtained with Triton X-100-solubilized enzyme preparation from washed particles. Hill plots of these data revealed multiple metal-nucleotide and free-metal binding sites. 2. Guanylate cyclase of supernatant fractions displayed typical Michaelis-Menten properties when enzyme required excess of (free) Mn2+ (over GTP) for maximal activities; Ka (free Mn2+) was about 0.15-0.25 mM at subsaturating concentrations of GTP. 4 MnATP, MnADP, and MnGDP were found to increase the activities of both particulate and superantant enzyme, when MnGTP concentration was below saturation and free Mn2+ ion concentration was low (less than 100 muM); MnATP (50muM-1 mM) inhibited both these activities at high free Mn2+ concentration (1.5 mM) and inhibition of the particulate enzyme was greater than that of supernatant enzyme. 5. Ca2+ ions stimulated supernatant-enzyme activity; the stimulatory concentration of Ca2+ ions depended on the concentration of Mn2+ and GTP. 6. A modest stimulation of particulate guanylate cyclase by pyrophosphate (0.02-1 mM) was observed; the pyrophosphate effect appeared to be competitive with respect to GTP. At a higher concentration (2 mM), pyrophosphate produced a marked inhibition of particulate enzyme; the nature of inhibitory effect appeared complex. 7. Inorganic salts (e.g. NaCl, KCl, LiBr, NaF) produced inhibition of particulate enzyme; the degree of inhibition of Triton X-100-stimulated activity was less than that of unstimulated activity. 9. Treatment of sarcolemmal or microsomal membranes with either phospholipase C or trypsin decreased, whereas phospholipase A increased, the activity of guanylate cyclase.
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PMID:Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver. 1 Aug 91

A macromolecule which binds intrinsic factor saturated with vitamin B12 has been solubilized from the guinea-pig ileum by homogenization followed by mechanical disruption without organic solvents or detergents. This intrinsic factor 'receptor' was further purified by precipitation with 30% saturated ammonium sulphate, centrifugation at 105000 g, and filtration through Sephadex G-200. Failure to precipitate the receptor following centrifugation at 105000 g for 3 h and filtration of the receptor with the included volumes through Sepharose 4B and 6B was evidence that it was solubilized. The purification of the receptor was monitored by a radiometric assay where the intrinsic factor-[57Co]vitamin-B12 complex coupled to the solubilized receptor precipitated at 15% sodium sulphate while intrinsic factor-[57Co]B12 alone remained soluble at this salt concentration. This radioassay also permitted the in vitro study of the interaction of the solubilized receptor and intrinsic factor saturated with [57Co]B12. The receptor did not bind intrinsic factor-[57Co]B12 below pH 5 while binding was observed to pH 9.0. Binding was equivalent at 37 degrees C and 25 degrees C, but was markedly reduced at 4 degrees C and 56 degrees C and was destroyed at 100 degrees C. The receptor resisted 60 min of digestion by trypsin, chymotrypsin, pronase and subtilisin. After 180 min digestion, pronase and subtilisin inactivated 90% and 41% of the receptor respectively, whereas trypsin and chymotrypsin inactivated only 21% and 23%. Trisodium EDTA inhibited the binding of intrinsic factor-[57Co]B12 to the receptor and this inhibition could be reversed by the addition of excess Ca2+. Mg2+ and Mn2+ were less effective than Ca2+ for the activity of the receptor. Kinetic analysis of the reaction indicated a maximum velocity of 0.083 nmole IF bound B12/min with a Km of 1.36 x 10(-10) M. The solubilized receptor had a greater affinity for intrinsic factor bound to vitamin B12 than for intrinsic factor free of vitamin B12. The solubilization of this intrinsic factor receptor without chemicals suggests that it is not an integral component of the microvillus membranes hydrophobically bonded to the lipid matrix, but rather a peripheral protein weakly associated with the membrane by non-covalent interaction.
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PMID:Solubilization, partial purification and radioassay for the intrinsic factor receptor from the ileal mucosa. 1 Sep 57

We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 X g, 1-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with alpha-methyl-D-mannoside. The alpha-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. Peroxidase activity corresponds with a glycoprotein band. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: alpha-methyl-D-mannoside greater than D-mannose greater than alpha-methyl-D-glucoside greater than D-glucose greater than D-galactose. This order of specificity is typical of Con A-sugar interactions. Furthermore, inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding. Reactivation of the carbohydrate binding site by the addition of Ca2+ and Mn2+ to demetallized Con A-agarose restores thyroid peroxidase binding. These and other experiments suggest that htyroid peroxidase is, like several other peroxidases, a glycoprotein. In addition, the interaction between thyroid peroxidase and Con A-agarose may provide a new purification tool for thyroid peroxidase.
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PMID:Interaction of thyroid peroxidase with concanavalin A covalently coupled to agarose. 1 48

1. Rat skeletal muscle was homogenized in 0.05M-Tris/HCl, pH 8.5, containing 1M-KCl. Myofibrillar proteins were precipitated by addition of (NH4)2SO4 (33% saturation). 2. The alkaline proteolytic activity that was precipitated with the myofibrillar proteins was solubilized with trypsin (conjugated to Sepharose) and further purified by affinity chromatography, ion-exchange chromatography and gel filtration. 3. The purified enzyme migrates as a single band in polyacrylamide-disc electrophoresis, and has optimum hydrolytic activity with azocasein and [14C]haemoglobin as substrates at pH 9.4 and 9.6 respectively. Its apparent molecular weight, as determined by gel filtration on Sephadex G-75, is 30800. 4. The purified alkaline proteinase is strongly inhibited by equimolar amounts of soya-bean trypsin inhibitor and ovomucoid, whereas di-isopropyl phosphorofluoidate and alpha-toluenesulphonyl fluoride have no effect. On the other hand N-ethylmaleimide and p-chloromercuribenzoate have inhibitory effects on the enzyme activity. 5. Bivalent metal ions (Fe2+, Co2+, Zn2+, Mg2+, Mn2+) diminish the proteolytic activity, at 1mM concentrations. Ca2+ ions and the metal-ion-chelating agent EDTA are without effect on enzyme activity. 6. The enzyme is part of the alkaline proteolytic activity that appears to be associated with myofibrillar proteins.
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PMID:Purification and some properties of an alkaline proteinase from rat skeletal muscle. 2 72

Large amounts of injected radiolabeled low density lipoproteins have been found by others to accumulate primarily in the liver and studies in various types of isolated cells, including hepatocytes, have indicated the presence of specific cell membrane recognition sites for lipoproteins. In the present studies, the high affinity binding of radiolabeled low density lipoproteins ([125I]LDL, d 1.020--1.063 g/mL) was measured in the major subcellular fractions of porcine liver homogenates. The nuclear and mitochondrial fractions were 1.9- and 1.4-fold enriched in binding activity with respect to unfractionated homogenates and contained 15% and 12% of the total binding activity, respectively. The microsomes, which contained most of the plasma membranes and endoplasmic reticulum, were approximately 4-fold enriched in binding and contained 73% of the binding activity. Microsomal subfractions obtained by differential homogenization and centrifugation procedures were 5.6--7.0-fold enriched in LDL binding and contained 54--58% of the homogenate binding activity. They were separated by discontinuous sucrose density gradient centrifugation into fractions which contained "light" and "heavy" plasma membranes and endoplasmic reticulum. The heavy membrane fraction was 2--4 fold in binding with respect to the parent microsomes (16--22 fold with respect to the homogenate). There was no enrichment of binding activity in the other two fractions. Two plasma membrane "marker" enzymes, nucleotide pyrophosphatase and 5'-nucleotidase, were also followed. Of the two, binding in the sucrose density gradient subfractions most closely followed nucleotide pyrophosphatase, which was also most highly enriched (3.2--3.3-fold) in the heavy membrane fraction, but did not follow it exactly. The enzyme was 2-fold richer in the light membranes than in the parent microsomes, though the light membrane binding activity was only 0.4--1.4 times that of the parent microsomes. High affinity binding was time and temperature dependent, saturable, and inhibited by unlabeled low density lipoproteins but not by unrelated proteins. Binding was stimulated 2--3 fold Ca2+, was not affected by treatment with Pronase or trypsin and was inhibited by low concentrations of phospholipids and high density lipoproteins (HDL). Heparin-Mn2+ treatment of HDL did not affect its ability to inhibit [125I] LDL binding. The LDL recognition site was distinct from the liver membrane asialoglycoprotein receptor; LDL binding was not inhibited by desialidated fetuin. We conclude that porcine liver contains a high affinity binding site that recognizes features common to both pig low density and high density lipoproteins. Further studies may elucidate the significance of this binding site in lipoprotein metabolism.
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PMID:Isolation of a porcine liver plasma membrane fraction that binds low density lipoproteins. 8 56

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg2+ dependent; Co2+ and Mn2+ but not Ca2+ could replace Mg2+ to some extent; the activation by Mg2+ was slightly antagonized by Ca2+. Even in the presence of Mg2+, Na+ or K+ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]0.5 v = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg2+. Solubilization, however, led to instability of the enzyme. The clostridial solubilized and membrane-bound ATPase showed different properties similar to the "allotopic" properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10(-4) M, led to 80% inhibition of the membrane-bound enzyme; oligomycin ouabain, or NaN3 had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment. Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H+ translocation. A H+-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prolaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H+-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.
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PMID:Properties and function of clostridial membrane ATPase. 13 64

The 20K dalton fragment of Ca2+ + Mg2+-ATPase obtained from th tryptically digested sarcoplasmic reticulum has been further purified using Bio-Gel P-100. This removed low-molecular-weight UV-absorbing and positive Lowry-reacting contaminants. The ionophoric activity of the 20K fragment in both oxidized cholesterol and phosphatidylcholine:cholesterol membranes is unaltered by this further purification. The 20K selectivity sequence in phosphatidylcholine:cholesterol membrane is Ba2+ greater than Ca2+ greater than Sr2+ greater than Mn2+ Mg2+. Digestion of intact sarcoplasmic reticulum vesicles with trypsin, which results in the dissection of the hydrolytic site (30K) from the ionophoric site (20K), is shown to disrupt energy transduction between ATP hydrolysis and calcium transport. This further implicates the 20K dalton fragment as a calcium transport site. These data and previous evidence are discussed in terms of a proposed model for the ATPase molecular structure and the mechanisms of cation transport in sarcoplasmic reticulum.
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PMID:Active calcium treatment transport via coupling between the enzymatic and the ionophoric sites of Ca2+ + Mg2+-ATPase. 14 15

The rapid formation of adhesions in suspension by lightly trypsinized BHK21 cells is not dependent on protein synthesis, and only in part on cellular metabolism, although it is completely inhibited by heat- and aldehyde-fixation of the cells. A requirement for protein synthesis becomes evident only if cells are exposed to high levels of trypsin for long periods. Formation of adhesions does not require addition to the medium of divalent cations, although it is increased by divalent manganese and cobalt ions. It is promoted by cytochalasin B and by cyclic AMP and is not inhibited by p-mercuriphenylsulphonate. We discuss a possible relationship between aggregation and the formation of gap junctions.
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PMID:Trypsinized BHK21 cells aggregate in the presence of metabolic inhibitors and in the absence of divalent cations. 17 27

Studies have been conducted to characterize further the interaction between 125I-labeled bovine thyrotropin (TSH) and bovine thyroid plasma membranes. Sequential subcellular fractionation of thyroid homogenates yielded preparations of progressively greater specific binding activity, highest activity being found in fractions previously shown to contain predominately plasma membranes (Amir, S. M., Carraway, T.F., Kohn, L.D., and Winand, R.J. (1973) J. Biol. Chem. 248, 4092-4100). Although binding of 125I-TSH by plasma membranes was greatest at pH 6.0, studies were conducted at pH 7.45 as well as pH 6.0, and results obtained differed quantitatively, but not qualitatively. Binding was maximal at 0 degrees, 15 degrees, and 22 degrees and steady state values remained unchanged for at least 22 hours. At 37 degrees, binding was decreased by 40% at 1 hour; the loss was even greater (65%) at 50 degrees. A similar loss of binding was evident when membranes were preincubated without TSH at 37 degrees or higher and were then incubated with 125I-TSH at 0 degrees. Lineweaver-Burk analysis indicated that preincubation resulted in loss of receptor sites without change in affinity of residual receptors. Addition of Ca2+ (1 to 10 mM) to the preincubation medium prevented the effect of preincubation at 37 degrees by preserving the number of receptor sites without altering their affinity. Under similar conditions, Na+ and K+ were without protective effect. Membranes bound 45Ca2+ in a specific and saturable manner. Scatchard plots indicated a dissociatiion constant (Kd) of 9 X 10(-5) M and a capacity (n) of 54 nmol/mg of membrane protein. 45Ca2+ was also displaced from membranes by Mg2+ and Mn2+. Ca2+ had a biphasic effect on binding; low concentrations (1 to 10 muM) added to the incubation mixture stimulated binding, while higher concentrations (0.1 mM) caused inhibition. Mg2+ and Mn2+, at comparable concentrations, were also inhibitory, Na+ and K+ less so. In the case of Ca2+, both the stimulatory and inhibitory concentrations were lower than those required to achieve saturation of Ca2+-binding sites. Proteolytic enzymes (trypsin, alpha-chymotrypsin, and pronase) sharply reduced binding of 125I-TSH, owing to a decrease in receptor sites. Phospholipases A and C enhanced binding of TSH, while neuraminidase and beta-galactosidase were without measurable effect.
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PMID:Properties of the interaction between bovine thyrotropin and bovine thyroid plasma membranes. 18 81


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