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)

The liver serves as a storage organ for iron, 2-5 mg of Fe being exchanged between plasma and liver daily in the human being. Such iron transfer from serotransferrin (TF) to hepatocytes involves a TF receptor. We are reporting some of the properties of this receptor using isolated rat hepatocyte membranes as the receptor source. Iron-saturated 125I-rat TF was used for these studies. Specific versus nonspecific TF binding was evaluated by labelling the membranes with 125I-TF and then displacing specific binding with excess unlabelled TF. A mean residency time of 18-20 min was calculated for the TF on each receptor molecule. There were 31,000 +/- 17,000 receptors/cell with a dissociation constant of 0.3 X 10(-7) mol/l. The binding obeyed simple Michaelis-Menten kinetics. Specifically bound iron-saturated 125I-TF could best be eluted with cold Fe-saturated TF; cold apo- and 50% saturated TFs were less effective. The binding of apo- and 50% saturated TF was much less pronounced than that of Fe-saturated TF. The membrane receptor was moderately heat stable, extractable with detergent, and was trypsin sensitive. Studies with 125I/59Fe-TF and whole cells suggested that Fe-TF is not in a major way internalized during iron uptake. It is concluded that there are TF receptors present on the rat hepatocyte plasma membrane that may have a role in uptake by the cells without internalization of the TF molecule.
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PMID:The behavior of transferrin receptors in rat hepatocyte plasma membranes. 609 79

Noninbred Sprague-Dawley rats were maintained on a choline-deficient diet containing 0.05% ethionine. After 10-13 weeks, livers were dispersed with collagenase, lysozyme, collagenase and hyaluronidase. Pronase, or a selected batch of trypsin. The highest yield of cells with histochemically demonstrable gamma-glutamyl transpeptidase (GGT) was obtained with trypsin. After velocity sedimentation in an isokinetic gradient of Ficoll in tissue culture medium, two modal populations of cells with histochemically demonstrable GGT were observed. The first mode contained cells that were morphologically different from hepatocytes and that may be oval cells. The second, more rapidly sedimenting modal population of cells with GGT was morphologically similar to hepatocytes as assessed with Wright's stain; the location of this population in the gradient was the same as the location of cells with the appearance of hepatocytes that lacked iron and that had decreased glucose 6-phosphatase. In multiple experiments, the purest fractions contained 71.7 +/- 3.5% cells (mean +/- SD) with the appearance of hepatocytes with histochemically demonstrable GGT.
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PMID:Separation of two populations of cells with gamma-glutamyl transpeptidase from carcinogen-treated rat liver. 611 83

Colloidal iron staining, calcium binding and enzyme activities were studied in the isolated rat heart sarcolemma. Colloidal iron staining of the sarcolemma revealed a high density of negatively charged sites associated with the cell surface. This membrane fraction was found to have calcium binding activity at both low (0.1 mM) and high (1.25 mM) concentrations of calcium. Pretreatment of the sarcolemma with either trypsin, phospholipase C or neuraminidase, was associated with a reduction in colloidal iron staining as well as decreased calcium-binding activity at high concentrations of calcium. Calcium binding at low concentrations was decreased by both trypsin and neuraminidase. Mg2+ ATPase, Ca2+ ATPase, and Na+-K+ ATPase activities were altered by neuraminidase and trypsin treatments, whereas phospholipase C treatment altered Na+-K+ ATPase only. It is concluded that both surface negative charge and calcium-binding sites associated with the isolated rat heart sarcolemma are contributed by a mosaic of biomolecules including proteins, phospholipids and glycoproteins, and alterations in the surface charge may influence the activities of membrane-bound enzymes.
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PMID:Negatively charged sites and calcium binding in the isolated rat heart sarcolemma. 616 50

Iron-starved meningococci grown at either pH 7.2 or 6.6 were capable of removing and incorporating iron from human transferrin by a saturable, cell surface mechanism that specifically recognized transferrin rather than iron. The maximum expression of the iron uptake system occurred after 4 h of iron starvation. The uptake of the iron was dependent upon a functioning electron transport chain and was sensitive to 60 degrees C and trypsin. Cells grown under iron-sufficient conditions were incapable of accumulating iron from transferrin. No evidence was found for a primary role for cell-free soluble siderophores in the removal of iron from transferrin. The nonpathogenic neisseriae, Neisseria flava and N. sicca, were unable to utilize iron on transferrin.
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PMID:Expression of a high-affinity mechanism for acquisition of transferrin iron by Neisseria meningitidis. 621 Jun 35

The alpha-oxidation of [1-14C]phytanic acid of high specific activity was studied in postnuclear and various subcellular fractions from rat liver. alpha-Oxidation in the postnuclear fraction required ATP, Mg2+, nicotinamide, and molecular oxygen for activity. alpha-Oxidation was inhibited by iron-specific chelating agents and respiratory chain inhibitors. Partial inhibition by carbon monoxide indicated a possible involvement of cytochrome P-450. However, phenobarbital-treated rat liver postnuclear fraction did not stimulate phytanic acid alpha-oxidation above that of control. Subcellular fractionation indicated that in addition to the mitochondrial fraction, cytosol was required for activity. The cytosolic factor appeared to be dialyzable; it was inactivated by heat treatment, but not affected by trypsin digestion. NAD, CoA, ascorbic acid, and catalase did not replace cytosolic activity nor did the recently characterized heat-stable factors in brain hydroxylation, namely, adenosine nucleotides and glutamate.
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PMID:Phytanic acid alpha-oxidation in rat liver. Requirement of cytosolic factor. 623 33

Since leukocytes comprise a major portion of staphylococcal abscesses, the properties of the bactericidal material in abscess homogenates were compared with those of bactericidal systems associated with leukocyte lysosomes. The bactericidal material in abscess homogenates was distinguished from the myeloperoxidase system by its resistance to heat (100 degrees C, 30 min), lack of solubility in dilute acid (0.005 N HCl), resistance to strong acid (pH 1), and insensitivity to catalase. It was differentiated from the cationic proteins by its lack of solubility in dilute acid, insensitivity to iron (0.1 mM) or trypsin (5 mg/ml), and greater activity in solutions of increased ionic strength. These characteristics, together with its sensitivity to Ca2+ or albumin, suggested that the material might be lipid. Subsequent studies revealed that all the bactericidal activity resided in the lipid fraction recovered after extraction of abscess homogenates by the Dole procedure.
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PMID:Partial characterization of a bactericidal system in staphylococcal abscesses. 625 79

The rate of hydrolysis of the synthetic trypsin substrate N alpha-benzoyl-L-arginine-p-nitroanilide was determined from turnover rate curves, when the trace elements zinc, iron, cobalt, and nickel were added in the concentration range from 0.9 . 10(-7) to 0.9 . 10(-3) mol Men+/l. The enzyme substrate ratio was 1:50. An influence on the activity of trypsin, depending on the element used and on its concentration could be determined. Cobalt and iron accelerated the enzyme's capacity of hydrolysis at all concentrations used, whereas addition of higher but not toxic amounts of zinc or nickel resulted in an inhibition.
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PMID:[Influence of various trace elements on tryptic hydrolysis]. 626 Nov 95

Complex III was isolated and purified from bakers' yeast by ammonium sulfate fractionation and column chromatography on Ultrogel AcA 34. The purified complex contained 7.03 nmol/mg of protein and 4.24 nmol/mg of protein of cytochromes b and c1, respectively. The specific activity of the complex was 17.1 mumol/min/mg of protein, using the decyl analog of coenzyme Q as substrate. Electrophoresis of the purified complex revealed the presence of seven polypeptides with molecular weights ranging from 15,500 to 50,000. Polypeptides having molecular weights lower than 15,000 were not observed, except when the complex was dissociated in the absence of proteolytic inhibitors, suggesting that these low molecular weight species arise as a result of proteolytic digestion of the complex. The isoelectric points of the subunits of complex III and their stoichiometry wee determined. Trypsin and chymotrypsin digestion of the oxidized and reduced forms of the isolated complex suggested that the two high molecular weight core proteins are embedded within the complex and hence are inaccessible to the exogenous proteases, while cytochromes b and c1, the iron-sulfur protein, and the 17,500-dalton subunit are substantially exposed to the surface of the complex. The iron-sulfur protein appears to undergo a conformational change upon reduction of the complex, rendering it less susceptible to trypsin digestion. The core proteins and the iron-sulfur protein were purified, and antibodies against these proteins were raised. Immunoinhibition studies with these antibodies also indicated that the antigenic sites of the core proteins were embedded in the complex.
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PMID:Purification and polypeptide characterization of complex III from yeast mitochondria. 628 57

HeLa cells were found to have a single class of non-interacting receptors specific for transferrin. Both apotransferrin and diferric transferrin competed equally with 125I-diferric transferrin for receptor binding. Transferrin binding was temperature-dependent and reversible. Binding of transferrin to cells exhibited a KD of 27 nM with a maximum binding capacity of 1.8-3.7 x 10(6) molecules/cell. Cells grown in the presence of diferric transferrin or in the presence of ferric ammonium citrate exhibited a concentration- and time-dependent decrease in 125I-diferric transferrin binding. The decrease in binding activity reflected a reduction in receptor number rather than an alteration in ligand receptor affinity. Growth of cells in saturating concentrations of apotransferrin did not cause a decrease in receptor number. When iron-treated cells were removed to media free of ferric ammonium citrate, the receptor number returned to control values by 40 h. When receptors were removed with trypsin, cells grown and maintained in ferric ammonium citrate-supplemented media demonstrated a rate of receptor reappearance 47% that of control cells grown in ferric ammonium citrate-free media. Cells grown in media supplemented with diferric transferrin or ferric ammonium citrate exhibited an increase in cytosolic iron content. The transferrin receptor number returned to normal after cells were removed to unsupplemented media, despite persistent elevation of cytosolic iron content. Increased iron content did not appear to be the sole factor determining receptor number.
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PMID:Regulation of HeLa cell transferrin receptors. 628 49

The subcellular location of radiolabeled transferrin (125I-Tf), internalized during cellular iron uptake, and the cellular distribution of transferrin (Tf) receptors were studied in cultured HeLa cells. Cells were incubated at 37 degrees C with 125I-Tf(Fe)2. Forty per cent of the labeled ligand was associated with cell surface receptors. The remaining 60% was internalized as shown by the inability to dissociate 125I-Tf from cells by competition with excess Tf(Fe)2 or treatment of cells with 0.2 M acetic acid containing 0.5 M NaCl. Subcellular fractionation studies using sucrose density gradients indicated that internalized Tf was localized in a membranous vesicle distinct from lysosomes, Golgi apparatus, endoplasmic reticulum, or plasma membranes. The subcellular distribution of Tf receptors was studied using an assay for detergent solubilized receptors. Even without preincubation with ligand, the majority of cellular Tf receptors were localized intracellularly in a vesicle with the same buoyant density as the vesicle containing internalized 125I-Tf. Using an assay for occupied receptors, we demonstrated that the same vesicle contained both internal receptors and internalized ligand. A portion (20%) of the intracellular receptor pool was insensitive to trypsin treatment of whole cells at 37 degrees C suggesting that during the experimental time period (20-30 min) this portion did not recycle to the cell surface. We propose that during cellular iron uptake, Tf receptor-ligand complexes are internalized and directed to a nonlysosomal compartment where iron is released, followed by recycling to the cell surface of an intact Tf receptor-apo-Tf complex.
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PMID:Internalization and subcellular localization of transferrin and transferrin receptors in HeLa cells. 630 99


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