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 surface anionic groups of Entamoeba invadens were analysed by cell electrophoresis, by ultrastructural cytochemistry, and by identification of sialic acids using paper and gas-liquid chromatography. Binding of colloidal iron hydroxide (CIH) and of cationized ferritin (CF) particles at pH 1.8 and 7.2, respectively, was observed on the cell surface. E. invadens has a highly negative surface charge (-0.96 microns s-1 V-1 cm). Treatment of the cells with trypsin and neuraminidase significantly reduced the electrophoretic mobility by 24% and 40%, respectively. Treatment of the amoebae with neuraminidase also markedly decreased the binding of CIH to the cell surface. This finding suggests that sialic acid residues are the major anionogenic groups exposed on the surface of E. invadens. Paper and gas-liquid chromatography showed that N-acetylneuraminic acid was the only derivative characterized in E. invadens.
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PMID:Sialic acid is a cell surface component of Entamoeba invadens trophozoites. 273 83

Adrenodoxin is an iron-sulfur protein which functions as a carrier of reducing equivalents in steroid hydroxylation reactions catalyzed by specific cytochromes P-450 in steroidogenic tissues such as adrenal cortex. Purified bovine adrenocortical adrenodoxin was shown to be selectively phosphorylated upon incubation with purified cAMP-dependent protein kinase, whereas other protein kinases were ineffective. The phosphorylation reaction was completed within 45 min at 30 degrees C and resulted in the optimal incorporation of 1 mol phosphate/mol adrenodoxin. Apoadrenodoxin, lacking the iron-sulfur cluster, was also phosphorylated under similar conditions. An apparent Km of 55 microM with a Vmax of 0.3 pmol 32P incorporated min-1 mg adrenodoxin-1 was calculated. Phosphorylation resulted in a striking change in several molecular properties of adrenodoxin, such as electrophoretic behavior and hydroxyapatite affinity, thus providing the possibility of clearly separating phosphorylated from unphosphorylated adrenodoxin. In addition, phosphoadrenodoxin became refractory to mild trypsin degradation, whereas this was not the case with apoadrenodoxin. The phosphorylated site of adrenodoxin was identified as a serine residue; study of peptide products resulting from CNBr and proteolytic cleavages of phosphoadrenodoxin suggested that Ser-88 was the target of the phosphorylation reaction. The influence of phosphorylation upon adrenodoxin activity was examined using cholesterol side-chain cleavage and 11 beta-hydroxylase (11 beta) systems, reconstituted from purified components. Phosphorylation of adrenodoxin resulted in an average twofold decrease in its Km values for the two specific cytochromes P-450 involved. This effect was paralleled by a positive relationship between the degree of adrenodoxin phosphorylation and its ability to support the overall activity of reconstituted side-chain cleavage and 11 beta-hydroxylase systems. Although it remains to be examined whether adrenodoxin is phosphorylated in the intact cell, the present observations suggest that it represents a potential target in the hormonal regulation of the adrenocortical differentiated functions, especially by stimulatory agents acting through a cyclic-AMP-dependent mechanism, such as adrenocorticotropin.
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PMID:Phosphorylation of bovine adrenodoxin. Structural study and enzymatic activity. 282 99

A human hepatic ferritin receptor has been isolated from human liver and has been purified using affinity chromatography. An affinity constant of 6.0 x 10(8) moles-1 liter was determined for the ferritin receptor. The molecular weight was estimated to be approximately 53,000 by gel electrophoresis. Binding of ferritin to the receptor coupled to a microparticulate support was specific for human liver ferritin with no binding of rat or porcine ferritin. Binding was unaffected by a 100-fold excess of human transferrin, human asialoorosomucoid and bovine albumin. After treatment of the receptor protein with trypsin, binding was not detected. The human hepatic ferritin receptor may play an important role in the uptake of iron into the hepatocyte in physiological and pathological conditions.
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PMID:Isolation of a human hepatic ferritin receptor. 283 1

Mixed-function oxidation of Escherichia coli glutamine synthetase by ascorbate, oxygen, and iron has previously been shown to cause inactivation of the enzyme and enhanced susceptibility to proteolytic attack by a variety of proteases. One of these proteases, from rat liver, is a high molecular weight cysteine proteinase which does not degrade native glutamine synthetase at neutral pH. Although inactive, the oxidized glutamine synthetase preparations used in this study were only partially degraded by this proteinase. Some of the subunits were degraded to acid soluble products with no detectable intermediates; the remaining subunits had not become susceptible to proteolytic attack during the limited exposure to the ascorbate mixed-function oxidation system. Several mammalian enzymes which are known to be inactivated by mixed-function oxidation were tested as substrates for the proteinase. Native rabbit muscle enolase and pyruvate kinase were resistant to degradation, but their oxidatively inactivated forms were degraded. Oxidized phosphoglycerate kinase and creatine kinase were also preferentially degraded. Moreover, trypsin degraded oxidized preparations of all of these enzymes faster than control preparations. Oxidative inactivation of superoxide dismutase by hydrogen peroxide caused a slight increase in susceptibility to proteolytic attack, but the enzyme was still relatively resistant to degradation both by the cysteine proteinase and by trypsin. Although oxidation conditions may not have been optimal for demonstrating enhanced proteolytic susceptibility, the results do indicate that mixed-function oxidation can render some mammalian enzymes, as well as bacterial glutamine synthetase, susceptible to degradation. Mixed-function oxidation of these proteins may be a mechanism of marking them for intracellular turnover.
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PMID:The effect of mixed-function oxidation of enzymes on their susceptibility to degradation by a nonlysosomal cysteine proteinase. 286 45

Monocytes and macrophages have receptors for the iron-binding protein lactoferrin. Lactoferrin acts as a potent inhibitor of granulocyte-macrophage colony stimulating factor production when it binds to these cells. Using a rosette assay and immunofluorescence, we have shown that cultured leukemia cells, including the human erythroid leukemia cell line K562, also have lactoferrin binding sites. The number of binding sites on K562 cells was estimated using soluble 59Fe-lactoferrin. Inhibition studies demonstrate that lactoferrin binding sites are distinct and unrelated to receptors for transferrin or the Fc portion of IgG, which are present on K562 cells. However, electrostatic forces may be important for lactoferrin binding, since other polycationic proteins (eg, protamine) inhibit lactoferrin binding. Prior treatment of K562 cells with trypsin nearly abolishes lactoferrin binding. However, these cells recover their ability to bind lactoferrin when trypsin is removed. Unlike transferrin receptors, the expression of lactoferrin binding sites is not regulated by cellular iron status. Cytosine arabinoside arrests the proliferation of K562 cells and simultaneously leads to a reduction in lactoferrin surface binding, suggesting that lactoferrin binding may be dependent on cell proliferation.
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PMID:Lactoferrin binding by leukemia cell lines. 303 77

Exposure of human erythrocyte ghosts to 0.2-5 mM periodate at 0 C for 15 min resulted in an increase of thiobarbituric acid-reactive substances and fluorescent materials in the membrane. This increase was suppressed by radical scavengers, butylated hydroxytoluene and thiourea, indicating that periodate caused lipid oxidation of ghosts. A role of hemoglobin in the periodate-induced lipid oxidation of ghosts was suggested by the fact that the oxidation was augmented by hemoglobin and inhibited by an iron chelator, desferrioxamine. Treatment of ghosts with periodate caused membrane protein cross-linking with and without disulfide bridge. Erythrocytes were also susceptible to lipid oxidation by periodate, but only disulfide-mediated protein cross-linking was observed. Erythrocytes treated with neuraminidase or trypsin were less susceptible, but those treated with neuraminidase along with galactose oxidase were nearly as susceptible as untreated cells. The effect of galactose oxidase was diminished by reduction of the enzyme-treated cells with borohydride. These results indicate that the aldehyde moieties generated by periodate at the sialyl residues or those generated by galactose oxidase at the terminal galactosyl or N-acetyl galactosaminyl residues of the membrane glycoconjugates play a stimulating role in the periodate-induced membrane lipid oxidation.
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PMID:Periodate-induced lipid oxidation of erythrocyte membranes. 303 33

Modification of carboxyl groups on putidaredoxin with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) resulted in loss of putidaredoxin reductase activity. The modification did not affect the visible absorption spectrum of putidaredoxin, indicating that the iron-sulfur center was not perturbed. In order to identify the carboxyl groups labeled by EDC, native and EDC-treated putidaredoxin were digested with a combination of trypsin and Staphylococcus aureus protease, and the resulting peptides were separated by high pressure liquid chromatography. The most heavily modified carboxyl groups were found to be those at residues 58, 65, 67, 72, and 77. These carboxyl groups are located in the same general region of the protein as those on adrenodoxin that have been shown to be involved in binding to both adrenodoxin reductase and cytochrome P-450scc. Chemical modification was also used to compare the role of lysine, arginine, and histidine residues on putidaredoxin and adrenodoxin. Modification of lysine and arginine residues had no effect on the reductase activity of either protein. The reductase activity of adrenodoxin was unaffected by labeling with 1 eq of diethyl pyrocarbonate/histidine residue, but labeling with a second equivalent completely abolished both activity and the iron-sulfur center spectrum. In contrast, modification of the 2 histidines in putidaredoxin with 1 eq each resulted in nearly complete loss of reductase activity. There was no significant activity for adrenodoxin in the putidaredoxin reductase assay or for putidaredoxin in the adrenodoxin reductase assay, demonstrating that, in spite of the structural similarity between the two proteins, they are not interchangeable functionally.
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PMID:The involvement of carboxylate groups of putidaredoxin in the reaction with putidaredoxin reductase. 309 90

The surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi was analysed by three approaches: a) visualization by electron microscopy of the binding of cationic particles (cationized ferritin at pH 7.2 and colloidal iron hydroxyde at pH 1.8) to the parasite's surface, b) visualization of the binding of fluorescein-labeled lectins (PNA and LPA) to the parasite's surface, and c) by cell electrophoresis. In all cases control, trypsin and neuraminidase-treated cells were analysed. The results obtained indicate that sialic acid residues located on the parasite's surface are responsible for the binding of cationic particles to it and the major component responsible for the net negative surface charge presented by T. cruzi. Phosphate groups, associated to phospholipids, also contribute to the negative surface charge. The effect of previous incubation of the parasites in the presence of lectins (ConA, WGA, PNA, RCA and LPA) on their surface charge was also analysed by cell electrophoresis.
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PMID:The surface charge of Trypanosoma cruzi: analysis using cell electrophoresis, lectins and ultrastructural cytochemistry. 309 34

The mechanism of iron translocation from intestinal lumen to portal plasma is poorly understood. To examine these processes, uptake of Fe2+ and Fe3+ by rat duodenal microvillous membrane vesicles prepared by a Ca2+ precipitation procedure was studied. Membrane aliquots were incubated with increasing concentrations of 59FeCl3 in the presence of a one-thousand-fold molar excess of citrate or 59FeSO4 with a twenty-fold molar excess of L-ascorbic acid. After various time intervals the incubation reaction was stopped by addition of 0.1 mM FeCl3 (4 degrees C), and uptake of 59Fe was determined by a vacuum filtration assay. Initial uptake velocity of 59FeCl3 and 59FeSO4 was determined from the slope of the cumulative uptake curves, which was linear for the first 60 s. Initial uptake rates of both, 59Fe3+ and 59Fe2+ revealed an identical saturable uptake component with a Km of 19-22 nM and Vmax of 8 pmol min-1 mg protein-1. In addition, transport of Fe2+ revealed a linear unspecific uptake phase, which was predominant at high substrate concentrations. Saturable uptake of Fe2+ and Fe3+ was temperature dependent, and significantly reduced by trypsin pretreatment of the microvillous membrane vesicles, indicating the involvement of a protein in the uptake process. This suggestion was pursued by isolation of an iron binding protein from duodenal brushborder membranes. After solubilization of microvillous plasma membranes with 1% Triton X 100, affinity chromatography of the membrane protein mixture over an iron chelate gel derived from epoxy activated Sepharose and elution with 50 mM EDTA yielded a single 52,000 dalton protein. The protein co-chromatographed over an Ultro-Pac TSK G 3000SW HPLC column together with 59FeCl3 and 59FeSO4. It showed no immunologic activity to rabbit antibodies against whole rat serum or rat transferrin. Furthermore, by photoaffinity labelling technique a single iron binding protein with a molecular weight of about 52,000 dalton was identified in microvillous membranes of the rat duodenum. These data are compatible with the hypothesis that intestinal iron absorption is mediated by a specific carrier-dependent transport system.
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PMID:Iron uptake by rat duodenal microvillous membrane vesicles: evidence for a carrier mediated transport system. 310 4

The study of guanidine-HCl or thermal denaturation of diferric ovotransferrin (Fe2Tf) has revealed a simultaneous unfolding of the two domains of the protein (Ikeda et al. (1985) FEBS Lett. 182, 305-309). In urea denaturation of Fe2Tf, however, two distinct steps of unfolding were observed in the urea concentration range from 4.5 to 9 M at pH 8.0 and 37 degrees C by measuring the residual iron-bound protein (absorbance at 465 nm) and the remaining folded structures (circular dichroism at 222 nm). From a study of urea denaturation of partially iron-saturated Tf whose iron preferentially occupied the N-domain, it was found that the first and the second steps of denaturation corresponded to those of the N-terminal (4.5-6 M urea) and C-terminal domains (over 7 M urea), respectively. The N-domain of Fe2Tf was selectively unfolded in 7 M urea and digested with trypsin to provide an iron-bound C-terminal fragment (42 kDa) in good yield (about 80% of theoretical). The kinetic analysis of the decrease in A465 of Fe2Tf in 9 M urea showed that the N-domain unfolded 3 x 10(2) times faster than the C-domain. With partially iron-saturated Tf, the decrease of A465 in 9 M urea also proceeded in a biphasic manner and the ratio, the decrement in A465 of the rapid phase/the decrement in A465 of the slow phase, gave the value of iron distribution as Fe at the N-site/Fe at the C-site.
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PMID:Different stability of N- and C-domain of diferric ovotransferrin in urea and application to the determination of iron distribution between the two domains. 318 52


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