Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Isolated rat liver lysosomes were incubated with [14C]methemoglobin under various conditions. Optimal pH for the in vitro proteolysis was found to be 4-5. To evaluate whether or not degradation of added proteins could be due to enzyme leakage the integrity of the lysosomes was measured. Isolated lysosomes were found to be stable for up to 10 min of incubation at pH 5.5 and for 30 min at pH 7. The degradation of three different proteins (methemoglobin, ovalbumin, and lysozyme) was analyzed. No correlation was detected between rate of breakdown and physical properties of the proteins. Leupeptin, chloroquine, and propylamine inhibited proteolysis of added proteins by 45-65% in both neutral and acid milieu. Possible energy requirement was tested by the addition of Mg2+ and ATP to the incubation medium. A dose-dependent increase in proteolytic rate was found when ATP was added to the lysosomal suspension, a finding most likely due to acidification of the lysosomes and ensuing increased degradation. GTP and ITP were somewhat less effective. The noncleavable ATP analogue 5'-adenylylimidodiphosphate gave no stimulation. The ATP-driven proteolysis was inhibited by ethylmaleimide. Isolated lysosomes were also incubated with ferritin in order to visualize a possible uptake process of a protein in the electron microscope. Following incubation, ferritin particles were seen inside intralysosomal vesicles which appeared to be formed by invagination of the lysosomal membrane, a process designated microautophagy. The results thus support the notion that isolated lysosomes may micropinocytose and degrade exogenously added proteins and that this process is ATP dependent.
Exp Mol Pathol 1985 Feb
PMID:Uptake--microautophagy--and degradation of exogenous proteins by isolated rat liver lysosomes. Effects of pH, ATP, and inhibitors of proteolysis. 396 51

Unlike the unactivated glucocorticoid-receptor complex, the thermally activated glucocorticoid-receptor complex was able to bind to Affigel blue (a matrix previously shown to bind proteins containing a dinucleotide fold region) under low ionic conditions (0.05 M K C1). Glucocorticoid-receptor complex binding capacity to Affigel blue was enhanced by increasing salt concentration. Optimal binding was obtained at 0.15 M K C1 and remained at a plateau level up to 0.4 M K C1. In contrast to Affigel blue binding, glucocorticoid-receptor complex binding to nuclei was optimum at low ionic strength buffer, declined at 0.15 M K C1 and became negligible at 0.4 M K C1. Interestingly, at physiological ionic strength (0.15 M K C1) both nuclei and Affigel blue bound to the glucocorticoid-receptor complex with almost identical capacity. Glucocorticoid-receptor complexes incubated 45 min at 25 degrees C (activation conditions) in the presence of 10 mM molybdate were unable to bind to Affigel blue (or isolated nuclei) as expected. The results obtained suggest that Affigel blue mimics isolated nuclei in the binding of activated glucocorticoid-receptor complexes under physiological (0.15 M K C1) conditions. In addition, Affigel blue may provide a rapid and easy method to study glucocorticoid-receptor complex activation and interaction with nuclear acceptor sites.
Mol Cell Biochem 1985 Jan
PMID:Interaction of the hepatic glucocorticoid-receptor complex with Affigel blue. 398 93

Enhanced reactivation (ER) and enhanced mutagenesis (EM) of herpes simplex virus type 1 were studied simultaneously in UV-irradiated stationary cultures of diploid normal human and xeroderma pigmentosum (XP) fibroblasts. Mutagenesis was assayed with unirradiated herpes simplex virus type 1 as a probe in a forward mutation assay (resistance to iododeoxycytidine). Dose-response studies showed that ER increased with the UV dose given to the virus. Optimal reactivation levels were obtained when normal cells and XP variant cells were exposed to a UV dose of 8 J . m-2 and the virus was irradiated with 150 J . m-2. Repair-deficient XP cells of complementation groups A, C, and D showed optimal reactivation levels with a UV dose to the cells of 1.0 J . m-2 and a UV dose to the virus of 40 J . m-2. The time course of appearance of ER and EM was also studied, both in the normal and XP cells. In all cell types except the XP variant cells, EM followed similar kinetics of appearance as did ER. Maximal activities occurred when infection was delayed 1 or 2 days after cell treatment. In XP variant cells, however, maximal expression of the EM function was significantly delayed with respect to ER. The results indicate that ER and EM are transiently expressed in normal and repair-deficient XP cells. Although both phenomena may be triggered by the same cellular event, ER and EM appear to be separate processes that occur independently of each other.
Mol Cell Biol 1984 Nov
PMID:Enhanced reactivation and enhanced mutagenesis of herpes simplex virus in normal human and xeroderma pigmentosum cells. 609 94

The effects of FSH on the aromatase activity of rat granulosa cells in culture were studied by measuring the stereospecific transfer of 3H from [1,2,6,7-3H]testosterone or [1 beta-3H]testosterone into 3H2O. The use of 3H2O release as a specific assay for aromatization in granulosa cells was validated by various means. 2 days after plating, cultures of granulosa cells released only minimal amounts of 3H2O from [1 beta-3H]testosterone during a 3-h incubation, whereas cells which had been treated with FSH, or with (Bu)2cAMP plus 3-isobutyl-1-methylxanthine (MIX), from the time of plating released considerable amounts of 3H2O into the culture medium. The release of 3H2O from [1 beta-3H]testosterone by cultured cells was inhibited by the aromatizable androgens, 19-hydroxytestosterone and 19-hydroxyandrostenedione, indicating the specificity of the method for aromatization. In order to study the mechanism by which FSH enhanced the release of 3H2O, optimal conditions for aromatization by cell-free sonicates were determined. Optimal aromatase activity was achieved by incubating cell-free sonicates at 37 degrees C in the presence of O2 in 20 mM phosphate buffer (pH 7.4) containing 5 mM dithiothreitol, 20 mM MgCl2, 0.5 mM NADP+, 20 mM glucose 6-phosphate and 2 U/ml glucose-6-phosphate dehydrogenase. A concentration of 0.25 microM testosterone and 0.1 microCi tritium was used in the standard assay. Under these conditions the assay was linear for 1 h with up to 150 microgram protein from granulosa cells having maximal aromatase activity. When cells in culture were stimulated with purified FSH for 48 h from the time of plating, the aromatase activity in cell-free sonicates increased in a dose-dependent fashion. The ED50 for Sairam's FSH S-1528 C2 was 12 ng/ml. It was concluded from these studies that FSH increases estrogen levels primarily by inducing an active aromatase rather than by influencing secretion or availability of substrate or cofactor for the aromatization reaction.
Mol Cell Endocrinol 1981 May
PMID:FSH induction of aromatase in cultured rat granulosa cells measured by a radiometric assay. 616 36

Epimastigote forms of Trypanosoma cruzi contain a soluble cAMP phosphodiesterase. Optimal activity was found at pH 8.0 and in the presence of 5 mM Mn2+. Other cations were less efficient and did not give rise to an additional stimulation when added in the presence of optimal concentrations of Mn2+. The enzyme is not Ca2+ dependent. The apparent Km of the enzyme for the substrate is 40 microM and no kinetic evidence for the existence of two enzymes has been found. Theophylline and caffein did not inhibit the T. cruzi cAMP phosphodiesterase. The enzyme activity does not change during cell growth suggesting that the fluctuation observed in the levels of cAMP are largely a response to variations in adenylyl cyclase activity. The intracellular concentrations of cAMP ranged between 0.04--0.15 microM. No evidence that the T. cruzi cAMP phosphodiesterase is regulated by an endogenous activator could be found. However, T. cruzi contains a heat-stable, low molecular weight, non-dialysable protein that activates mammalian cAMP phosphodiesterase in the presence of Ca2+. The properties so far studied of such an activator suggest that it might be equivalent to other Ca2+-dependent regulators described in vertebrate and invertebrate species.
Mol Biochem Parasitol 1980 Apr
PMID:cAMP phosphodiesterase and activator protein of mammalian cAMP phosphodiesterase from Trypanosoma cruzi. 625 27

[3H]Ecdysone, [3H]20-OH-ecdysone and [3H]ponasterone A were specifically bound by the 120 000 g supernatant of the homogenate from crayfish hypodermis. According to the HPLC test, the bound hormone was not metabolized. Analysis of the cytosol receptor by sucrose-density-gradient centrifugation, gel filtration and enzymatic degradation revealed the protein character of the receptor, a molecular weight of 70 000-80 000 D, and 5S. The binding capacity was rapidly lost after treatment with N-ethyl-maleimide or heating for 10 min at 60 degrees C. Optimal conditions for the demonstration of the receptor were incubation at 0-8 degrees C for 30 min and a salt concentration of 120 mM. A sequence of moulting hormones, and steroid hormones without moulting hormone activity were tested for ligand specificities. Estimation KD values yielded 3-6 x 10(-11) M for ponasterone A, 2-3 x 10(-9) M for 20-OH-ecdysone and 2 x 10(-8) M for ecdysone.
Mol Cell Endocrinol 1981 Dec
PMID:Characterization of cytoplasmic ecdysteroid receptors in the hypodermis of the crayfish, Orconectes limosus. 627 48

The effect of cerebroside sulfate, phosphatidylserine, and other phospholipids on opiate receptor function in neuroblastoma N18TG2 cells was studied by incorporation of lipids into the membrane bilayer of viable cells. A concentration- and time-dependent incorporation of sulfatide by N18TG2 cells was observed. The incorporated lipid was not metabolized during the incubation period of up to 48 hr at 37 degrees. Optimal conditions for lipid incorporation were determined to be 4 days after the cell seeding and in 1% fetal calf serum. The incorporated lipid was established to be associated with the plasma membrane fraction of the crude cell homogenate. Furthermore, increases in Vmax but not Km values of the adenylate cyclase for Mg2+, ATP, and prostaglandin E1 were observed in neuroblastoma N18TG2 cells exposed to cerebroside sulfate for 4--6 hr. The incorporation of cerebroside sulfate or phosphatidylserine by N18TG2 cells did not increase the number of opiate binding sites in this cell line as determined by [3H]naloxone, [3H]etorphine, or 3H-labeled D-Ala2-Met5-enkephalinamide binding. Although there was an increase in the affinity of [3H]naloxone binding, linear correlation between the amount of cerebroside sulfate incorporated and the quantity of binding increase was not observed. However, augmentation of both the potencies and the efficacies (maximal inhibitory level) of morphine and enkephalin to regulate adenylate cyclase activity was observed after sulfatide incorporation. At the maximal concentration of cerebroside sulfate used (67 microM) the opiate receptor activity in N18TG2 cells approached that of NG108-15 cells. Identical treatment of N18TG2 cells with cerebroside or psychosine sulfate did not produce any potentiation of the opiate inhibition of adenylate cyclase. Of all of the phospholipids tested--phosphatidylserine, phosphatidylinositol, and phosphatidylcholine--only phosphatidylcholine produced a potentiation of the opiate effect. Both synthetic dipalmitoyl phosphatidylcholine or brain phosphatidylcholine could elicit the potentiation.
Mol Pharmacol 1982 Mar
PMID:Potentiation of opiate action in neuroblastoma N18TG2 cells by lipid incorporation. 628 74

beta-N-Acetylglucosaminidase secreted by Entamoeba histolytica was extracted from the growth medium by affinity chromatography on CH-Sepharose 4 B coupled to p-aminophenyl-1-thio-beta-2-acetamido-2-deoxyglucopyranoside. The enzyme was further purified by isoelectric focusing, by sequential chromatography on DEAE-cellulose and Sephadex G-150, and by preparative disc gel electrophoresis. Chitobiose (betaGlcNAc1-4GlcNAc) derived from chitin as well as the oligosaccharides betaGlcNAc1-4 betaGlcUA1-3GlcNAc, betaGlcNAc1-4 betaGlcUA1-3 betaGlcAc1-4GlcUA, and betaGlcNAc1-4 betaGlc-UA1-3 betaGlcNAc1-4 betaGlcUA1-3 betaGlcNAc1-4GlcUA derived from hyaluronic acid were tested as potential physiological substrates. All these oligosaccharides are susceptible to action of beta-N-acetylglucosaminidase from E. histolytica. Under identical conditions chitobiose is cleaved 38-48 times faster than hyhyauronate oligosaccharides. No release of N-acetylglucosamine was observed when glycopeptides from ovalbumin were used as substrate. The pH optimum of hydrolase activity was 4.5 when chitobiose was used as substrate. Optimal hydrolysis of aluronate oligosaccharides was observed at pH 3.0 for trisaccharide and pH 2.0 for tetra- and hexasaccharide, respectively. Estimation of molecular weight by means of gel filtration gave values of 75 000. The isoelectric point was 5.02 beta-N-Acetylglucosaminidase from E. histolytica does not act on macromolecular chitin and hyaluronic acid.
Mol Biochem Parasitol 1983 Feb
PMID:Degradation of biogenic oligosaccharides by beta-N-acetyl-glucosaminidase secreted by Entamoeba histolytica. 630 11

In studies of the calcium paradox, the isolated perfused rat heart was used to characterize the relationship between myocardial protein leakage and the concentration of calcium antagonist (verapamil and D600) or calcium in the perfusion fluid during a cycle of calcium depletion and repletion. The results indicated a dose-dependency such that protein leakage could be progressively reduced by decreasing the concentration of calcium during calcium repletion and/or by increasing the concentrations of drug. Detailed dose-response studies with seven calcium antagonists (verapamil, D600, nifedipine, terodiline, diltiazem, fendiline and prenylamine) and a calcium concentration of 1.0 mmol/l during a 20 min period of calcium repletion following a 10 min period of calcium depletion revealed complex dose-response characteristics for each drug. In the dose range studied (0 to 40 mumol/l) all drugs with the exception of prenylamine were able to reduce protein leakage by up to 25 to 30% Optimal concentrations for verapamil, nifedipine, D600, diltiazem and terodiline were all between 2.0 and 4.0 mumol/l. With the exception of D600, which provided a constant reduction of protein leakage at all concentrations above this optimum, all drugs exhibited bell-shaped dose-response curves with a loss of efficacy at higher concentrations. Fendiline also had a bell-shaped dose-response curve with 23% as a maximal reduction of leakage; however, the optimal concentration for this drug was 21.0 mumol/l. Additional studies with verapamil revealed that the drug acted during the calcium repletion phase and did not influence events during calcium depletion. Simultaneous use of two drugs, verapamil and nifedipine, at their optimal concentrations failed to improve the reduction in protein leakage over and above that observed with one drug alone.
J Mol Cell Cardiol 1983 Jul
PMID:Slow calcium channel blockers and the calcium paradox: comparative studies in the rat with seven drugs. 631 58

In vitro growth and differentiation of granulocyte-macrophage progenitor cells (GM-CFU-C) requires colony-stimulating factors (CSF), and an in vivo role for CSF has also been proposed. Prostaglandins of the E series (PGE) have been reported to serve as negative feedback regulators of myelopoiesis. Here, we report evidence of augmented CSF secretion by mouse peritoneal Mo (macrophages) and bone marrow cells in vitro upon stimulation with various biological response modifiers (BRMs). Optimal induction of CSF secretion occurred after in vitro treatment of peritoneal Mo and mononuclear bone marrow cells with 50 micrograms/ml poly ICLC (polyriboinosinic-polycytidylic acid poly-L-lysine). 5 micrograms/ml lipopolysaccharide (LPS), or 500 U/ml interferon (IFN alpha,beta) for 2 days. The in vitro stimulation of CSF secretion was paralleled by an increase in PGE secretion by Mo and bone marrow cells. The PGE secretion could, however, be selectively blocked by preincubating the cells for 3 h with indomethacin (10(-7) Mol) leaving CFS production intact. In vivo treatment of mice with either maleic anhydride divinyl ether copolymer (MVE-2; 25 mg/kg) or poly ICLC (2 mg/kg) significantly increased levels of CSF in serum, as well as in culture supernatants of in vivo-treated peritoneal Mo and bone marrow cells. The increase in serum CSF levels and in secretion of CSF by peritoneal Mo and bone marrow cells was followed by a dose-dependent increase in GM-CFU-C, in nucleated bone marrow cells, and in peripheral blood leukocytes. The same BRMs also stimulated the secretion of PGE by in vivo-activated peritoneal Mo, but not by bone marrow cells. Pretreatment of the mice with indomethacin (4 mg/kg) almost completely suppressed PGE secretion by peritoneal Mo, but did not change the CSF secretion by peritoneal Mo or bone marrow cells and had no significant effect on bone marrow cellularity. Therefore, MVE-2 and poly ICLC, in addition to their immunomodulatory activity, can also have stimulatory effects on myelopoiesis, presumably mediated through secretion of CSFs. Protection and/or restoration of bone marrow function could thus either provide the opportunity for more extensive chemotherapy or could increase the number of Mo effector cells available for activation against tumor targets.
 ...
PMID:Comparison of in vitro and in vivo modulation of myelopoiesis by biological response modifiers. 633 54


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