Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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This report describes the first isolation and molecular characterization of the mitochondrial F1-ATPase from Trypanosoma brucei. The isolation procedure utilized is a modified chloroform extraction procedure. In contrast to earlier reports on the F1-ATPase from other trypanosomatids, the F1-ATPase we have isolated from the procyclic form of T. brucei a complex composed of five distinct subunits. Apparent molecular weights of these subunits are 55,000 [alpha], 42,000 [beta], 32,000 [gamma], 22,000 [delta], and 17,000 [epsilon]. The F1 moiety which possesses the active site of the H(+)-ATPase has an ATPase activity in the standard Tris-HCl coupled enzyme assay with a Vmax of 22.96 mumol min-1 (mg protein)-1 and a Km value of 0.60 mM. This ATPase activity is cold labile and is not susceptible to oligomycin inhibition as is the membrane bound enzyme. Upon reconstitution with F1-ATPase depleted membranes (urea particles) the ATPase regains oligomycin sensitivity to the same extent as that found in the intact inner membrane vesicles. ATP synthesis is also restored to these particles upon reconstitution with F1. These results indicate that this F1-ATPase as isolated is intact with respect to all the critical H(+)-ATPase functions.
Mol Biochem Parasitol 1990 Nov
PMID:The mitochondrial ATP synthase of Trypanosoma brucei: isolation and characterization of the intact F1 moiety. 214 43

1. The histone complement of oligodendrocyte chromatin at different stages of brain development was studied after acid extraction of nuclei. 2. HCl-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1(0) with differentiation. 4. The increase in H1(0) is accompanied by a concomitant decrease in the total amount of the ubiquitinated form of histone H2A (A24).
Cell Mol Neurobiol 1990 Jun
PMID:The relative proportion of H1(0) and A24 is reversed in oligodendrocytes during rat brain development. 216 55

One of the commonest methods of denaturing nucleic acids, denaturation by heat, was found to be ineffective for double-stranded (ds)RNA when RNA preparations contain 200 mM or higher concentrations of NaCl. We report acid denaturation of dsRNA (incubation for 10 min in the presence of 80 mM HCl) was particularly useful for such preparations. When genomic dsRNAs of rotavirus were extracted by ion-exchange chromatography columns (Extractor) and then denatured by acid, the detection of rotavirus RNA in stool specimens by the previously reported dot-blot hybridization assay (Yamakawa, K. et al. (1989). Molecular and Cellular Probes 3, 397-401.) was significantly improved.
Mol Cell Probes 1990 Oct
PMID:Improved detection of rotavirus RNA in dot-blot hybridization assay by chromatographic extraction and acid denaturation of double-stranded RNA. 217 47

Several new 4,19-substituted steroids and previously synthesized corticosteroids were assayed for affinity to type 1 receptors in human mononuclear leukocytes. 11 beta,19-epoxy-4,21-dihydroxypregn-4-ene-3,20-dione (2) was hydrogenated with Pd-C to yield a mixture of all four dihydro derivatives 5, accompanied by 4,21-diacetoxy-11 beta,19-epoxy-3-hydroxypregnan-20-one (6) and 21-acetoxy-11 beta,19-epoxy-4-hydroxypregnane-3,20-dione (7). With hot acetic + p-toluenesulfonic acid 5 underwent rearrangement to 21-acetoxy-11 beta,19-epoxypregn-5-ene-4,20-dione (8) Pd-C hydrogenation of 3,21-diacetoxy-5 beta,19-cyclopregna-2,9(11)-diene-4,20-dione (10) gave 3,21-diacetoxy-5 beta,19-cyclopregn-5-ene-4,20-dione (11) and the 9,11-dihydro derivative of the latter. Treatment of 10 with warm HCl furnished 19-chloro-4,21-dihydroxypregna-4,9(11)-diene-3,20-dione (13). Pd-C hydrogenation of its diacetate 14 afforded the 4,5-dihydro derivative 18, 19-chloro-21-acetoxypregn-9(11)-en-20-one (15), its 4-acetoxy derivative 16 and the 3,4-diacetoxy derivative 17. When tested in a radioreceptor assay in human mononuclear leukocytes the synthesized compounds showed only low relative binding affinities (RBA) to type 1 receptor, the highest being 0.72% for 13 (aldosterone = 100%). For comparison, other RBA in this system were: 19-noraldosterone, 20%; 18-deoxyaldosterone, 5.8%; 18-deoxy-19-noraldosterone, 4.7%; 18,21-anhydroaldosterone, 0.37%; 17-isoaldosterone, 7.6% and apoaldosterone, 4.3%
J Steroid Biochem Mol Biol 1990 Oct
PMID:Synthesis of 4,19-disubstituted derivatives of DOC. Radioreceptor assay of some corticosteroid derivatives in human mononuclear leukocytes. 226 58

The novel reversible and selective inhibitor of monoamine oxidase-A (MAO-A) Ro 41-1049 [N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide HCl] shows inhibition characteristics similar to those of the structurally related reversible MAO-B inhibitors Ro 16-6491 and Ro 19-6327. In the present study, tritiated Ro 41-1049 was used as a high affinity ligand to study the binding characteristics of this inhibitor to MAO-A and its interactions with the enzyme. An homogeneous population of high affinity binding sites for [3H]Ro 41-1049 was found in membrane preparations from human frontal cortex and placenta (Kd = 16.5 +/- 1.4 and 64.4 +/- 19.2 nM, respectively). In frontal cortex the Bmax value for [3H]Ro 41-1049 (2.6 +/- 0.4 pmol/mg of protein) was about one third of the Bmax calculated for the MAO-B-selective ligand [3H]Ro 16-6491. The density of [3H]Ro 41-1049 binding sites in human placenta varied greatly in the different tissue samples investigated, showing an average Bmax of 101.7 +/- 36.5 pmol/mg of protein. Apparent binding equilibrium was reached after 1 hr of incubation at 37 degrees. At this temperature the binding was reversible, with a dissociation t 1/2 of about 35 min. At lower temperatures the radioactivity dissociation was much slower. Among the various drugs tested, only inhibitors of MAO-A were able to effectively prevent [3H]Ro 41-1049 specific binding. As previously reported for the MAO-B ligands [3H]Ro 16-6491 and [3H]Ro 19-6327, the analysis of the membrane-bound radioactivity showed that [3H]Ro 41-1049 was entirely recovered in the form of its aldehyde derivative, indicating that Ro 41-1049 was deaminated by MAO-A. The existence of a Ro 41-1049 adduct reversibly bound to the enzyme active site might explain the inhibition mechanism of this compound. The exposure of the radioligand-enzyme complex to NaBH3CN at pH 4.5 caused the irreversible covalent incorporation of about 70% of the specifically bound radioactivity into a 60-kDa polypeptide. This incorporation was dependent on the pH and on the amount of NaBH3CN added. The presence of MAO-A- but not MAO-B-selective inhibitors prevented the covalent incorporation of [3H]Ro 41-1049. The present results indicate that [3H]Ro 41-1049 is incorporated into a subunit of MAO-A, in the presence of NaBH3CN, and modifies a protein domain that is essential for the enzyme activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1990 Mar
PMID:Characterization of the binding of [3H]Ro 41-1049 to the active site of human monoamine oxidase-A. 231 88

Using monoclonal antibody 19-9, elevated levels of the sialyl Lea antigen (NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNac beta 1-3R) are detected in serum from most cystic fibrosis patients. We now report further characterization of the serum antigen and evidence that it is on a mucin glycoprotein and not on glycolipids. The antigen has an apparent molecular weight greater than 2 X 10(6) by gel filtration on Sephacryl S-400. On density gradient centrifugation, the antigen has a density of 1.54 g/ml in cesium chloride and 1.42 g/ml in cesium chloride/4 M guanidine HCl. Immunostaining with monoclonal antibody 19-9 of lipid extracts from cystic fibrosis patient serum and erythrocytes does not detect any antigen on glycolipids. The antigen was purified by gel filtration and density gradient centrifugation. After tritium labeling of the sialic acid residues, sodium dodecyl sulfate gel electrophoresis separates two subunits with apparent molecular weights of 200,000 and 400,000. All of the labeled sialic acid is released as low molecular weight oligosaccharides after mild alkaline borohydride degradation. The purified antigen contains fucose, galactosamine, glucosamine, and galactose but no mannose and is enriched in the amino acids threonine, serine, glycine, proline, and alanine. The purified antigen binds several antibodies recognizing epitopes common to many mucins. Thus, the physical, biochemical, and immunochemical properties of the purified antigen indicate that the sialyl Lea antigen is present on mucins in the serum of cystic fibrosis patients.
Am J Respir Cell Mol Biol 1990 Apr
PMID:Isolation and characterization of mucin from the serum of cystic fibrosis patients. 232 69

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1990 May 05
PMID:Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora. 233 12

The conformation of apomyoglobin under acidic conditions depends on both pH and salt conditions. Using KCl and HCl, a phase diagram for the pH and ionic strength-dependent conformational states was constructed based on the change in the far-ultraviolet circular dichroism. Acid-denatured apomyoglobin consists of two conformational states, the largely unfolded state (UA) and the relatively compact, acidic intermediate state (A). The UA state is stable below pH 3 and at low ionic strength. High ionic strength favors the A state. Because the minimal ionic strength increases significantly with a decrease in pH below 2, the UA state is limited to only a small region between pH 1 and 3.
J Mol Biol 1990 Aug 20
PMID:Phase diagram for acidic conformational states of apomyoglobin. 238 68

SM-10661 [(+/-)-(cis)-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one HCl] displayed marked in vitro inhibition of rabbit platelet aggregation induced by 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkyl-PAF), 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16-PAF), and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine, with IC50, values of 5.50, 5.94, and 3.68 microM, respectively. It also inhibited alkyl-PAF-induced aggregation of human platelets with an IC50 of 3.00 microM, but it did not inhibit platelet aggregation induced by ADP, collagen, arachidonic acid, the thromboxane A2 agonist U46619, or the Ca ionophore A23187, at concentrations up to 400 microM. Furthermore, SM-10661 antagonized [3H]-C16-PAF binding to rabbit platelets competitively, with an IC50 of 1.0 microM. SM-10661 protected against alkyl-PAF-induced lethality in mice with an ID50 of 6.0 mg/kg intravenously or 24 mg/kg orally. In guinea pig, SM-10661 inhibited the alkyl-PAF (0.1 micrograms/kg)-induced increase in bronchial pressure, with an ID50 of 0.7 mg/kg intravenously or 15 mg/kg orally. Bronchial hyperreactivity to bombesin after the infusion of alkyl-PAF was also inhibited dose-dependently by the infusion of SM-10661, with an ID50 of 25 mg/kg. In addition, SM-10661 inhibited alkyl-PAF (0.01 micrograms/kg)-induced hypotension in rats, with an ID50 of 0.36 mg/kg intravenously or 33 mg/kg orally. SM-10661, when given orally, showed rapid absorption and good duration of pharmacological activity in rats and rabbits.
Mol Pharmacol 1990 Sep
PMID:Biological effect of orally active platelet-activating factor receptor antagonist SM-10661. 240 27

Rapid restitution of the gastric and intestinal epithelium after acute injury involves emigration of cells from the gastric glands and basal half of the intestinal villi. An intact basal lamina is prerequisite to the restitution process. The present study was performed to determine the effects of acid on the rat gastric and duodenal basal lamina. The basal lamina was denuded in vitro by ultrasonic vibration. The tissue was then immersed in 0.2 M mannitol (control) or in HCl (5-50 mM) for 10 min. Samples of the tissues were examined by transmission and scanning electron microscopy. Some samples were stained with ruthenium red to demonstrate glycosaminoglycans. The lower concentrations of acid (5 and 10 mM) had little or no effect on the structure of the basal lamina. However, exposure to 20 and 50 mM HCl caused extensive damage to the basal lamina and exposed the underlying connective tissue matrix of the lamina propria. Ruthenium red staining demonstrated differences in size and location of glycosaminoglycans within the basal laminae of stomach and intestine. Exposure to acid at concentrations of 20 or 50 mM caused total loss of ruthenium red staining in both intestinal and gastric basal laminae. Exposure to 10 mM acid resulted in loss of the outermost (luminal) layer of anionic sites from the gastric basal lamina. These studies demonstrate that brief exposure to acid, in concentrations which are necessary for the formation of hemorrhagic erosions in the stomach, caused damage to the basal lamina. This damage may impair epithelial restitution and thus account, in part, for the role of acid in ulcerogenesis.
Virchows Arch B Cell Pathol Incl Mol Pathol 1985
PMID:Effects of acid on the basal lamina of the rat stomach and duodenum. 241 80


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