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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Layer by layer uncoating of influenza A and B viruses with non-ionic detergent (NP-40) at fixed pH was developed. Treatment of virions with NP-40 at neutral or alkaline pH solubilized the lipoprotein envelope and the surface glycopolypeptides HA1 and HA2, but the internal core structures containing matrix protein M1 remained. Exposition of the cores in acidic media (pH 4,5 and lower) selectively solubilized protein M1 and released viral ribonucleoprotein (RNP). The resulting M1 sedimented in a glycerol gradient with a coefficient of 2.8 S and most probably exists as a monomer of 27,000 Da polypeptide. Neutralization of protein M1 with Tris-HC1 at pH 7.0 did not cause aggregation of M1 polypeptides. The described method of viron layer by layer uncoating with non-ionic detergent at fixed pH is suitable for isolation of subvirus structures and individual viral proteins.
Mol Biol (Mosk)
PMID:[Influenza virus proteins: preparation of a soluble M1 polypeptide by means of a stepwise deproteination of virions]. 188 94

When Eubacterium sp. 144 was grown in the presence of progesterone, extracts of these cells contained a 4-ene-3-ketosteroid-5 alpha-reductase (5 alpha-reductase). No evidence for the presence of a 5 beta-steroid-reductase or a 5 alpha to 5 beta-steroid-isomerase was found. 5 alpha-Reductase activity was dependent on reduced methyl viologen as the electron donor and this could be generated biologically by adding pyruvate or H2 to cell extracts or chemically by adding sodium dithionite. NADH or NADPH with or without flavin nucleotides were not electron donors for 5 alpha-reductase. Most of the 5 alpha-reductase activity (60-65%) of crude extracts was located in the membrane fraction and the enzyme was solubilized by treatment with 1% Triton X-100. Optimum 5 alpha-reductase activity occurred at pH 7.0-7.5 in potassium phosphate buffer but was stimulated by Tris-HCl buffer (pH 8.0-9.0). 5 alpha-Reductase activity was highest at 10% (v/v) methanol and was progressively inhibited by higher methanol concentrations. Sulfhydryl reagents strongly inhibited 5 alpha-reductase but the enzyme was not affected by other metabolic inhibitors. Extracts prepared from cells induced with 16-dehydroprogesterone and grown without hemin contained 5 alpha-reductase and 16-dehydroprogesterone-reductase activities equivalent to those found in extracts of induced cells grown with hemin. This indicates that hemin is not required for the synthesis of active steroid double bond-reductases in strain 144.
J Steroid Biochem Mol Biol 1991 Sep
PMID:Properties of a 4-ene-3-ketosteroid-5 alpha-reductase in cell extracts of the intestinal anaerobe, Eubacterium sp. strain 144. 191 27

The gelatin-binding region of fibronectin is isolated easily as a stable and functional 42 kDa fragment containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9. This fragment exhibits a single reversible melting transition near 64 degrees C in TBS buffer (0.02 M-Tris buffer containing 0.15 M-NaCl, pH 7.4). The transition is characterized by a calorimetric to van't Hoff enthalpy ratio of 1.6, suggesting a complex domain structure. A 30 kDa fragment with the same NH2 terminus (I6-II1-II2-I7) melts reversibly near 65 degrees C with delta Hcal/delta HvH = 1.3, also consistent with the presence of more than one domain. To elucidate further the domain structure, three non-overlapping subfragments were prepared and characterized with respect to their unfolding induced by heat and guanidinium chloride. The three subfragments, each containing two modules, are designated from amino or carboxyl-terminal location as 13 kDa (I6-II1) 16 kDa (II2-I7) and 21 kDa (I8-I9) according to their apparent Mr in SDS/polyacrylamide gel electrophoresis. All three subfragments exhibited reversible transitions in TBS buffer, behaving in the calorimeter as single co-operative units with delta Hcal/delta HvH close to unity. However, the specific enthalpies and changes in heat capacity associated with the melting of all fragments and subfragments in TBS buffer were low compared to those of most compact globular proteins, suggesting that not all modules are represented. When titrated with guanidinium chloride at 25 degrees C, all fragments exhibited monophasic reversible unfolding transitions detected by changes in fluorescence. Heating in the presence of 6 M-guanidinium chloride revealed three additional transitions not seen in the absence of denaturants. These transitions have been assigned to three of the four type I finger modules (I6, I7 and I9), one of which (I6) was isolated and shown to retain a compact structure as stable as that observed for this module within the parent fragments. Two other modules (II2 and I7) are destabilized when separated from their neighbors. Thus, despite their small size (50 to 60 amino acid residues), all six of the modules in the gelatin-binding region of fibronectin form independently folded domains, three of which (I6, I7 and I9) are unusually stable. Evidence is provided that four of the six modules interact with each other in the parent fragment. This interaction may explain previously noted disruptions in the otherwise uniform strand-like images seen in electron micrographs of fibronectin.
J Mol Biol 1991 Feb 05
PMID:Domain structure and interactions of the type I and type II modules in the gelatin-binding region of fibronectin. All six modules are independently folded. 199 38

The focus of this study has been to determine the conformation of the holoprotein of recombinant flavodoxin from Desulfovibrio vulgaris with the FMN in each of its three oxidation states. The structures of the oxidized state of the wild-type flavodoxin at 2.0 A from D. vulgaris was used as a starting model for refinement. Diffraction experiments were conducted at low temperature (-150 degrees C) in order to maintain the oxidation state of interest throughout the intensity data collection. yellow bipyramids by the standard hanging-drop method from 3.2 M-ammonium sulfate in 0.1 M-Tris-HCl buffer at pH 7.0 with protein concentrations ranging from 0.7% to 0.9%. The reduced states of the crystals were achieved through the addition of sodium dithionite at pH 7.0 for the semiquinone (semi-reduced) and at pH 9.0 for the hydroquinone (fully reduced). Data sets consisting of one at room temperature (oxidized state) and three at low temperature (each oxidation state) were collected on a Nicolet P3F/Xentronics area detector X-ray diffractometer system. The four structures, hydroquinone at 2.25 A resolution and all others at 1.9 A resolution, were refined by the restrained parameter least-squares program PROLSQ. The final crystallographic R-values converged to 0.21 (hydroquinone), 0.20 (semiquinone), 0.20 (oxidized, low temperature), and 0.17 (oxidized, room temperature). The reduced states of flavodoxin show a different conformation of the protein polypeptide chain (Asp61-Gly62) in the vicinity of NH(5) of the isoalloxazine group relative to the oxidized state. However, there are only slight conformational differences between the semiquinone and hydroquinone states. In this report, structural comparisons of the three are made, with particular emphasis on the features that might be related to the difference in temperature of the diffraction data collections and differences in the oxidation state of the FMN.
J Mol Biol 1991 Mar 05
PMID:Comparison of the crystal structures of a flavodoxin in its three oxidation states at cryogenic temperatures. 200 3

Post mitochondrial supernatants (S-12 extracts) were prepared from Phycomyces blakesleeanus by grinding washed and frozen mycelial cakes in fine sand and extracting the paste produced with buffer containing Tris-HCl pH 7.8 (0.1 M), EDTA (0.01 M), dithiothreitol (5 mM) and glycerol (10% v/v). The S-12 extracts, obtained in this way, reproducibly hydroxylated progesterone, producing 7 alpha- and 15 beta-hydroxyprogesterone the major products of whole-cell transformation. Cell-free progesterone hydroxylation was found to be approximately linearly dependent on extract concentration, to require reduced NADP (partly replaceable by NADH), and to be dependent on progesterone (apparent Km calculated to be 4 mM). K+ and Mg2+ were found not to be required. Maximum progesterone hydroxylation occurred after 2 h at pH 7.8 and at 24 degrees C. Using optimum conditions S-12 extracts were capable of hydroxylating between 5 and 15% of added progesterone (0.2 mM). Hydroxylation was found to be partially inhibited by carbon monoxide (ca 40%) and almost completely inhibited by azoles, ketoconazole and diconazole. The NADPH and molecular oxygen requirements were replaceable by NaIO4. These findings strongly suggest that hydroxylation was being catalyzed by cytochrome P-450. This was confirmed by preparing progesterone-hydroxylating microsomes and Triton N-101-solubilized microsome extracts, and by obtaining a dithionite-reduced carbon monoxide-difference absorption spectrum peak at 455 nm in the solubilized microsome extracts.
J Steroid Biochem Mol Biol 1991 Feb
PMID:Microbial transformation of steroids--VII. Hydroxylation of progesterone by extracts of Phycomyces blakesleeanus. 200 46

16-Dehydroprogesterone reductase (16-DHPR) activity was present in cell extracts of Eubacterium sp. strain 144 only when the organism was grown in the presence of steroids containing a delta 16-17 double bond and C-20-ketone. Cells grown with 16-dehydropregnenolone contained 16-DHPR activity but lacked delta 4-5-3-keto steroid reductase activity. Pyruvate or sodium dithionite served as electron donors for 16-DHPR and both reactions required methyl viologen as an electron carrier. Neither NADH nor NADPH, with or without flavin nucleotides, were used by 16-DHPR. Enzyme activity was detected in the cytoplasmic fraction (40%) and membrane fraction (20%) of crude cell extracts, but 40% of the activity was unaccounted for following ultracentrifugation. 16-DHPR activity was unaffected by pH in potassium phosphate buffer over the range 5.0 to 8.5, but was inhibited by Tris-HCl above pH 7.0. 16-DHPR activity was inhibited by sulfhydryl reagents, but inhibitors of electron transport reactions or metal chelators did not affect the enzyme.
J Steroid Biochem Mol Biol 1991 Feb
PMID:Characteristics of 16-dehydroprogesterone reductase in cell extracts of the intestinal anaerobe, Eubacterium sp. strain 144. 200 47

The preparation of hybrid histone octamers with wheat histone H2A variants replacing chicken H2A in the chicken octamer is described. The fidelity of the reconstituted hybrid octamers was confirmed by dimethyl suberimidate cross-linking. Polyglutamic-acid-mediated assembly of these octamers on long DNA and subsequent micrococcal nuclease (MNase) digestion demonstrated that, whereas chicken octamers protected 167 base-pairs (representing 2 full turns of DNA), hybrid histone octamers containing wheat histone H2A(1) with its 19 amino acid residue C-terminal extension protected an additional 16 base pairs of DNA against nuclease digestion. The protection observed by hybrid histone octamers containing wheat histone H2A(3) with both a 15 residue N-terminal and a 19 residue C-terminal extension was identical with that observed with H2A(1)-containing hybrid histone octamers with only the 19 residue C-terminal extension. These results suggest that the role of the C-terminal extension is to bind to DNA of the "linker" region. The thermal denaturation of chicken and hybrid core particles was identical in 10 mM-Tris.HCl.20 mM-NaCl, 0.1 mM-EDTA, confirming that there was no interaction between the basic C-terminal extension and DNA of the core particle. Denaturation in EDTA, however, showed that hybrid core particles had enhanced stability, suggesting that the known conformational change of core particles at very low ionic strength allows the C-terminal extension to bind to core particle DNA under these conditions. A model accounting for the observed MNase protection is presented.
J Mol Biol 1991 Apr 20
PMID:Extended C-terminal tail of wheat histone H2A interacts with DNA of the "linker" region. 202 50

Human sperm nuclei were isolated with mixed alkyltrimethylammonium bromide and dithiothreitol (MATAB/DTT) and decondensed by treatments with lithium diiodosalicylate (LIS), sodium chloride, or Tris salts. Concentrations as low as 1 mM LIS induced measurable nuclear swelling compared to 600 mM required for the other two salts. As measured by image analyses, the projected nuclear area increased linearly up to approximately fivefold with LIS concentrations up to 10 mM. Swollen nuclei also maintained the elliptical shapes characteristic of the human sperm head. Expanded sperm nuclei of three men were hybridized with a fluorescently labeled 3.4 kb Y chromosome-specific repetitive DNA probe; 50.1% of the nuclei of each semen sample showed fluorescent labeling over a part of the nucleus indicating presence of the Y chromosome. In comparison, unswollen sperm did not yield reliable hybridization signals. This procedure is suitable for determining the proportion of human sperm with Y chromosomes and can be used to evaluate sperm separation techniques. The availability of probes specific for most human chromosomes suggests that this procedure may find general application in studies of sperm chromosomal constitution.
Mol Reprod Dev 1990 Nov
PMID:Fluorescence in situ hybridization to Y chromosomes in decondensed human sperm nuclei. 207 35

Hypothyroid-induced atrophy of cardiac myocytes was examined in adult female rats in an effort to correlate hemodynamic and cellular changes associated with this disorder. Additional rats were studied 6 weeks after discontinuing antithyroid treatment to determine if structural and functional changes were completely reversible. To induce hypothyroidism, rats were injected daily with propylthiouracil (PTU) for 4 weeks. Control animals were injected similarly with Tris buffer. At the end of the treatment period, hemodynamic measurements were made prior to obtaining isolated myocytes. Cell volume, length, and cross-sectional area were obtained from the septum, and left and right ventricles of treated and untreated rats. After four weeks treatment with PTU, body weight was unchanged but heart weight was significantly reduced by 24%. Characteristic hemodynamic changes associated with hypothyroidism in the rat were noted (eg. reduced heart rate, cardiac output, dP/dtmax, and ventricular pressure). Cell volume was significantly smaller in hypothyroid rats primarily due to a reduction in myocyte cross-sectional area. The hemodynamic and cellular response to hypothyroidism was similar in the right and left ventricle. Six weeks after discontinuing PTU treatment, cellular and hemodynamics changes had returned to normal. It was concluded that hypothyroidism caused a true cardiac atrophy which was reversible. Reduced myocyte cross-sectional area was responsible for most of the myocyte atrophy.
J Mol Cell Cardiol 1990 Dec
PMID:Influence of hypothyroidism and the reversal of hypothyroidism on hemodynamics and cell size in the adult rat heart. 208 54

The formation of active subtilisin E from pro-subtilisin E requires the removal of the N-terminal pro-sequence of 77 residues. Pro-subtilisin E produced in Escherichia coli using a pINIII-ompA vector was first extracted with 6 M guanidine-HCl and 5 M urea and purified to homogeneity in the presence of 5 M urea. Upon drop dialysis against 0.2 M sodium phosphate buffer (pH 6.2), the purified pro-subtilisin in 5 M urea was processed to active subtilisin of which the N-terminal sequence and migration in SDS-polyacrylamide gel electrophoresis were identical to those of authentic active subtilisin E. This process was found to be very sensitive to the ionic strengths and anions used. Under the optimum conditions (dialysis against 0.5 M (NH4)2SO4 and 1 mM CaCl2 in 10 mM Tris-HCl buffer (pH 7.0) at 4 degrees C for 1 h), approximately 20% of pro-subtilisin E was converted to active subtilisin E. The activation process was not inhibited by Streptomyces subtilisin inhibitor, and pro-subtilisin E in which the active site was mutated (Asp32 to Asn) was unable to be processed under the optimum conditions. These results confirmed the previous hypothesis that the processing of pro-subtilisin occurs by an intramolecular, autoprocessing mechanism.
Mol Microbiol 1990 Feb
PMID:Pro-subtilisin E: purification and characterization of its autoprocessing to active subtilisin E in vitro. 211 Sep 97


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