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101.
102.
Estradiol 17 beta-dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, oxidoreductase activities copurified from the cytosol of human-term placenta as a homogeneous protein (native enzyme), were reactivated at equal rates to 100% activity following complete inactivation in the presence of cofactor (NADPH) with the affinity alkylator estrone 3-(bromoacetate). Reactivation was accomplished by base-catalyzed hydrolysis of steroidal ester-amino acid linkages in the enzyme active site. The rate of enzyme reactivation was pH dependent. In identical studies without NADPH, only 12% of the original enzyme activity was restored. Completely reactivated enzyme was repurified by dialysis. Enzyme in control mixtures (control enzyme) that contained estrone in place of alkylator was treated the same as the reactivated enzyme. Reactivated enzyme exhibited a 6.0-fold lower affinity for common substrates, a 1.8-fold lesser affinity for NAD+ and NADH, and the same affinity for NADP+ and NADPH compared to control enzyme. In incubations that included NADPH, the reactivated enzyme maintained full activity during a 20-h second exposure to estrone 3-(bromoacetate), but in identical incubations without NADPH, the reactivated enzyme was rapidly inactivated at the same rate as the control and native enzymes. The control and reactivated enzymes were inactivated at equal rates by 16 alpha-(bromoacetoxy)estradiol 3-(methyl ether) in the presence or absence of cofactor (NADP+) and exhibited similar Kitz and Wilson inhibition constants for this affinity alkylator. Estrone 3-(bromo[2'-14C]acetate) incubated with native enzyme and NADPH produced radiolabeled 3-(carboxymethyl)histidine and S-(carboxymethyl)cysteine.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
103.
104.
Jaagsiekte sheep retrovirus Env protein stabilizes retrovirus vectors against inactivation by lung surfactant, centrifugation, and freeze-thaw cycling 下载免费PDF全文
Jaagsiekte sheep retrovirus (JSRV) replicates in the lungs of sheep and causes the secretion of copious lung fluid containing the virus. Adaptation of JSRV to infection and replication in the lung and its apparent resistance to the denaturing activity of lung fluid suggest that vectors based on JSRV would be useful for gene therapy targeted to the lung. We show here that a retrovirus vector bearing the JSRV Env is stable during treatment with lung surfactant while an otherwise identical vector bearing an amphotropic Env is inactivated. Furthermore, the JSRV vector was stable during centrifugation, allowing facile vector concentration, and showed no loss of activity after six freeze-thaw cycles. However, the JSRV vector was inactivated by standard disinfectants, indicating that JSRV vectors pose no unusual safety risk related to their improved stability under other conditions. 相似文献
105.
Ceccarelli C Liang ZX Strickler M Prehna G Goldstein BM Klinman JP Bahnson BJ 《Biochemistry》2004,43(18):5266-5277
The crystal structure of NAD(+)-dependent alcohol dehydrogenase from Bacillus stearothermophilus strain LLD-R (htADH) was determined using X-ray diffraction data at a resolution of 2.35 A. The structure of homotetrameric htADH is highly homologous to those of bacterial and archaeal homotetrameric alcohol dehydrogenases (ADHs) and also to the mammalian dimeric ADHs. There is one catalytic zinc atom and one structural zinc atom per enzyme subunit. The enzyme was crystallized as a binary complex lacking the nicotinamide adenine dinucleotide (NAD(+)) cofactor but including a zinc-coordinated substrate analogue trifluoroethanol. The binary complex structure is in an open conformation similar to ADH structures without the bound cofactor. Features important for the thermostability of htADH are suggested by a comparison with a homologous mesophilic enzyme (55% identity), NAD(+)-dependent alcohol dehydrogenase from Escherichia coli. To gain insight into the conformational change triggered by NAD(+) binding, amide hydrogen-deuterium exchange of htADH, in the presence and absence of NAD(+), was studied by HPLC-coupled electrospray mass spectrometry. When the deuteron incorporation of the protein-derived peptides was analyzed, it was found that 9 of 21 peptides show some decrease in the level of deuteron incorporation upon NAD(+) binding, and another 4 peptides display slower exchange rates. With one exception (peptide number 8), none of the peptides that are altered by bound NAD(+) are in contact with the alcohol-substrate-binding pocket. Furthermore, peptides 5 and 8, which are located outside the NAD(+)-binding pocket, are notable by displaying changes upon NAD(+) binding. This suggests that the transition from the open to the closed conformation caused by cofactor binding has some long-range effects on the protein structure and dynamics. 相似文献
106.
Navaneetham D Jin L Pandey P Strickler JE Babine RE Abdel-Meguid SS Walsh PN 《The Journal of biological chemistry》2005,280(43):36165-36175
Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 angstroms (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI loop 1 11TGPCRAMISR20 and loop 2 34FYGGC38 were tested for their ability to inhibit FXIa. The P1 site mutation R15A completely abolished its ability to inhibit FXIa. IC50 values for the wild type protein and the remaining mutants were as follows: PN2KPI WT, 1.28 nM; P13A, 5.92 nM; M17A, 1.62 nM; S19A, 1.86 nM; R20A, 5.67 nM; F34A, 9.85 nM. The IC50 values for the M17A and S19A mutants were not significantly different from those obtained with wild type PN2KPI. These functional studies and activated partial thromboplastin time analysis validate predictions made from the PN2KPI-FXIac co-crystal structure and implicate PN2KPI residues, in descending order of importance, Arg15, Phe34, Pro13, and Arg20 in FXIa inhibition by PN2KPI. 相似文献
107.
Efficient expression of the yeast metallothionein gene in Escherichia coli. 总被引:2,自引:0,他引:2 下载免费PDF全文
The yeast metallothionein gene CUP1 was cloned into a bacterial expression system to achieve efficient, controlled expression of the stable, unprocessed protein product. The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, and zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP1 acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium. 相似文献
108.
Perception of inert particles by calanoid copepods: behavioral observations and a numerical model 总被引:2,自引:0,他引:2
Bundy Marie H.; Gross Thomas F.; Vanderploeg Henry A.; Rudi Strickler J. 《Journal of plankton research》1998,20(11):2129-2152
High-resolution video showed freely swimming Diaptomus sicilisattacking and capturing inert 5O µm polystyrene beadsthat were outside the influence of the copepod feeding current.The beads were frequently more than half a body length awayand were attacked after the bow wake of the movingcopepod displaced the bead away from the copepod. To investigatethe hypothesis that deformation of streamlines around the copepodand its first antennae stimulated the attack response, a finiteelement numerical model was constructed. The model describedthe fluid interactions between a large object approaching asmaller object in a laminar flow at Reynolds number 5, whichis characteristic of the fluid regime experienced by foragingcopepods. The model revealed that fluid velocity fluctuationsand streamline deformations arose in the region between thetwo objects as separation distance between the objects decreased.The video observations and the model results support the hypothesesthat chemoreception is not required for the detection and captureof large phytoplankton cells [Vanderploeg et ai, in Hughes,R.N. (ed.), Behavioral Mechanisms of Food Selection. NATO ASISeries G20, 1990; DeMott and Watson, /. Plankton Res., 13, 1205-1222,1991], and that swimming behavior plays an integral role inprey detection.
4Present address: Academy of Natural Sciences Estuarine ResearchCenter, 10545 Mackall Road, St Leonard, MD 20685, USA 相似文献
109.
James L. Thomas Brett W. Evans Gustavo Blanco Robert W. Mercer J. Ian Mason Stuart Adler William E. Nash Keith E. Isenberg Ronald C. Strickler 《The Journal of steroid biochemistry and molecular biology》1998,66(5-6):327-334
3β-Hydroxysteroid dehydrogenase/steroid Δ5 → 4-isomerase (3β-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3β-HSD/isomerase (monomeric Mr=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.01). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3β-HSD activity, whereas the Km and Vmax values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The Vmax (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean Vmax (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3β-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar Vmax values for substrate oxidation by the 3β-HSD activity. The 3β-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD+ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD+ was dramatically decreased. Based on these kinetic measurements, His261 appears to be a critical amino acid residue for the 3β-HSD activity, and Tyr253 or Tyr254 participates in the isomerase activity of human type I (placental) enzyme. 相似文献
110.
In human placenta, 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric Mr = 41,000), gel filtration at constant specific enzyme activity (Mr = 77,000), and a single NH2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone (Km = 1.6 microM, Vmax = 48.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.4 microM, Vmax = 48.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.3 microM, Vmax = 914.2 nmol/min/mg) and 5-androstene-3,17-dione (Km = 27.6 microM, Vmax = 888.4 nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and androstenedione, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD+) reduction (mean Km +/- SD using C-19 and C-21 dehydrogenase substrates = 26.4 +/- 0.8 microM, mean Vmax = 73.2 +/- 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37 degrees C), stability in storage and solution, effects of divalent cations, and the single NH2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles. 相似文献