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101.
Prolonged agonist exposure often induces downregulation of G protein-coupled receptors (GPCRs). Although downregulation of the prototypical beta(2)-adrenergic receptor (beta(2)AR) has been extensively studied, the underlying mechanisms have yet to be resolved. As even less is known about the beta(1)-subtype, we investigated the downregulation of human beta(1)AR stably expressed in Chinese hamster fibroblasts in response to the agonist isoproterenol or the cell-permeable, chlorophenylthio-cAMP (CPT-cAMP). While either effector mediated decreases in both beta(1)AR binding activity and steady-state beta(1)AR mRNA levels, there were significant differences in their actions. Whereas agonist-mediated downregulation of beta(1)AR followed first-order kinetics, that induced by CPT-cAMP was delayed for several hours and approximately 50% of the former. Furthermore, agonist but not CPT-cAMP induced beta(1)AR internalization, and inhibiting internalization also suppressed agonist-mediated downregulation. The latter, however, was more sensitive than the former to agonist concentration (EC(50) of 0.3 vs 48 nM). Thus, at < or =1 nM agonist, downregulation occurred without internalization and with a pattern similar to that mediated by CPT-cAMP. The amounts of beta(1)AR downregulated or internalized were proportional to initial receptor levels but reached saturation at approximately 2 and 3 pmol/mg of protein, respectively. The fate of beta(1)AR protein during downregulation was determined by immunoblotting with anti-C-terminal antibodies. In agonist-treated cells, beta(1)AR protein disappeared with time and without any immunoreactive degradation products. Agonist-mediated downregulation of the human beta(1)AR appears to be a complex process that consists of both agonist- and cAMP-specific components. The former involves both receptor internalization and degradation whereas the latter involves a reduction in receptor mRNA. 相似文献
102.
2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain (where its levels are 170-folds higher than those of anandamide), is produced by neurons in an activity- and calcium-dependent manner, and is rapidly eliminated. The mechanism of 2-AG inactivation is not completely understood, but is thought to involve carrier-mediated transport into cells followed by enzymatic hydrolysis. We examined the possible role of the serine hydrolase, monoglyceride lipase (MGL), in brain 2-AG inactivation. We identified by homology screening a cDNA sequence encoding for a 303-amino acid protein, which conferred MGL activity upon transfection to COS-7 cells. Northern blot and in situ hybridization analyses revealed that MGL mRNA is unevenly present in the rat brain, with highest levels in regions where CB1 cannabinoid receptors are also expressed (hippocampus, cortex, anterior thalamus and cerebellum). Immunohistochemical studies in the hippocampus showed that MGL distribution has striking laminar specificity, suggesting a presynaptic localization of the enzyme. Adenovirus-mediated transfer of MGL cDNA into rat cortical neurons increased the degradation of endogenously produced 2-AG in these cells, whereas no such effect was observed on anandamide degradation. These results indicate that hydrolysis via MGL may be a primary route of 2-AG inactivation in intact neuronal cells. 相似文献
103.
Temporal genetic variation in Aedes aegypti populations in Ho Chi Minh City (Vietnam) 总被引:3,自引:0,他引:3
Aedes aegypti, the main vector of dengue viruses in Asia, displays variation in population density over time. The larval habitats of this species being unevenly distributed and transient (depending on cycles of drought and flood), the forces generating temporal variation in gene frequencies in populations are studied. We sampled seven mosquito populations from Ho Chi Minh City (Vietnam) and its suburbs on five occasions between April 1999 and August 2000. We investigated genetic variation by studying isoenzyme and microsatellite polymorphism and susceptibility to a dengue 2 virus strain. Ae. aegypti populations collected during the dry season (January-April) showed genetic differentiation (F(ST) = 0.016, P < 10(-6) for isoenzymes) and showed more differentiated infection rates of the dengue 2 virus. The genetic structure of the population is less marked during the rainy season (F(ST) = 0.081, P < 10(-6)). Thus, environmental factors, such as rainfall and factors related to human activity, such as breeding site density and insecticide treatment, control the genetic structure of Ae. aegypti populations in the short term. The implications of studies of this kind for the design of future control programmes are discussed. 相似文献
104.
Lipopolysaccharyl alpha-galactosyltransferase from Neisseria meningitidis catalyzes the transfer of a galactosyl moiety from the activated donor UDP-Gal to glycoconjugates to yield an elongated saccharide product with net retention of anomeric configuration relative to the donor substrate. Through kinetic analyses in which the concentrations of both substrates are independently varied and through inhibition studies with dead-end analogues of both substrates and with the oligosaccharide product, we have demonstrated that this enzyme follows an ordered bi-bi kinetic mechanism. Various aspects of the chemical mechanism including the possible formation of a covalent glycosyl-enzyme intermediate were also probed using an assortment of strategies. While the results of these investigations were unable to clearly delineate the chemical mechanism of this enzyme, they provide important insights into the catalytic machinery surrounding the events involved in catalysis. 相似文献
105.
Kuznetsov VY Ivanov YD Bykov VA Saunin SA Fedorov IA Lemeshko SV Hoa HB Archakov AI 《Proteomics》2002,2(12):1699-1705
The application of atomic force microscopy (AFM) technique in proteomic research, identification and visualization of individual molecules and molecular complexes within the P450cam containing monooxygenase system was demonstrated. The method distinguishes between the binary protein complexes and appropriate monomeric proteins and, also, between the binary and ternary complexes. The AFM images of the components of a cytochrome P450cam containing monooxygenase system - cytochrome P450cam (P450cam), putidaredoxin (Pd) and putidaredoxin reductase (PdR) - were obtained on a mica support. The molecules of P450cam, Pd and PdR were found to have typical heights of 2.6 +/- 0.3 nm, 2.0 +/- 0.3 and 2.8 +/- 0.3 nm, respectively. The measured heights of the binary Pd/PdR and P450cam/PdR complexes were 4.9 +/- 0.3 nm and 5.1 +/- 0.3 nm, respectively. The binary P450cam/Pd complexes were found to have a typical height of about (3.9 / 5.7 nm) and the ternary PdR/Pd/P450cam complexes, a typical height of about 9.1 +/- 0.3 nm. 相似文献
106.
107.
To promote studies of cytochrome c (Cyt c) ranging from apoptosis to protein folding, a system for facile mutagenesis and high-level expression is desirable. This work used a generally applicable strategy for improving yields of heterologously expressed protein in Escherichia coli. Starting with the yeast Cyt c plus heme lyase construct of Pollock et al. [Pollock, W. B., Rosell, F. I., Twitchett, M. B., Dumont, M. E., and Mauk, A. G. (1998) Biochemistry 37, 6124-6131], an E. coli-based system was designed that consistently produces high yields of recombinant eucaryotic (equine) Cyt c. Systematic changes to the ribosome binding site, plasmid sequence, E. coli strain, growth temperature, and growth duration increased yields from 2 to 3 mg/L to as much as 105 mg/L. Issues related to purification, fidelity of heme insertion, equilibrium stability, and introduction and analysis of mutant forms are described. As an example, variants tailored for folding studies are discussed. These remove known pH-dependent kinetic folding barriers (His26 and His33 and N-terminus), reveal an additional kinetic trap at higher pH due to some undetermined residue(s), and show how a new barrier can be placed at different points in the folding pathway in order to trap and characterize different folding intermediates. In addition, destabilizing glycine mutants in the N-terminal helix are shown to affect the fractional yield of a heme inverted Cyt c isoform. 相似文献
108.
High pressure is an interesting and suitable parameter in the study of the dynamics and stability of proteins. The effects of pressure on proteins delineates its volumic (deltaV degrees ) and energetic (deltaG degrees ) parameters. An enormous amount of effort has been invested by several laboratories in developing basic theory and high pressure techniques that allow the determination of barotropic parameters. Cytochrome P450s, one of the largest super families of heme proteins, are good models for high pressure studies. Two distinct pressure-induced spin transitions of the heme iron in the active site and a P450 to P420 inactivation process have been characterized. The obtained reaction volumes of these two processes for a series of analog-bound cytochrome P450s are compared. We have shown that both the spin volume and the inactivation volume are dependent on the substrate analogs which are known to modulate the polarity and hydration of the heme pocket. Several linear correlations were found between these reaction volumes and the physico-chemical properties of the heme protein such as the polarity-induced exposure of tyrosines, the hydration of the cytochrome CYP101 heme pocket, and the mobility and binding of the substrates indicate that they constitute the main contribution to the complex thermodynamic reaction volume parameters. This interpretation allows us to conclude that cytochrome CYP101, CYP2B4 and CYP102 possess a similar mechanism of substrate binding. Interestingly the barotropic behaviors of monomeric cytochrome P450s are quite different from those of oligomeric and hetorooligomeric cytochrome P450s. The interactions of heterooligomeric subunits influence the stability of individual cytochrome P450s and the asymmetric organization of subunits which can control and modulate the activity and the recognition with NADPH-cytochrome P450 reductase. 相似文献
109.
Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes 总被引:2,自引:0,他引:2
Ward DM Shiflett SL Huynh D Vaughn MB Prestwich G Kaplan J 《Traffic (Copenhagen, Denmark)》2003,4(6):403-415
The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size. 相似文献
110.
Homoisoflavonoids from Ophiopogon japonicus Ker-Gawler 总被引:5,自引:0,他引:5
From the ethyl acetate extract of the tuberous roots of Ophiopogon japonicus (Liliaceae) eight known and five new homoisoflavonoidal compounds were isolated. The new compounds are 5,7-dihydroxy-8-methoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (1), 7-hydroxy-5,8-dimethoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (2), 5,7-dihydroxy-6,8-dimethyl-3-(4'-hydroxy-3'-methoxybenzyl)chroman-4-one (3), 2,5,7-trihydroxy-6,8-dimethyl-3-(3',4'-methylenedioxybenzyl)chroman-4-one (4) and 2,5,7-trihydroxy-6,8-dimethyl-3-(4'-methoxybenzyl)chroman-4-one (5). Their structures have been elucidated by mass and NMR spectroscopy. Compounds 4 and 5 are the first isolated homoisoflavonoids with a hemiacetal function at position 2. 相似文献