Binding of the transition state analog MgADP-fluoroaluminate to F1-ATPase.
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Escherichia coli F1-ATPase from mutant betaY331W was potently inhibited by fluoroaluminate plus MgADP but not by MgADP alone. beta-Trp-331 fluorescence was used to measure MgADP binding to catalytic sites. Fluoroaluminate induced a very large increase in MgADP binding affinity at catalytic site one, a smaller increase at site two, and no effect at site three. Mutation of either of the critical catalytic site residues beta-Lys-155 or beta-Glu-181 to Gln abolished the effects of fluoroaluminate on MgADP binding. The results indicate that the MgADP-fluoroaluminate complex is a transition state analog and independently demonstrate that residues beta-Lys-155 and (particularly) beta-Glu-181 are important for generation and stabilization of the catalytic transition state. Dicyclohexylcarbodiimide-inhibited enzyme, with 1% residual steady-state ATPase, showed normal transition state formation as judged by fluoroaluminate-induced MgADP binding affinity changes, consistent with a proposed mechanism by which dicyclohexylcarbodiimide prevents a conformational interaction between catalytic sites but does not affect the catalytic step per se. The fluorescence technique should prove valuable for future transition state studies of F1-ATPase. (+info)
The thrombospondin receptor integrin-associated protein (CD47) functionally couples to heterotrimeric Gi.
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Integrin-associated protein (IAP; CD47) is a thrombospondin receptor that forms a signaling complex with beta3 integrins resulting in enhanced alphavbeta3-dependent cell spreading and chemotaxis and, in platelets, alphaIIbbeta3-dependent spreading and aggregation. These actions of CD47 are all specifically abrogated by pertussis toxin treatment of cells. Here we report that CD47, its beta3 integrin partner, and Gi proteins form a stable, detergent-soluble complex that can be recovered by immunoprecipitation and affinity chromatography. Gialpha is released from this complex by treatment with GTP or AlF4. GTP and AlF4 also reduce the binding of CD47 to its agonist peptide (4N1K) derived from thrombospondin, indicating a direct association of CD47 with Gi. 4N1K peptide causes a rapid decrease in intraplatelet cyclic AMP levels, a Gi-dependent event necessary for aggregation. Finally, 4N1K stimulates the binding of GTPgamma35S to membranes from cells expressing IAP and alphavbeta3. This functional coupling of CD47 to heterotrimeric G proteins provides a mechanistic explanation for the biological effects of CD47 in a wide variety of systems. (+info)
The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions.
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Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes. (+info)
Agonist-induced translocation of Gq/11alpha immunoreactivity directly from plasma membrane in MDCK cells.
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Both Gsalpha and Gqalpha are palmitoylated and both can move from a crude membrane fraction to a soluble fraction in response to stimulation with agonists. This response may be mediated through depalmitoylation. Previous studies have not demonstrated that endogenous guanine nucleotide-binding regulatory protein (G protein) alpha-subunits are released directly from the plasma membrane. We have examined the effect of agonist stimulation on the location of Gq/11alpha immunoreactivity in Madin-Darby canine kidney (MDCK) cells. Bradykinin (BK; 0.1 microM) caused Gq/11alpha, but not Gialpha, to rapidly translocate from purified plasma membranes to the supernatant. AlF and GTP also caused translocation of Gq/11alpha immunoreactivity from purified plasma membranes. BK caused translocation of Gq/11alpha immunoreactivity in intact cells from the basal and lateral plasma membranes to an intracellular compartment as assessed by confocal microscopy. Thus Gq/11alpha is released directly from the plasma membrane to an intracellular location in response to activation by an agonist and direct activation of G proteins. G protein translocation may be a mechanism for desensitization or for signaling specificity. (+info)
A mutation in the heterotrimeric stimulatory guanine nucleotide binding protein alpha-subunit with impaired receptor-mediated activation because of elevated GTPase activity.
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It has been reported that substitution of Arg258, a residue within the GTPase domain of the heterotrimeric guanine nucleotide binding protein (G protein) alpha-subunit (alphas), to alanine (alphas-R258A) results in decreased activation by receptor or aluminum fluoride (AlF4-) and increased basal GDP release. Arg258 interacts with Gln170 in the helical domain, and, presumably, loss of this interaction between the GTPase and helical domain leads to more rapid GDP release, resulting in decreased activation by AlF4- and increased thermolability. In this study, we mutate Gln170 to alanine (alphas-Q170A) and demonstrate that this mutant, like alphas-R258A, has decreased activation by AlF4-, increased thermolability (both reversed in the presence of excess guanine nucleotide), and an increased rate of GDP release. However, unlike alphas-R258A, alphas-Q170A does not have impaired receptor-mediated activation. Therefore, this interdomain interaction is critical to maintain normal guanine nucleotide binding (and hence normal activation by AlF4-) but is not important for receptor-mediated activation. In single turnover GTPase assays, the catalytic rate for GTP hydrolysis of alphas-R258A was 14-fold higher than normal whereas that of alphas-Q170A was unaffected. Examination of the alphas crystal structure suggests that Arg258, through interactions with Glu50, might constrain the position of Arg201, a residue critical for catalyzing the GTPase reaction. This is an example of a mutation in a heterotrimeric G protein that results in an increased intrinsic GTPase activity and provides another mechanism by which G protein mutations can impair signal transduction. (+info)
MgATP-independent hydrogen evolution catalysed by nitrogenase: an explanation for the missing electron(s) in the MgADP-AlF4 transition-state complex.
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When the MoFe (Kp1) and Fe (Kp2) component proteins of Klebsiella pneumoniae nitrogenase are incubated with MgADP and AlF4(-) in the presence of dithionite as a reducing agent, a stable putative transition-state complex is produced [Yousafzai and Eady (1997) Biochem. J. 326, 637-640]. Surprisingly, the EPR signal associated with reduced Kp2 is not detectable, but Kp1 retains the S=3/2 EPR signal arising from the dithionite reduced state of the MoFe cofactor centre of the protein. This is consistent with the [Fe4S4] centre of the Fe protein in the complex being oxidized, and similar observations have been made with the complex of Azotobacter vinelandii [Spee, Arendsen, Wassink, Marritt, Hagen and Haaker (1998) FEBS Lett. 432, 55-58]. No satisfactory explanation for the fate of the electrons lost by Kp2 has been forthcoming. However, we report here that during the preparation of the MgADP-AlF4 K. pneumoniae complex under argon, H2 was evolved in amounts corresponding to one half of the FeMoco content of the Kp1 (FeMoco is the likely catalytic site of nitrogenase with a composition Mo:Fe7:S9:homocitrate). This is surprising, since activity is observed during incubation in the absence of MgATP, normally regarded as being essential for nitrogenase function, and in the presence of MgADP, a strong competitive inhibitor of nitrogenase. The formation of H2 by nitrogenase in the absence of AlF4(-) was also observed in reaction mixtures containing MgADP but not MgATP. The reaction showed saturation kinetics when Kp1 was titrated with increasing amounts of Kp2 and, at saturation, the amount of H2 formed was stoichiometric with the FeMoco content of Kp1. The dependence of the rate of formation of H2 on [MgADP] was inconsistent with the activity arising from MgATP contamination. We conclude that MgATP is not obligatory for H+ reduction by nitrogenase since MgADP supports a very low rate of hydrogen evolution. (+info)
Propofol inhibits muscarinic acetylcholine receptor-mediated signal transduction in Xenopus Oocytes expressing the rat M1 receptor.
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The effects of propofol, 2,6-diisopropylphenol, an intravenous general anesthetic, on signal transduction mediated by the rat M1 muscarinic acetylcholine (ACh) receptor (M1 receptor) were examined in electrophysiological studies by analyzing receptor-stimulated, Ca2+-activated Cl--current responses in the Xenopus oocyte expression system. In oocytes expressing the M1 receptor, ACh induced the Ca2+-activated C1- current, in a dose-dependent manner (EC50= 114 nM). Propofol (5-50 microM) reversibly and dose-dependently inhibited induction of the Ca2+-activated Cl- current by ACh (100 nM) (IC50=5.6 microM). To determine a possible site affected by propofol in this signal transduction, we tested the effects of this anesthetic (10 microM) on the activation of current by injection of CaCl2 and aluminum fluoride (AlF4-). Propofol did not affect activation of the current by the intracellular injected Ca2+, or activation of the current by the intracellular injected AlF4-. These results indicate that propofol does not affect G protein, the inositol phosphate turnover, release of Ca2+ from Ca2+ store or the Ca2+-activated Cl- channel. Propofol apparently inhibits the M1 receptor-mediated signal transduction at the receptor site and/or the site of interaction between the receptor and associated G protein. (+info)
Elastin calcification and its prevention with aluminum chloride pretreatment.
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Elastin, an abundant structural protein present in the arterial wall, is prone to calcification in a number of disease processes including porcine bioprosthetic heart valve calcification and atherosclerosis. The mechanisms of elastin calcification are not completely elucidated. In the present work, we demonstrated calcification of purified elastin in rat subdermal implants (Ca(2+) = 89.73 +/- 9.84 microgram/mg after 21 days versus control, unimplanted Ca(2+) = 0.16 +/- 0.04 microgram/mg). X-ray diffraction analysis along with resolution enhanced FTIR spectroscopy demonstrated the mineral phase to be a poorly crystalline hydroxyapatite. We investigated the time course of calcification, the effect of glutaraldehyde crosslinking on calcification, and mechanisms of inhibition of elastin calcification by pretreatment with aluminum chloride (AlCl(3)). Glutaraldehyde pretreatment did not affect calcification (Ca(2+) = 89.06 +/- 17.93 microgram/mg for glutaraldehyde crosslinked elastin versus Ca(2+) = 89.73 +/- 9.84 microgram/mg for uncrosslinked elastin). This may be explained by radioactive ((3)H) glutaraldehyde studies showing very low reactivity between glutaraldehyde and elastin. Our results further demonstrated that AlCl(3) pretreatment of elastin led to complete inhibition of elastin calcification using 21-day rat subdermal implants, irrespective of glutaraldehyde crosslinking (Ca(2+) = 0.73-2.15 microgram/mg for AlCl(3) pretreated elastin versus 89.73 +/- 9.84 for untreated elastin). The AlCl(3) pretreatment caused irreversible binding of aluminum ions to elastin, as assessed by atomic emission spectroscopy. Moreover, aluminum ion binding altered the spatial configuration of elastin as shown by circular dichroism (CD), Fourier transform infrared (FTIR), and (13)C nuclear magnetic resonance (NMR) spectroscopy studies, suggesting a net structural change including a reduction in the extent of beta sheet structures and an increase in coil-turn conformations. Thus, it is concluded that purified elastin calcifies in rat subdermal implants, and that the AlCl(3)-pretreated elastin completely resists calcification due to irreversible aluminum ion binding and subsequent structural alterations caused by AlCl(3). (+info)