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Stimulation of B1 and B2 kinin receptors on cultured rabbit superior mesenteric artery smooth muscle cells with des-Arg9-bradykinin (DBK) and bradykinin (BK), respectively, results in significantly different patterns of intracellular Ca2+ mobilization. Single-cell fluorescence imaging of Fura-2-loaded cells revealed that although both DBK and BK initially triggered similar rapid increases in cytosolic free Ca2+, the DBK response was biphasic and sustained, whereas the BK response was transient. The DBK response was maintained for > or = 20 min with the second phase characterized by an elevated plateau and/or base-line oscillations. The BK response was limited to an initial transient peak with the exception of a few cells, which after a prolonged latency period, exhibited weak but regular base-line oscillations. The initial BK- and DBK-stimulated rises in cytosolic free Ca2+ were dependent on the release of Ca2+ from intracellular stores that seemed to be common for the two agonists. On the other hand, the continuation of the sustained phase of the DBK response required the influx of extracellular Ca2+, as well as continuous receptor occupancy by the agonist. Stimulation of cells with DBK followed by washing and restimulation with the same agonist within < or = 2 min resulted in a second B1 receptor response that was not significantly different from the first response. In contrast, the same protocol with BK yielded a dramatically decreased second B2 receptor response. This attenuation did not seem to be due to a lack of Ca2+ in the agonist-sensitive intracellular stores because DBK elicited a full response after BK stimulation. This study shows that in single cultured RSMA smooth muscle cells, agonist stimulation of B1 receptors generates a sustained intracellular Ca2+ signal, whereas stimulation of B2 receptors promotes rapid and homologous desensitization, resulting in a transient Ca2+ signal. These distinct receptor-specific patterns of Ca2+ mobilization imply significantly different roles for B1 and B2 kinin receptors in vascular smooth muscle cells.

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The infectious pattern of mycoplasmas (Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma genitalium) in mammalian cells was examined using confocal microscopy and flow cytometry combined with cell fractionation and mycoplasma viability determinations. Within 2 h postinfection mycoplasmas parasitize cell surfaces, enter the intracellular spaces and locate throughout the cytoplasmic and perinuclear regions. These mycoplasmas can be cultivated from cytoplasmic and nuclear fractions 96 h later and continue to persist intracellularly for at least 7 days, suggesting a much more active intracellular role for mycoplasmas than had been considered previously.

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The enzyme rhodanese (EC 2.8.1.1) could be reversibly refolded from urea in the presence of lauryl maltoside, beta-mercaptoethanol, and sodium thiosulfate. The unfolding/folding transition monitored using intrinsic fluorescence was resolved into two two-state transitions with midpoints at 3.6 and 5.0 M urea. The analysis assumed an intermediate with an emission maximum at 345 nm. Monitoring anisotropy of intrinsic fluorescence also gave an asymmetric transition. Activity followed one two-state transition centered at 3.6 M urea with no major change of secondary structure. Without thiosulfate or mercaptoethanol, there was one two-state transition at 5.0 M urea giving a species, in dilute urea, with a fluorescence maximum at 345 nm. This intermediate slowly relaxed toward 335 nm (t1/2 = 85 min) if only thiosulfate was absent but without regaining activity. Subsequent addition of thiosulfate led to a first-order recovery of activity (t1/2 = 75 min). Thus, a possible folding intermediate can be trapped which displays increased access of water and solutes to its fluorescent tryptophans. This intermediate conformer, which is flexible, has considerable secondary structure, is inactive, has exposed hydrophobic surfaces, and requires specific reducing conditions to regain full activity. Refolding probably involves an initial, rapid, hydrophobic collapse with acquisition of secondary structure to form the intermediate, followed by slower adjustment to the native global conformation. Final reactivation requires reduction localized at the active site.

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The enzyme rhodanese in the form without transferred sulfur, (E), was inactivated by carboxymethylation with iodoacetic acid (E.IAA), and its conformation was compared with that of E inactivated by oxidative processes (Eox). Formation of E.IAA led to the exposure of binding sites for the fluorescent apolar probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (BisANS). The dissociation constant for BisANS decreased as the concentration of E.IAA decreased and ranged from approximately 200 microM at 1 mg/ml protein to approximately 2 microM at protein concentrations below 0.1 mg/ml. Centrifugation confirmed that E.IAA, but not the underivatized enzyme, could associate. E.IAA was proteolyzable by chymotrypsin or endoproteinase Glu C (V8), while rhodanese containing bound sulfur, ES, was totally refractory, and E was only clipped to a small extent. This constellation of consequences was only previously observed with oxidatively inactivated rhodanese. Fluorescence depolarization measurements of bound BisANS were consistent with exposure of apolar surfaces and association of the protein. The fluorescence spectra of BisANS bound to E.IAA or Eox were identical and distinct from the spectrum of BisANS bound to phenylglyoxal-inactivated ES. Digestion with chymotrypsin was followed using protein and BisANS fluorescence and showed a similar response for E.IAA and Eox. These results indicate that the consequences of forming Eox and E.IAA are very similar. Thus, reaction of the active site sulfhydryl group apparently triggers a conformational change leading to increased protein flexibility and increased exposure of hydrophobic surfaces. In the case of oxidation, the trigger might involve initial formation of an active site sulfenic acid which ultimately gives higher oxidation states that could include disulfides.

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The conformations of sulfur-free and sulfur-containing rhodanese were followed with and without the detergent lauryl maltoside after guanidinium chloride (GdmCl) addition to 5 M to study the apparent irreversibility of denaturation. Without lauryl maltoside, sulfur-containing rhodanese denatured in a transition giving, at approximately 2.3 M GdmCl, 50% of the total denaturation induced change observed by activity, CD, or intrinsic fluorescence. Sulfur-free rhodanese gave more complex behavior by intrinsic fluorescence and CD. CD showed loss of secondary structure in a broad, complex, and apparently biphasic transition extending from 0.5 to 3 M GdmCl. The interpretation of the transition was complicated by time-dependent aggregation due to noncovalent interactions. Results with the apolar fluorescence probe 2-anilinonaphthalene-8-sulfonic acid, implicated apolar exposure in aggregation. Sulfhydryl reactivity indicated that low GdmCl concentrations induced intermediates affecting the active site conformation. Lauryl maltoside prevented aggregation with no effect on activity or any conformational parameter of native enzyme. Transitions induced by GdmCl were still observed and consistent with several phases. Even in lauryl maltoside, an increase in apolar exposure was detected by 2-anilinonaphthalene-8-sulfonic acid, and by protein adsorption to octyl-Sepharose well below the major unfolding transitions. These results are interpreted with a model in which apolar interdomain interactions are disrupted, thereby increasing active site accessibility, before the intradomain interactions.

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Fluorescence studies have been performed on yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) as a function of temperature. Observations of both the intrinsic protein fluorescence and the fluorescence of the noncovalently bound apolar probe 2-(p-toluidinyl)naphthalene-6-sulfonic acid under conditions where hexokinase is monomeric, indicate that significant thermal structural transitions occur in the protein over the physiological range of temperature (0 degrees-40 degrees C) and that there are different temperature-dependent forms of the enzyme. Thermal transitions between these forms are affected by the binding of the substrates D-glucose and ATP-Mg. It therefore appears that catalysis connects conformers that differ in stability and the present results are consistent with models in which hexokinase function is linked to changes in the interactions between the domains into which this protein is folded.

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Studies have been performed to quantitate the binding of the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid (2,8-ANS) to catalytic intermediates of the enzyme rhodanese: the sulfur-substituted form (ES) and the sulfur-free form (E). The molecular 2,8-ANS has not been extensively used for protein studies, and some characterization is presented to demonstrate its usefulness as a probe for apolar binding sites. The molecule 2,8-ANS binds to at least two classes of sites on rhodanese. One class (class 1) is present in the ES form and has a Kd of 1.7 mM. The E form of rhodanese appears to have a second class of sites (class 2) in addition to the class 1 sites. Two independent fluorometric methods of analyzing the class 2 binding of 2,8-ANS to the E form gave an average value for Kd congruent to 179 microM. These fluorometric titrations, together with a Job plot, clearly indicate that 2,8-ANS binds to more than one site on the E form of rhodanese. The apparent apolarity is slightly higher for class 2 sites than for the class 1 sites, but both give Z factors of greater than 85. The substrate thiosulfate is able to displace the probe that is bound to the class 2 sites on the E form of the enzyme. Further, 2,8-ANS is found to be a competitive inhibitor of the catalyzed reaction with an apparent Kd of 170 microM. Circular dichroism measurements detect no significant changes in the average conformation of rhodanese that can be ascribed to the presence of 2,8-ANS.(ABSTRACT TRUNCATED AT 250 WORDS)

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The intrinsic fluorescence of the enzyme rhodanese is quenched by as much as 30% when sulfur is transferred to the free enzyme form, E, giving the sulfur-substituted enzyme, ES. This fluorescence change (lambda ex = 295 nm and lambda em = 335 nm) has been used to quantitate the E and ES forms which are isolatable, obligatory intermediates in rhodanese catalysis. Fluorescence titration was performed using cyanide to irreversibly remove sulfur from ES. The results show a stoichiometry corresponding to 1 bound sulfur/molecule of the ES form of rhodanese (Mr = 33,000). The fluorescence changes were used to measure the concentrations of E and ES when these were in reversible equilibria induced by interactions with the substrates S2O3(2-) and SO3(2-). These results were compared with an equilibrium constant derived from published kinetic studies for the reaction (formula; see text) The very close agreement between the physical and kinetic methods indicate that there are no significant concentrations of intermediates other than E and ES. Overall, the results are compatible with the formation of a persulfide intermediate in rhodanese catalysis and are consistent with conclusions from x-ray crystallography and absorption spectroscopy. In addition, these procedures offer a facile method to measure equilibria between catalytic intermediates in the rhodanese reaction using functionally relevant concentrations.

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The active-site sulfhydryl group in the enzyme thiosulfate sulfurtransferase (rhodanese; thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) is alkylated rapidly by iodoacetic acid in the free enzyme form, E, with complete loss of sulfurtransferase activity. Iodoacetic acid is completely ineffective with the sulfur-substituted form of the enzyme, ES. Iodoacetamide, on the other hand, has no effect on either enzyme form. The competitive enzyme inhibitor, toluenesulfonic acid, protects against inactivation in a strictly competitive way and analysis gives an apparent binding constant for toluenesulfonic acid of 12.5 mM, which is in agreement with studies of its effect on the catalyzed reaction. These results are taken to indicate that iodoacetic acid is an affinity analog for the substrate, thiosulfate, and inactivates because it can use the specific thiosulfate binding interactions, correctly orient its reactive center and displace intraprotein interactions which appear to protect the active-site sulfhydryl group in the E form.

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