The prototype for a new series of ratio-mode fluorescence indicators of cytosolic free Ca2+ concentration ( [Ca2+Ii) has been developed. The fluorophore, termed FluoRhod,S is a hybrid containing elements of the closely related fluorescein and rhodamine structures. The novel feature of the prototype indicator, FluoR hod-2, which incorporates a tetracarboxylate chelating element similar to that of 1,2bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid (BAPTA), is that it shifts from rhodamine-to fluorescein-like fluorescence on binding Ca2? The synthesis and properties of a series of tetracarboxylate derivatives of the FluoRhod fluorophore are described which led to the FluoRhod-2 structure with an apparent dissociation constant for Ca2+ and pK, adjusted to meet the requirements for a [Ca2+Ii indicator in the ratio mode. The excitation and emission maxima of FluoRhod-2 are 537 nm and 566 nm in the absence of Ca2+ and 480 nm and 537 nm in the presence of Ca2+. The indicator can be used in either the dual excitation or dual emission mode or the ratio of these two ratios can be used by making dual excitation and dual emission measurements. The brightness of FluoRhod-2 is comparable to that of fura-2 with the advantage of excitation in the visible range. FluoRhod-2 is insensitive to pH from 6.5 to 7.5 when used in the ratio mode, irrespective of the extent to which the indicator is complexed to Ca2?The uses and limitations of the current generation of fluorescent indicators of cytosolic free Ca2 + concentration ([Ca2 'Ii) are well established. Fura-2 has been most used; it is about 30 times brighter than the prototype indicator, quin2.' The calibration problem is overcome because the excitation spectrum of fura-2 shifts when Ca2+ is bound and therefore allows [Ca2+Ii to be measured directly from the ratio of fluorescent emission intensities at two excitation wavelengths (usually 340 and 380 nm).2 The limitations of fura-2 are mainly the short wavelengths of the excitation maxima and the insolubility of the acetoxymethyl ester (AME) derivative 3,4 used to achieve cell permeation. To overcome this the indicator is frequently dispersed in a surfactant, pluronic F-127.4*5 Fura-2 AME is hydrolysed more slowly than quin2 AME and under some conditions bright spots of particulate fluorescence are observed in Both the slow hydrolysis and the association of the indicator with intracellular structures may be due to the hydrophobicity of fura-2 AME. The most recent versions of fluorescent indicators for [Ca2 'Ii are based on the fluorescein or rhodamine chromophores incorporated into BAPTA derivatives to give fluo-3 and rhod-2 with excitation wavelengths at 499 nm and 521 nm respectively.8 Calcium binding causes an increase in fluorescence emission but as the unchelated indicators are not significantly fluorescent the indicators cannot be used for ratio measurements of [Ca2 'Ii. There are, however, major potential advantages of using the fluorescein or rhodamine fluorophores for [Ca2 +Ii indicators t Abbreoiations used: AME, ace...
The effect of increased benzo substitution on the cation-binding strength of diaza-18-crown-6 has been investigated. The planarity constraint imposed by increased substitution reduces the effective cavity size thus giving rise to an increased selectivity for sodium over potassium, magnesium and calcium that is sufficient to provide a class of indicators suitable for measuring intracellular free sodium. The cation binding parameters and the associated spectral changes of a selection of compounds with varied aryl fluorination patterns have shown that there is very little electronic interaction between the aromatic rings and the bound cation through the heteroatoms of the crown ether. Only small adjustments to the cation affinity can therefore be achieved via alteration of the aryl substitution patterns. Aryl substitution adjacent to the hetero atoms gives a reduction in the cation affinity of the tribenzo-crown ether, similar to that found on fluorination of the calcium chelator BAPTA ortho to the oxyethylene bridge. These observations have led to the preparation of a new indicator for intracellular sodium that we have called FCrown-1. The new indicator has a dissociation constant for sodium of 11 mmol dm" and a selectivity for sodium over potassium of 173-fold. The sodium concentration is reported by the chemical shift (fast exchange) of one of the two types of fluorine present with a maximum sodium-induced shift of 4.6 ppm downfield. The resonance arising from the other fluorine substituents is insensitive to sodium and acts as an internal chemical shift reference at 8.8 to 13.4 ppm upfield from the reporting signal.Paper 2/05554F
The menadiol oxidase activity of Arum maculatum mitochondria has been solubilized and fractionated. A preparation has been obtained which has an increased specific activity and a greatly decreased polypeptide composition when compared to the mitochondria. This preparation retains normal inhibitor sensitivities in that the oxidation of menadiol remains insensitive to cyanide and is inhibited by aromatic hydroxamates. Metal analyses of the preparation showed that only iron was closely correlated with the oxidase activity. No unusual lipid components were detected in the preparation. The results are discussed in relation to chemical quinol oxidation mechanisms and to several recent hypotheses concerning the nature of the higher plant alternative oxidase.The phenomenon of thermogenesis in plants has been documented for over 200 years but it was not until the 1930s that van Herk (28) was able to ascribe the process to the presence of a cyanide-insensitive respiratory pathway which subsequently became known as the alternative pathway. The molecular nature of this pathway has however remained obscure, because of the apparent lack of any distinguishing spectroscopic features of the components involved. The diverse suggestions of its possible nature have been the subject of several relatively recent reviews (1,10,13,23 quinol oxidase preparation. These results are discussed in relation to the above-mentioned suggestions as to the nature of the oxidase.MATERIALS AND METHODS Preparations. Mitochondria were prepared from the unopened spadices ofArum maculatum by a standard procedure (3). They were either used immediately or stored in liquid N2 until required. Protein content of the mitochondria was estimated from the amount of Cyt b present and assuming 0.4 nmol Cyt b/mg protein. The Cyt b was estimated from a dithionite-reduced minus oxidized difference spectrum and with an extinction coefficient at 560 nm of 20 mm-' cm-'.Menadiol was prepared from the quinone by a method described previously (16). The final solid was a light purple color and was stable for several months under N2. A stock solution of 50 mm in 96% ethanol with 10 mM HCI was used. This solution was colorless and stable for several weeks at -20°C. It was discarded as soon as any coloration was noted.Quinol Oxidase Assay. Activity was measured under dark conditions with a Clark-type 02 electrode in a buffer containing 50 mM Mes, 2 mm EDTA, and 1 mm KCN at 25°C and pH 6.0; 0.5 mM menadiol was added and the basal autoxidation rate was noted. The nonenzymic rate was always in the range of 5 to 10 nmol 02 consumed/ml min. The reaction was then started by addition of an appropriate amount of enzyme. That the stimulation observed was caused by quinol oxidase was confirmed by inhibition with 1 mm SHAM.2 Rates are pseudo-first order with quinol and hence the reaction profiles are curved. Rates were estimated from the initial part of these curves. Units of oxidase activity are defined as nmol 02 consumed/min at 25C and 0.5 mM menadiol.Spectra.
The effects of alkyl substitution in the aminodiacetic groups of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) have been examined systematically with the aim of increasing the affinity for Ca2+ of novel 19F N M R and fluorescent Ca2+ indicators based on the BAPTA structure into the range required to measure the cytosolic free Ca2+ concentration ( [Ca2+Ii). Single methylation on the j.3-carbon of each aminodiacetic acid group of BAPTA derivatives gave increases in the association constant for Ca2+ [K(Ca*+)] of up to 22-fold, depending o n the structure of the parent BAPTA derivative. This was shown to be the most effective alkylation for BAPTA derivatives to raise K(Ca2+) with a minimal increase in pK,, and no loss of selectivity for Ca2+, so that the insensitivity of Ca2+ binding t o p H and [ Mg2+] over the physiological ranges was maintained.(1) Single methylation on the P-carbon of each aminodiacetic acid group of BAPTA derivatives symmetrically substituted with single fluorines at the 4-position (4FBAPTA) or at the 5-position (5FBAPTA) raised log K(Ca2+) from 5.61 to 6.81 and 6.12 to 7.47, respectively. The corresponding pK, , values (average of the t w o highest pK, values in the molecule) were increased from 4.50 to 6.02 and 5.85 t o 6.59. The 19F N M R spectroscopic properties of the methylated derivatives were unchanged compared with the parent compounds except for slower exchange rates. These new methylated derivatives of 4FBAPTA and 5FBAPTA therefore extend the range of K( Ca2+) available for 19F NMR indicators of [Ca2+Ii.(2) Single methylation o n the P-carbon of each aminodiacetic acid group of BAPTA derivatives symmetrically substituted at the 5-position on the aromatic rings with trifluoromethyl groups increased log K(Ca2+) from 4.83 to 6.14 and the pK, , from 5.39 to 5.90, respectively. There is a Ca2+-induced chemical shift of the 19F N M R resonance of 0.81 ppm upfield with slow exchange at 376 M H z and 30 "C. This compound was selected as a prototype for 19F N M R indicators with enhanced sensitivity through the incorporation of two trifluoromethyl groups to replace the t w o single fluorine substituents in the current 19F N M R indicators.
Beef heart mitochondrial bc 1 complex (ubiquinone-cytochrome c oxidoreductase) has been assayed by its ability to catalyse the reaction of duroquinol reduction of ferricytochrome c. When the isolated complex is reincorporated into lipid vesicles, the enzyme-catalysed electron transfer rate becomes uncoupler-sensitive. Initial experiments suggest that a protonmotive force is generated across the vesicles when electron transfer is initiated. Both A* and ApH components of this protonmotive force then influence an internal rate constant of the bcl complex. Mitochondrial bc complexLipid vesicle
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