Azide Binding Research Articles

Azide inhibition of mitochondrial ATPase

In intact mitochondrial systems azide has been reported as an inhibitor of cytochrome oxidase (Keilin, 1936; Yonetani and Ray, 1965), an uncoupler of oxidative phosphorylation (Loomis and Lipmann, 1949; Judah, 1951; Slater, 1955), and an inhibitor of ATPase (Robertson and Boyer, 1955). Judah (1951) and Robertson and Boyer (1955) reported that azide inhibition of the exchange reactions was associated with energy transfer. Palmieri and Klingenberg (1967) have suggested that azide acts on cytochrome 5 only. Results presented here show that the amount of azide required to inhibit succinate oxidation in tightly coupled mitochondria respiring in state 3 is also sufficient to inhibit purified ATPase. Furthermore, azide inhibition of ATPase activity is enhanced in the presence of ADP to an extent unaccountable on the basis of mass action alone. These findings indicate that azide has an activity on energy transfer which is distinct from azide binding wi.th cytochrome a.

Kinetics of the ferrimyoglobin-azide-system

A novel freeze-quench instrument with a characteristic «dead-time» of 137±18 μs is reported. The prototype has several key features that distinguish it from conventional freeze-quench devices and provide a significant improvement in time resolution: (a) high operating pressures (up to 400 bar) result in a sample flow with high linear rates (up to 200 m s−1); (b) tangential micro-mixer with an operating volume of ∼1 nl yields short mixing times (up to 20 μs); (c) fast transport between the mixer and the cryomedium results in short reaction times: the ageing solution exits the mixer as a free-flowing jet, and the chemical reaction occurs “in–flight” on the way to the cryomedium; (d) a small jet diameter (∼20 μm) and a high jet velocity (∼200 m s−1) provide high sample-cooling rates, resulting in a short cryofixation time (up to 30 μs). The dynamic range of the freeze-quench device is between 130 μs and 15 ms. The novel tangential micro-mixer efficiently mixes viscous aqueous solutions, showing more than 95% mixing at η≤4 (equivalent to protein concentrations up to 250 mg ml−1), which makes it an excellent tool for the preparation of pre-steady state samples of concentrated protein solutions for spectroscopic structure analysis. The novel freeze-quench device is characterized using the reaction of binding of azide to metmyoglobin from horse heart. Reaction samples are analyzed using 77 K optical absorbance spectroscopy, and X–band EPR spectroscopy. A simple procedure of spectral analysis is reported that allows (a) to perform a quantitative analysis of the reaction kinetics and (b) to identify and characterize novel reaction intermediates. The reduction of dioxygen by the bo3-type quinol oxidase from Escherichia coli is assayed using the MHQ technique. In these pilot experiments, low-temperature optical absorbance measurements show the rapid oxidation of heme o3 in the first 137 μs of the reaction, accompanied by the formation of an oxo-ferryl species. X-band EPR spectroscopy shows that a short-living radical intermediate is formed during the oxidation of heme o3. The radical decays within ∼1 ms concomitant with the oxidation of heme b, and can be attributed to the PM reaction intermediate converting to the oxoferryl intermediate F. The general field of application of the freeze-quench methodology is discussed.

CATION TRANSPORT IN YEAST

1. The distribution of azide added to suspensions of bakers' yeast was studied under various conditions. The recovery of azide was estimated in the volume of water into which low concentrations of electrolytes can readily diffuse (anion space). Considerable azide disappeared from this anion space. 2. The incomplete recovery of azide in the anion space is due to its uptake by the cells. This uptake occurs against a concentration gradient at 0 degrees C., and is attributed to binding of azide by cell constituents. 3. Confirmatory evidence is presented that one such constituent is the K carrier in the cell membrane. The azide inhibition of K transport is not mediated by inhibition of cytochrome oxidase in the mitochondria. 4. From the amount of combined azide and the experimentally determined dissociation constant of the K carrier-inhibitor complex, the maximum value for the concentration of this carrier is calculated as 0.1 microM/gm. yeast. 5. The addition of glucose and PO(4) causes a secondary K uptake which is not azide-sensitive and is clearly distinct from the primary, azide-sensitive mechanism. 6. The existence of a separate carrier responsible for Na extrusion is reconsidered. It is concluded that present evidence does not necessitate the assumption that such a carrier is active in yeast.