Biochemical and biophysical studies on cytochrome c oxidase. XIII. Effect of cholate on the enzymic activity.

(1) Cholate is a mixed-type inhibitor of the enzymic activity of cytochrome c oxidase. The rate equations for mixed-type inhibition of the enzyme have been derived, based on Minnaert's Mechanism IV (1961, Biochim. Biophys. Acta 50, 23-34). The Ki of cholate for the free enzyme (E) and for the complexes of the enzyme with cytochrome c (ES and EP) was determined, being 125 and 190 microM, respectively. (2) Comparison of the properties of cholate and the intrinsic inhibitor of cytochrome c oxidase (Van Buuren et al. (1971) Biochim. Biophys. Acta 243, 468-480) with respect to their type of inhibition and their affinity for enzyme, reveals that they are identical.

The kinetics of the reduction of cytochrome c by the superoxide anion radical.

1. At neutral pH ferricytochrome c is reduced by the superoxide anion radical (O2-), without loss of enzymatic activity, by a second order process in which no intermediates are observed. The yield of ferrocytochrome c (82-104%), as related to the amount of O2- produced, is slightly dependent on the concentration of sodium formate in the matrix solution. 2. The reaction (k1 equals (1.1+/-0.1) - 10(6) M-1 - s-1 at pH 7.2, I equals 4 mM and 21 degrees C) can be inhibited by superoxide dismutase and trace amounts of copper ions. The inhibition by copper ions is removed by EDTA without interference in the O2- reduction reaction. 3. The second-order rate constant for the reaction of O2- with ferricytochrome c depends on the pH of the matrix solution, decreasing rapidly at pH greater than 8. The dependence of the rate constant on the pH can be explained by assuming that only the neutral form of ferricytochrome c reacts with O2- and that the alkaline form of the hemoprotein is unreactive. From studies at pH 8.9, the rate for the transition from the alkaline to the neutral form of ferricytochrome c can be estimated to be 0.3 s-1 (at 21 degrees C and I equals 4 mM). 4. The second-order rate constant for the reaction of O2- with ferricytochrome c is also dependent on the ionic strength of the medium. From a plot of log k1 versus I1/2-(I + alphaI1/2)-1 we determined the effective charge on the ferricytochrome c molecule as +6.3 and the rate constant at I equals 0 as (3.1+/-0.1) - 10(6) M-1 - s-1 (pH 7.1, 21 degrees C). 5. The possibility that singlet oxygen is formed as a product of the reaction of O2- with ferricytochrome c can be ruled out on thermodynamic grounds.

The mechanism of reduction of cytochrome c as studied by pulse radiolysis.

1. The reaction of hydrated electrons with ferricytochrome c was studied using the pulse-radiolysis technique. 2. In 3.3 mM phosphate-buffer (pH 7.2), 100 mM methanol and at a concentration of cytochrome c of less than 20 muM the reduction kinetics of ferricytochrome c by hydrated electrons is a bimolecular process with a rate constant of 4.5-10-10 M-1-S-1 (21 degrees C). 3. At a concentration of cytochrome c of more than 20 muM the apparent order of the reaction of hydrated electrons with ferricytochrome c measured at 650 nm decreases due to the occurrence of a rate-determining first-order process with an estimated rate constant of 5-10-6s-1 (pH 7.2, 21 degrees C). 4. At high concentration of cytochrome c the reaction-time courses measured at 580 and 695 nm appear to be biphasic. A rapid initial phase (75% and 30% of total absorbance change at 580 and 695 nm, respectively), corresponding to the reduction reaction, is followed by a first-order change in absorbance with a rate constant of 1.3-10-5 S-1 (pH 7.2, 21 degrees C). 5. The results are interpreted in a scheme in which first a transient complex between cytochrome c and the hydrated electron is formed, after which the heme iron is reduced and followed by relaxation of the protein from its oxidized to its reduced conformation. 6. It is calculated that one of each three encounters of the hydrated electron and ferricytochrome c results in a reduction of the heme iron. This high reaction probability is discussed in terms of charge and solvent interactions. 7. A reduction mechanism for cytochrome c is favored in which the reduction equivalent from the hydrated electron is transmitted through a specific pathway from the surface of the molecule to the heme iron.

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis.

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa3 were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa3. The bimolecular rate constant for this reaction is 2 x 10(7) M-1 . s-1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20 degrees C). (2) The ionic strength dependence of the cytochrome c-cytochrome aa3 interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa3 complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (epsilon) of the reaction medium on the reaction of cytochrome C with cytochrome aa3 was investigated. With increasing epsilon the second-order rate constant decreased.

Determination of alpha 1-adrenoceptor subtype selectivity by [3H]-prazosin displacement studies in guinea-pig cerebral cortex and rat spleen membranes.

1. [3H]-prazosin homogeneously labels alpha 1-adrenoceptors in guinea-pig cerebral cortex and rat spleen membranes with dissociation constants of 1.28 and 1.49 x 10(-10) M respectively. 2. Phentolamine and WB 4101 displacement studies show that guinea-pig cerebral cortex contains 30% alpha 1A- and 70% alpha 1B-adrenoceptor subtypes, whereas rat spleen contains a virtually homogeneous alpha 1B-adrenoceptor subtype population. The alpha 1-adrenoceptor population of rat thoracic aorta is predominantly of the alpha 1A-adrenoceptor subtype, and in guinea-pig thoracic aorta it is mainly of the alpha 1B-adrenoceptor subtype. 3. Half of the compounds displacing [3H]-prazosin bound to guinea-pig cerebral cortex membranes display alpha 1A-adrenoceptor selectivity. Among these compounds, WB 4101 and methoxamine are most selective, displaying selectivity ratios of approximately 38 and approximately 26 respectively. 4. The affinity constants of the non-selective compounds for the alpha 1-adrenoceptor in guinea-pig cerebral cortex membranes correlate well with the affinity constants obtained for alpha 1B-adrenoceptors in rat spleen membranes. The affinities of selective compounds for the alpha 1B-adrenoceptor subtype in guinea-pig cerebral cortex correlate very well with their affinity for alpha 1B-adrenoceptor in the rat spleen homogenate. Both regression lines coincide with the line of identity. The affinity constants of selective compounds for the alpha 1A-adrenoceptors in guinea-pig cerebral cortex only apparently correlate with the affinity for either the alpha 1B-adrenoceptors in guinea-pig cerebral cortex or in the rat spleen. Regression analyses indicate a straight line relationship (r2>0.9) between pKEA and Pk1B but the regression lines deviate from the line of identity.

Isolation and characterization of an enkephalin-hydrolyzing dipeptidyl-aminopeptidase from calf-brain striatum.

Cytosolic dipeptidyl-aminopeptidase with a high affinity for Leu-enkephalin (Km = 5-7 microM) was partially purified from the 25,000 g supernatant of calf-brain striatum. The procedure included pH 4.5 denaturation, DEAE-cellulose chromatography and Blue Sepharose CL-6B chromatography and resulted in preparations that are free from other enkephalin-hydrolyzing enzymes. This enzyme, which is called enkephalinase B, has a positively charged group in its active site and presumably also a Zn atom since the loss in activity induced by EDTA treatment can be restored without loss of substrate affinity by low concentrations of ZnSO4.

Synthesis of enkephalinase B inhibitors, and their activity on isolated enkephalin-degrading enzymes.

Compounds in which a dipeptide moiety is linked to a metal chelating mercapto group were synthesized to obtain effective enkephalinase B inhibitors. Inhibitors containing two hydrophobic amino acid side-chains decrease enkephalinase B activity with a potency depending on the length of the spacer connecting the mercapto group and the dipeptide (IC50 values vary between 0.35 and 14 microM) and they also inhibit enkephalinase A and aminopeptidase activity. Compounds lacking the carboxy terminal side-chain are not recognized by enkephalinase B or aminopeptidase but are potent inhibitors of enkephalinase A. Our most potent enkephalinase B inhibitor is mercaptoacetyl-Phe-Phe (designated phelorphan), having an IC50 value of 0.35 microM for enkephalinase B. This compound also effectively inhibits enkephalinase A (IC50 = 0.02 microM) and aminopeptidase activity (IC50 = 13 microM). Phelorphan can therefore be considered as a complete inhibitor of enkephalin biodegradation.

Effect of various enkephalin analogs and dipeptides on the enzymatic activity of enkephalinase B isolated from calf-brain striatum.

The inhibitory potency of several enkephalin analogs and dipeptides on the calf-brain enkephalinase B activity was established with the aim to characterize its active site. Highest potency was measured for dipeptides with a large side chain on both amino acids. The nature of the distal amino acid is of minor importance, provided it is not a glycine. Free carboxylic function is required for good interaction, whereas the stereochemical configuration of the dipeptide is less so. Enkephalinase B has only little affinity for D-Ala2-Leu-enkephalin. The data are to be used for the design of new enkephalinase B inhibitors.

Inhibitors of calf-brain enkephalinase A and B.

Enkephalinase A and B isolated from calf-brain striatum have comparable substrate specificity (Km for Leu-enkephalin = 1-3.10(-5) microM) but a quite different affinity for certain inhibitors: phosphate, Secobarbital and Thiorphan are effective inhibitors for Enkephalinase A (IC50 of 2.5 mM, 30 microM and 4 nM respectively), while Enkephalinase B does not react with any of these compounds. Both enzymes are inhibited by 1 mM EDTA and o-phenanthroline indicating the presence of a metal atom in or near their active sites. Although with different abilities, both enzymes recognize dipeptides having at least one hydrophobic amino-acid side chain. The potency of such dipeptides can be used for a description of the active site.

Effect of inhibitors of enkephalin degradation in the isolated guinea-pig ileum.

Bestatin and high concentration of puromycin increase the depressing effect of [Met] enkephalin on the twitch response of the electrically stimulated guinea-pig ileum. Thiorphan (enkephalinase A inhibitor) is hardly effective, but phelorphan (mercapto-acetyl-Phe-Phe) a newly synthesized enzyme-inhibitor which effectively inhibits the enkephalinase A, enkephalinase B and soluble aminopeptidase activity, potentiates the effect of enkephalin dose-dependently and in low concentrations (0.01-1 microM). Enkephalinase A, though present in these tissues, is not functional under the conditions of the test, because it is inhibited by the physiological buffer itself. These results demonstrate that enkephalinase B and the membrane bound aminopeptidase, but not the soluble aminopeptidase or enkephalinase A hydrolyse enkephalins in the isolated guinea-pig ileum.