The inhibition of caeruloplasmin by azide

1. The inhibition of the oxidase activity of caeruloplasmin by azide was investigated at 25 degrees and 7.5 degrees . 2. The inhibition is reversible on dilution or Sephadex treatment, indicating a caeruloplasmin-azide complex. 3. The enzyme is protected against azide inhibition by chloride, acetate or EDTA, the last-named acting not by chelation but by a non-specific effect similar to that of acetate. 4. Lineweaver-Burk plots with different concentrations of azide are parallel. This may occur either when the enzyme-substrate complex or when a subsequent intermediate structure of the enzyme forms the inhibited complex. 5. At 7.5 degrees inhibition may be shown not to occur until after the initial reaction of enzyme with substrate. 6. At 7.5 degrees , the inhibition is of the mutual-depletion type, inhibitory concentrations of azide being comparable with the concentration of caeruloplasmin. It is shown that the binding of a single azide group completely inhibits a caeruloplasmin molecule. 7. An arrangement of the four valence-changing copper atoms of caeruloplasmin is proposed in which they are so close together in the cuprous form that reoxidation may occur by the simultaneous transfer of four electrons from the copper atoms to a single oxygen molecule.     

The inhibition of lipid autoxidation by human caeruloplasmin.

1. Purified caeruloplasmin was shown to inhibit lipid autoxidation induced by ascorbic acid or inorganic iron in the following systems: (a) an emulsion of linolenic acid in water; (b) an untreated ox brain homogenate in phosphate buffer; (c) a similar homogenate whose susceptibility to autoxidation had been abolished by dialysis or by heating and then restored by the above pro-oxidants. 2. The optimum conditions for this antioxidant activity were studied. 3. Caeruloplasmin did not inhibit autoxidation by u.v. irradiation in dialysed or preheated homogenates. 4. The apoprotein (without copper) had no antioxidant activity, whereas CuSO4 alone was much less effective as an antioxidant. 5. Iron-free transferrin also had some antioxidant activity.     

Non-classical inhibition of uricase by cyanide.

The existence of unoccupied nuclear oestradiol-receptor sites in normal human endometrium was investigated. Nuclei were prepared from endometrial samples obtained by curettage and exposed to [3H]oestradiol, which became maximmaly bound at 0 degrees C within 1 h. This result contrasted with the binding kinetics of oestradiol--receptor complexes, since the exchange of hormone took at least 3 h at 30 degrees C and no displacement occurred at 0 degrees C. Before concluding that the nuclear sites were unoccupied, the presence of endogenous low-affinity ligands was excluded, because the association rate of oestradiol was unchanged after nuclei were stripped from their putative ligands, and the displacement of oestrone bound to nuclear receptor by oestradiol was very slow at 0 degrees C. The available sites had high affinity for oestradiol (KD 1.3 nM) and binding-specificity characteristics of oestradiol receptors. Similar results were observed with crude and purified nuclear preparations. It was concluded that a significant proportion of nuclear oestradiol receptors in normal human endometrium is unoccupied by endogenous hormones.     

The oxidation of NN-dimethyl-p-phenylenediamine by oxidizing agents and by caeruloplasmin

1. The oxidation of NN-dimethyl-p-phenylenediamine (DPD) by inorganic oxidants and by caeruloplasmin was studied. Some experiments were also made with NNN'N'-tetramethyl-p-phenylenediamine (TPD). 2. E(mM) (550) of the first free radical oxidation product of DPD (DPD(+)) was 9.8 and E(mM) (563) of the corresponding product of TPD (TPD(+)) was 12.5. 3. The non-enzymic decomposition of DPD(+) was studied with respect to temperature, pH, concentration and DPD/DPD(+) ratio, thus defining conditions for enzyme experiments under which DPD(+) extinction at 550mmu was proportional to enzyme activity. 4. Rates of oxidation of DPD to DPD(+) by caeruloplasmin were constant over a range of DPD concentrations. At low DPD concentrations a lag period occurred, which was eliminated by addition of DPD(+). 5. A lag period was not observed with TPD, but at low TPD concentrations the rate of TPD(+) formation was greater when TPD(+) was added. This suggests that TPD(+) may compete weakly as a substrate with TPD and may be oxidized further by the enzyme before a non-enzymic reaction with TPD to form more TPD(+). 6. With DPD sulphate or acetate or TPD sulphate as substrate, Lineweaver-Burk plots were curved. With DPD hydrochloride the chloride ion caused inhibition at higher concentrations, opposing the curvature. 7. Curved Lineweaver-Burk plots were interpreted in terms of two types of substrate binding site with different K(m) values but similar V(max.) values. 8. The apparent thermodynamic changes associated with enzyme-substrate-complex formation at the sites with higher K(m) suggest that considerable conformational change may occur on binding at these sites. 9. With substrate concentrations at which only the low-K(m) sites are involved 2mol. of DPD(+)/mol. of caeruloplasmin are formed before a steady state is established. At higher substrate concentrations up to 3.2mol. of DPD(+)/mol. of caeruloplasmin are formed at this initial stage. 10. Results are discussed in relation to caeruloplasmin structures in which (a) two valence-changing and two permanently cuprous copper atoms are more accessible than the remaining four copper atoms or (b) binding of substrate at one site hinders access of substrate to another site.     

Glutathione peroxidase: inhibition by cyanide and release of selenium.

Treatment of highly purified ovine erythrocyte glutathione peroxidase with KCN resulted in loss of enzyme activity and release of selenium from the enzyme. The inactivation by cyanide was time-dependent and the rate was strongly influenced by temperature, concentration of cyanide, oxidation state of the enzyme, and pH. The pH effect could be explained on the basis of the increasing proportion of cyanide ion with increasing pH. Inhibition could be prevented by prior reduction of the purified enzyme with glutathione, dithiothreitol, or dithionite. Oxidation with cumene hydroperoxide was necessary to demonstrate cyanide inhibition of glutathione peroxidase activity in rat liver cytosol. These observations explain why cyanide inhibition of glutathione peroxidase has not been noted previously and provide new approaches for studying the chemical nature of the enzyme-selenium.     

Mechanism of cyanide inhibition of Phloem translocation

Petiolar application of potassium cyanide inhibited ¹⁴C-assimilate translocation without affecting source leaf photosynthesis or phloem loading of sucrose in Phaseolus vulgaris. The inhibition of transport was correlated with disruption of the structural integrity of the sieve tubes (sieve pore blockage) rather than impairment of a metabolic process in the translocation path driving translocation.     

Respiration of the tissues of some invertebrates and its inhibition by cyanide

A study of the metabolism of Bermuda marine invertebrates at 25 degrees C. shows that the respiratory rates of many of the tissues approximate those of vertebrate tissues at the same temperature. There is no apparent correlation between respiratory rate and phylogenetic development: tissues from some of the simpler forms use as much oxygen per unit weight as those from certain of the more highly developed animals. Cyanide inhibition experiments reveal a great variation in the amount of oxygen consumption which is dependent upon sensitive heavy metal systems. Three types of tissues, the jellyfish Cassiopea frondosa, the branchial tree of the sea cucumber, Stichopus m?bii, and two kinds of tunicates, were completely unaffected by even 10(-2)M HCN. Other tissues such as sea urchin sperm, squid gills, and lobster nerve and muscle were almost completely inhibited by much lower concentrations. Most of the materials retained 20 to 40 per cent of the normal respiratory rate in 10(-2)M HCN. The possibility that vanadium may play a part in the oxidation-reduction systems of the completely resistant animals is discussed. There is a thousandfold variation in the concentration of cyanide required to produce 50 per cent inhibition of respiration in the different tissues. Sea urchin sperm is 50 per cent inhibited by 10(-6)M HCN: the sea fan requires 10(-3)M for the same effect. Other tissues lie at intermediate points. When the logarithm of the ratio of the inhibited to the uninhibited respiration is plotted against the concentration of cyanide the resulting line has a slope which in most cases approximates 1. This indicates that one mole of enzyme ordinarily combines with one mole of inhibitor. Eggs of the sea urchin, Tripneustes esculentus, show a three- to fivefold increase in the rate of oxygen uptake on fertilization. The respiration of both the fertilized and unfertilized eggs is almost entirely inhibited by 10(-4)M HCN. Cell division in the fertilized eggs is blocked by somewhat less than 10(-5)M cyanide, a concentration which reduces respiration to 40 per cent of the normal level.     

Inhibitors of caeruloplasmin

1. A method is described by which substances inhibiting caeruloplasmin oxidase activity directly may be distinguished from those acting on stimulatory contaminant iron or on the product of enzyme action. 2. Many previously reported inhibitors, including saturated aliphatic carboxylates, hydrazines, 1,10-phenanthroline, borate and various psycho-active drugs, are found either not to act on the enzyme or to inhibit it only weakly. 3. A series of inorganic anions are compared as inhibitors. Anions such as azide and cyanide with strong copper-binding properties are the most effective inhibitors. There is a general inverse relationship between anion size and inhibitory power. Iodide is anomalous, the order of effectiveness of halides being F(-)>I(-)[unk]Cl(-)>Br(-). 4. Multidentate copperchelating ligands have little inhibitory effect. 5. A group of substances containing the structural unit [unk]C=[unk].CO(2)H, including fumarate and benzoate, cause inhibition. 6. Relative inhibitions by a series of mono-substituted benzoates are inversely related to molecular size. 7. Results are discussed in relation to earlier work on the disposition and function of the copper atoms of caeruloplasmin.     

Nickel-specific, slow-binding inhibition of carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide

The inhibition of purified carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide was investigated in both the presence and absence of CO and electron acceptor. The inhibition was a time-dependent process exhibiting pseudo-first-order kinetics under both sets of conditions. The true second-order rate constants for inhibition were 72.2 M-1 s-1 with both substrates present and 48.9 and 79.5 M-1 s-1, respectively, for the reduced and oxidized enzymes incubated with cyanide. CO partially protected the enzyme against inhibition after 25-min incubation with 100 microM KCN. Dissociation constants of 8.46 microM (KCN) and 4.70 microM (CO) were calculated for the binding of cyanide and CO to the enzyme. Cyanide inhibition was fully reversible under an atmosphere of CO after removal of unbound cyanide. N2 was unable to reverse the inhibition. The competence of nickel-deficient (apo) CO dehydrogenase to undergo activation by NiCl2 was unaffected by prior incubation with cyanide. Cyanide inhibition of holo-CO dehydrogenase was not reversed by addition of NiCl2. 14CN- remained associated with holoenzyme but not with apoenzyme through gel filtration chromatography. These findings suggest that cyanide is a slow-binding, active-site-directed, nickel-specific, reversible inhibitor of CO dehydrogenase. We propose that cyanide inhibits CO dehydrogenase by being an analogue of CO and by binding through enzyme-bound nickel.     

Formate dehydrogenase. Subunit and mechanism of inhibition by cyanide and azide.

Formate dehydrogenase (EC 1.2.1.2) prepared from peas (Pisum sativum) was a two-subunit enzyme. The enzyme accelerated the formation of an NAD+-cyanide compound having an adsorption band at 330 nm. The enzyme was able to bind one NAD+ molecule per each subunit but only 1 mole of NAD+-cyanide compound was formed per two subunits. The complex of NAD+, cyanide, and the enzyme was very stable and had no catalytic activity. Azide inhibited the formate dehydrogenase reaction in two different ways. By incubation of the enzyme with azide in the presence of NAD+, half of its catalytic activity was lost. The remaining activity was also inhibited by azide but this inhibition was removed competively by formate. Contrary to the case of cyanide the inhibition by azide could be removed by dialysis and no spectral species due to the addition compound of NAD+ and azide could be observed. The data from double recipricol plots of the initial velocity and the formate concentration led to a conclusion that formate dehydrogenase has two sites with about equal catalytic activity. The Km for formate was different for the two catalytic sites (1.67 and 6.25 mM) but the difference was not noticeable in the case of the Km for NAD+.     

An investigation of the decolorization of caeruloplasmin by acid

1. The acid decolorization of caeruloplasmin was studied at various temperatures and pH values. 2. Two decolorization reactions may be distinguished: (i) an irreversible reaction with the thermodynamic characteristics of a protein denaturation; (ii) the attainment of an equilibrium between blue and colourless forms of caeruloplasmin with apparent pK 3.7 at 25 degrees . 3. The low temperature-dependence of the equilibrium suggests that it does not involve a gross denaturative change. 4. The reversible decolorization occurred both aerobically and anaerobically, indicating that the change does not involve dissociation of a copper-oxygen complex. 5. Spectral changes during the decolorization are described. 6. The changes occurring during acid decolorization are discussed in relation to a formal model of the gross structure of caeruloplasmin.