π-Cation interactions as the origin of the weak absorption at 532 nm observed in tryptophan-containing polypeptides.

We have previously reported that bovine serum albumin (BSA) and other proteins that do not contain prosthetic groups exhibited a weak light absorption in the visible, only detectable by pulsed laser-induced optoacoustic spectroscopy (LIOAS). Human serum albumin (HSA) exhibited signals 25% higher than those observed with BSA. Signals comparable to those obtained with BSA were observed with poly(L-Trp, L-Lys), poly(L-Trp, L-Arg) or poly(L-Trp, L-Orn) at pH 7.0. No signals were obtained when tryptophan was replaced by other amino acids or when free tryptophan or the tripeptide Lys-Trp-Lys was assayed (pH 7.0). Tryptophan in HCl 5 N produced LIOAS signals similar to those produced by tryptophan-containing copolymers. Moreover, the absorption peak could be observed in a UV-VIS spectrophotometer. Therefore, the LIOAS signals obtained with BSA, HSA, and tryptophan-containing random copolymers may be attributed to a new transition of the indole moiety of their tryptophan residues when "protonated". Tryptophan residues of proteins are known to participate in π-cation interactions, which are important in protein stability and function. As a matter of fact, HSA and BSA contain an internal tryptophan in close proximity to lysine and arginine residues and therefore suitable for π-cation interactions. The strength of this type of interaction strongly depends on distances and relative orientations of both amino acid residues. Accordingly, these interactions should be highly sensitive to conformational changes. Based on preliminary results that have shown that LIOAS signal at 532 nm depended on the aggregation state of BSA and/or on the oxidation state of its Cys-34, we postulate that the LIOAS signal observed with proteins and tryptophan-containing polypeptides are related to Trp-Lys or Trp-Arg interactions and that the intensity of the signal depends on the strength of such interactions.

Inhibition of steady-state mitochondrial ATP synthesis by bicarbonate, an activating anion of ATP hydrolysis.

Bicarbonate, an activating anion of ATP hydrolysis, inhibited ATP synthesis coupled to succinate oxidation in beef heart submitochondrial particles but diminished the lag time and increased the steady-state velocity of the (32)Pi-ATP exchange reaction. The latter effects exclude the possibility that bicarbonate is inducing an intrinsic uncoupling between ATP hydrolysis and proton translocation at the level of F(1)F(o) ATPase. The inhibition of ATP synthesis was competitive with respect to ADP at low fixed [Pi], mixed at high [Pi] and non-competitive towards Pi at any fixed [ADP]. From these results we can conclude that (i) bicarbonate does not bind to a Pi site in the mitochondrial F(1); (ii) it competes with the binding of ADP to a low-affinity site, likely the low-affinity non-catalytic nucleotide binding site. It is postulated that bicarbonate stimulates ATP hydrolysis and inhibits ATP synthesis by modulating the relative affinities of the catalytic site for ATP and ADP.

Synthesis and elastase inhibitory activity of 6 alpha-chloro-2,2-dimethyl-3 alpha-(pivaloyloxy)methylpenam sulfone, 6 alpha-chloro-2,2-dimethyl-3-exo-methylenepenam sulfone, benzyl and methyl 6 alpha-substituted penicillanate sulfones.

The triflates and pivalates of 3 alpha-hydroxymethyl-6-substituted-2,2-dimethylpenam sulfones 3, 5; methyl and benzyl 6-substituted penicillanates 6-9 and 3-exo-methylene-6-substituted-2,2-dimethylpenam sulfone 4 were synthesized. These novel compounds were evaluated as elastase inhibitors using porcine pancreatic elastase. The effects that structural modifications of substituents on C-3 and C-6 in the penam nucleus have on elastase activity were examined and several similarities and distinctions were identified when compared to the reported penicillin esters and amides elastase inhibitors.

Synthesis and beta-lactamase inhibitory activity of 6-fluoropenicillanic acids.

The benzyl 6-fluoro-penicillanate sulfides 4a, 6a, 7a; and sulfones 6c, 7d were synthesized. The conversion to their free acids 4b, 6b, 6d, 7b, 7e and potassium salts 7c, 7f are described. These acids and salt 7c were evaluated as beta-lactamase inhibitors using beta-lactamase I from Bacillus cereus. The data indicate that substitution of the 6 alpha-hydrogen by a 6 alpha-fluorine atom on 6 beta-bromopenicillanic acid (1), leads to loss of beta-lactamase inhibitory activity. In the case of the isomers 6 beta- and 6 alpha-fluoropenicillanic acids the 6 beta-enantiomer proved to be considerably more potent. Potassium salts of 6 beta-fluoropenicillanate sulfide and sulfone were unstable in solid state and in water solution. The fragmentation of the sulfone in two parts in water solution is consistent with the hydrolytic behavior to the penicillanic acid sulfone (2) with 0.5 N NaOH.

Inhibition by suramin of mitochondrial ATP synthesis.

Suramin, a drug intensively used in the chemotherapy of African trypanosomiasis and onchocerciasis, is currently being tested in clinical trials for AIDS treatment. Its effects on mitochondrial energy metabolism in mammals were studied. At low concentrations it inhibited ATP synthesis and ATPase activity in submitochondrial particles, as well as ADP-stimulated oxygen consumption and the uncoupler-stimulated ATPase activity in intact rat liver mitochondria. At higher concentrations it also inhibited uncoupled electron transport in both submitochondrial particles and intact mitochondria. From comparison of the kinetic patterns of those inhibitions, evidence suggesting that the adenine nucleotide translocase may be another target for the action of suramin was obtained. The relevance of these findings to the understanding of the biochemical basis of suramin toxicity is discussed.

Inhibition of energy metabolism by benzoxazolin-2-one.

The effects of the title compound (BOA) on energy-linked reactions in mitochondria were studied. BOA inhibited electron transfer between the flavin and ubiquinone in Complex I, and ATP synthesis at the F1 moiety of the ATPase complex. These results are discussed in relation to the toxicity of BOA towards a wide range of aerobic organisms.

Inhibition of mitochondrial energy-linked reactions by 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), a hydroxamic acid from Gramineae.

DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) is the main hydroxamic acid isolated from maize extracts. It inhibited reversibly ATP synthesis, Pi-ATP exchange reaction and ATPase activity in submitochondrial particles from bovine heart. Half-maximal effects were obtained with 4, 2, and 6 mM DIMBOA respectively. At higher concentrations it also inhibited mitochondrial electron transport (I50 = 11 mM). Irreversible inactivation of mitochondrial electron transport, Pi-ATP exchange reaction and 8-anilino-1-naphthalene sulfonate energy-dependent fluorescence enhancement was also observed. These effects of DIMBOA on energy-linked mitochondrial reactions may explain the inhibitory action of DIMBOA on several aerobic organisms.

Steady-state kinetics of F1-ATPase. Mechanism of anion activation.

The kinetic behaviour of the ATPase activity of beef heart F1 depends largely on the exposure of the enzyme to some anionic ligands such as sulphate and/or EDTA. F1 prepared in the presence of such anions exhibited a triphasic kinetic pattern whereas F1 from which those anions were removed by dialysis exhibited only two Km values for ATP. Conversely to what has been previously reported, bicarbonate did not linearize F1-ATPase kinetics. Moreover, anion activation cannot be simply explained by promotion of ADP release but mainly by an increase in affinity of the third catalytic site for ATP.

A new plot for the kinetic analysis of dead-end enzyme inhibitors.

A new procedure to characterize reversible dead-end inhibitors is presented. Preliminary identification of the inhibitor type is made by plotting vo/vi against the inhibitor concentration at different substrate concentrations. The inhibition constants for competitive, uncompetitive and mixed dead-end inhibitors are determined by secondary plots of l/(slope) vs [S], l/(slope) vs l/[S] and (slope)(Ks + [S] vs [S] respectively. These secondary plots render straight lines only for their corresponding type of inhibitor. For noncompetitive inhibitors all the secondary plots used yield straight lines. Therefore, the application of this plotting procedure leads to unambiguous diagnosis of the inhibitor type. An important feature of the procedure presented here is that the variable used (vo/vi) is independent on Vmax values. Therefore, experimental values obtained from enzyme preparations showing significant differences in their specific activities -i.e. enzyme coming from different purification steps- can be used.

Effects of gramine on energy metabolism of rat and bovine mitochondria.

The indole alkaloid gramine is found in several plant families. Its effects on mammalian mitochondria and submitochondrial particles were studied. Low concentrations of gramine slightly stimulated basal electron transport, totally inhibited the Ca2+-induced respiratory control and partially abolished the enhancement of 8-anilino-1-naphthalene sulfonate fluorescence induced by proton translocation coupled to ATP hydrolysis or to succinate oxidation. At higher concentrations gramine inhibited specifically the electron transport at the level of Complex I of the respiratory chain. The I50 values (2-6 mM) were dependent on the presence of uncouplers. Higher concentrations of the alkaloid also inhibited coupled succinate oxidation and ATP hydrolysis (I50 = 10 mM). Possible explanations for these effects are discussed.

Inhibition by suramin of oxidative phosphorylation in Crithidia fasciculata.

1. The ADP plus Pi-stimulated oxidation of succinate by mitochondria from the insect trypanosomatid Crithidia fasciculata was maximally inhibited (64%) by suramin at a concentration (60 microM) which did not affect the electron transport uncoupled by FCCP. Inhibition of the latter required a considerably higher concentration of the drug, 50% inhibition being attained at about 0.8 mM. 2. ATP synthesis by mitochondrial particles was inhibited by suramin, 50% inhibition being attained at about 50 microM. This inhibition was strictly competitive towards ADP, but it was not linearly competitive, since a secondary plot of apparent Km values vs concentration of the drug was strongly concave upwards. 3. The FCCP-stimulated ATPase activity of the mitochondrial particles was completely abolished either by oligomycin (20 micrograms/ml) or by 200 microM suramin. 4. The results suggest that oxidative phosphorylation may be a primary target for the trypanocide effect of suramin on organisms having, like C. fasciculata, a well-developed respiratory chain.

The pre-steady state and steady-state kinetics of the ATPase activity of mitochondrial F1.

1. The lag time before maximum velocity of ATP hydrolysis is reached upon mixing ATP with F1 is much greater than can be explained by a simple Michaelis-Menten mechanism, and must be due to an activation reaction. The lag time is dependent on the concentration of MgATP (half-maximal at 30 microM) and is equal to 30 ms at infinite MgATP concentration. The initial rate of hydrolysis by nucleotide-depleted F1 is much greater than with normal F1. It is tentatively suggested that the activation reaction with normal preparations is due to replacement of firmly bound ADP by MaATP. 2. After the initial time lag, the reaction follows very closely first-order kinetics provided that the concentration of MgATP is much less than the Km and the reaction is completed within 2 s. This is not expected if the dissociation constant of the enzyme-MgADP complex, an intermediate in the enzymic reaction, is much lower than the Km as has been reported in the literature. The value of V/Km, calculated from the exponential decay, is very close to that calculated from independent measurements of V and Km. 3. The low values for Ki(ADP) reported in the literature were found to be due to a slow (in the order of seconds) formation of an inhibited MgADP-enzyme complex. Dissipation of this inhibited complex by ATP requires seconds. The dissociation constant of the MgADP-enzyme complex that is an intermediate in the enzyme reaction was found to be 150 microM. 4. ADP but not ATP becomes firmly bound to nucleotide-depleted F1 in the absence of Mg2+.

Spegazzinine, a new inhibitor of mitochondrial oxidative phosphorylation.

The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied. Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I50 was 120 microM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca2+ and the proton ejection associated with the ATP-dependent Ca2+ uptake were not affected by the alkaloid. Oxidative phosphorylation and the Pi-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I50, 50 microM) while the ATP-dependent reactions, reduction of NAD+ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I50, 125 microM). Oxygen uptake by submitochondrial particles was not affected. The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 microM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine. The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.