Selective inactivation of MAO-B by benzyl-dimethyl-silyl-methanamines in vitro

The alpha-silyl amines benzyl-dimethyl-silyl-methanamine and the p-fluoro and p-chloro derivatives are potent time-dependent inhibitors of rat brain MAO-B. The inhibition exhibits saturation kinetics, takes place in the enzyme active-site and is irreversible. The most potent inhibitor in the series is 4-fluorobenzyl-dimethyl-silyl-methanamine (KI = 11 microM, tau 1/2 = 2.3 min). Its selectivity for the B-form relative to the A-form of rat brain MAO is higher than 10(4). Benzyl-dimethyl-silyl-methanamines may represent a new family of anti-Parkinsonian agents.     

Selective inactivation of heterokaryons of Candida albicans by anaerobiosis

Heterokaryons (hets), but not monokaryons of Candida albicans die when grown anaerobically on minimal medium. Their rates of inactivation increase with decreases in growth temperatures from 37°C to 25°C. At 10°C, however, anaerobiosis is not lethal and suppresses the inactivation which normally occurs among hets cultured aerobically at that temperature. Killing of hets by anaerobiosis can be altered significantly by certain exogenously provided amino acids or intermediates of oxidative respiration. Aspartic acid alone promotes inactivation whereas alanine, glutamic acid or lysine individually have no effects. However, glutamate and lysine combined afford slight protection against inactivation while aspartate and glutamate combined, with or without lysine, are highly protective: the activity of the aspartate-glutamate combination is completely negated by the addition of alanine. Other common amino acids have no effects on het responses to anaerobiosis other than the ability, when combined, to relieve the antagonism of alanine for the aspartate-glutamate combination. Anaerobic survivals are also enhanced by oxalacetic acid or -ketoglutaric acid, and even more so by a combination of these two intermediates. The resistances to inactivation elicited by the oxalacetate -ketoglutarate or aspartate-glutamate combinations are not additive. These relationships are interpreted to signify that inactivation of hets by anaerobic growth is largely, if not exclusively, due to depletion of their oxalacetic acid and -ketoglutaric acid contents for amino acid biosyntheses, and the unique inability of het cells to replenish those keto acids upon subsequent return to aerobic conditions. The observations are consistent with previous indications that mitochondria formed by hets are functionally abnormal.     

Selective inactivation of cytochrome P-450 isozymes by suicide substrates

The autocatalytic destruction of cytochrome P-450 by the following six substrates has been investigated in vivo and in vitro with microsomal and purified, reconstituted rat liver enzymes: 2-isopropyl-4-pentenamide (AIA), 1-ethinylcyclopentanol, 17α-propadienyl-19-nortestosterone, fluroxene, 5,6-dichloro-1,2,3-benzothiadiazole (DCBT), and 1-aminobenzotriazole (ABT). Administration of the first three substrates to rats pretreated with either phenobarbital (Pb) or 3-methylcholanthrene (3-MC), or their incubation with hepatic microsomes from such rats, produced a larger decrease in cytochrome P-450 levels in the membranes from Pb- than 3-MC-treated rats. Comparable losses, however, were observed in microsomes from rats pretreated with both Pb and 3-MC when the last three agents were used. Similar experiments were carried out using the major cytochrome P-450 isozymes purified from liver microsomes of Pb- or 3-MC-treated rats. The Pb isozyme was inactivated during catalytic turnover of all six substrates while only three substrates (DCBT, ABT, and fluroxene) were found to inactivate the 3-MC isozyme. Oxygen consumption studies with purified enzymes have shown that AIA is not a measurable substrate for the 3-MC isozyme, a fact which explains its failure to inactivate this isozyme. Similar studies with the Pb isozyme establish that one enzyme molecule is inactivated for approximately every 230–320 AIA molecules processed by the enzyme.     

Selective inactivation of muscarinic receptor subtypes

Muscarinic receptors exist in multiple subtypes, denoted as M₁, M₂ M₃ and M₄, encoded by four distinct but related genes. A fifth gene product, m5, has also been predicted although this sequence awaits a pharmacological equivalent. Many tissues express more than one muscarinic receptor subtype, which may couple to different intracellular effectors and thus have different physiological roles. One way to characterize the role of each receptor is to selectively inactivate one receptor population, thus pharmacologically ‘isolating’ the muscarinic receptor subtype of interest. Selective receptor inactivation can be achieved using either a selective, irreversible antagonist, or protection using a selective, reversible antagonist against a non-selective irreversible antagonist. Therefore, combination of these two approaches may provide optimal selective inactivation. Several muscarinic alkylating agents have been identified, including phenoxybenzamine, EEDQ (N-Ethoxycarbonyl-1-ethoxy-1,2-dihydroquinoline) and propylbenzilylcholine mustard. These irreversible antagonists do not, in general, discriminate between muscarinic receptor subtypes and are frequently used to estimate the affinity and relative efficacy of muscarinic agonists. Consequently, use of these irreversible antagonists provides estimations of the ‘receptor reserve’ associated with a response mediated by muscarinic receptor activation. In contrast, 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl)piperidine) selectively inactivates M₃ receptors, but will not discriminate between M₁ M ₂ or M₄ receptors. In the absence of highly selective alkylating agents, receptor protection by reversible antagonists may be used. Thus, reversible antagonists, such as pirenzepine, methoctramine or para-fluorohexahydrosiladifenidol, at appropriate fractional receptor occupancies, may protect M₁ M₂ or M₃ receptors against alkylation by phenoxybenzamine. Selective alkylation of M₃ receptors by 4-DAMP mustard is enhanced with concurrent M₂ protection. This approach has been applied to defining the role of these muscarinic receptor subtypes in the control of ileal smooth muscle tone. These data suggest that, in ileum, M₂ receptors may act to inhibit β-adrenoceptor activation, thereby offsetting relaxation, while M₃ receptors directly mediate contraction.     

The inactivation of rhodanese by nitrite and inhibition by other anions in vitro

Cyanide detoxification in mammals occurs, in part, by sulfur transfer by rhodanese to form the less toxic thiocyanate. Thiosulfate and nitrite are often used in combination for the treatment of cyanide intoxication. This report shows that nitrite can inhibit the rate of sulfur transfer by rhodanese in vitro. Nitrate, chloride, sulfate, and acetate were also examined as inhibitors. Inhibition by nitrite appeared to be more complex than for the other anions tested. Closer examination showed that nitrite can inactivate the sulfur-free rhodanese. Our observation leads to the suggestion that, in vivo, either rhodanese is maintained in its more stable sulfur-substituted form or cellular compartmentalization prevents inactivation by nitrite.     

Fatty acid synthetase complex: Selective inactivation by phenylmethylsulphonyl fluoride

Reaction of pigeon and rat liver fatty acid synthetases with phenylmethylsulphonyl fluoride at pH 7.0 results in rapid and complete loss of activity for fatty acid synthesis. Acetyl and malonyl transacylation, two reductions, dehydration and condensation-CO₂ exchange reactions are not appreciably altered in the modified enzyme. However, the deacylation of palmityl CoA is completely inhibited. Complete inactivation results in the incorporation of about 1.9 moles of ¹⁴C-phenylmethylsulphonyl groups/mole of the enzyme complex. These results suggest that either two moles of a fatty acyl deacylase or two deacylases with different fatty acyl chain length specificities may be functional in the enzyme complex.     

Selective inactivation of the infectivity of freeze-dried Sendai virus by heat

The infectivity of freeze-dried Sendai virus was destroyed after heating at 100 ° C for 20 min while the hemagglutinin (HA) titer and the hemolytic (HL) activity were not affected. The HA titer was unaltered after heating at up to 140 ° C for 30 min. The HL activity was increased after freeze-drying, further increased after heating of freeze-dried virus at 115 ° C for 20 min, but was destroyed after heating for 30 min at 140 ° C.The selective heat inactivation of freeze-dried Sendai virus could be of use in the production of myxovirus vaccines and inactivated virus for cell-fusion studies.     

Virus inactivation by anilinonaphthalene sulfonate compounds and comparison with other ligands

Bis-(8-anilinonaphthalene-1-sulfonate) (bis-ANS) causes inactivation of vesicular stomatitis virus (VSV) at micromolar concentrations while butyl-ANS and ANS are effective at concentrations one and two orders of magnitude higher, respectively. VSV fully inactivated by the combined effects of 10 microM bis-ANS and 2.5 kbar hydrostatic pressure elicited a high titer of neutralizing antibodies. Incubation of VSV with >/=2 M urea at atmospheric pressure caused very little virus inactivation, whereas at a pressure of 2.5 kbar, 1 M urea caused inactivation that exceeded by more than two orders of magnitude the sum of the inactivating effects produced by urea and pressure separately. Measurements of bis-ANS fluorescence showed that increasing the urea concentration reduces the pressure required to disrupt the structure. We conclude that anilinonaphthalene sulfonate compounds inactivate VSV by a mechanism similar to that produced by pressure. The most effective antiviral compound was bis-ANS which can be used for the preparation of safe viral vaccines or as an antiviral drug eventually.     

Selective inactivation of rabbit reticulocyte initiation factor eIF-3 by helenalin

Helenalin, a sesquiterpene lactone which reacts primarily with exposed sulfhydryl groups, was shown to be an effective inhibitor of protein synthesis in rabbit reticulocyte lysates. Optimal inhibition required a 30 min preincubation in the absence of any added thiol compound. β-Mercaptoethanol was more effective than reduced glutathione in protecting enzyme sulfhydryl groups from inactivation by helenalin. Using partially fractionated systems, it was possible to show that helenalin had no effect on the elongation reactions or on the formation of the ternary initiation complex. However, the conversion of the ternary complex to the 48 S initiation complex was strongly inhibited. In this assay, only the initiation factor(s) were sensitive to helenalin. Using an assay system which requires all the initiation factors for optimal activity it was possible to show that the 0–40% ammonium sulfate cut of intiation factors (containing eIF-3 and eIF-4B) was sensitive to helenalin, while the 40–50% ammonium sulfate cut (containing eIF-2 and eIF-5) was not. Both ammonium sulfate cuts were equally sensitive to inhibition by the sulfhydryl reagent N-ethylmaleimide. Three purified rabbit reticulocyte initiation factors were then tested in the same assay system. Only eIF-3 showed appreciable sensitivity to helenalin, while eIF-2, eIF-3 and eIF-4B were all sensitive to inactivation by N-ethylmaleimide. These data suggest that helenalin may possess a relatively high degree of specificity as a sulfhydryl reagent.     

Selective inactivation of various acyl-CoA dehydrogenases by (methylenecyclopropyl)acetyl-CoA

Inactivation of five distinct acyl-CoA dehydrogenases by (methylenecyclopropyl)acetyl-CoA (MCPA-CoA), the toxic metabolite of hypoglycin from unripe ackee fruit, was investigated using purified enzyme preparations. Short-chain acyl-CoA (SCADH), medium-chain acyl-CoA (MCADH) and isovaleryl-CoA (IVDH) dehydrogenases were severely and irreversibly inactivated by MCPA-CoA, while 2-methyl-branched chain acyl-CoA dehydrogenase (2-meBCADH) was only slowly and mildly inactivated. Long-chain acyl-CoA dehydrogenase (LCADH) was not significantly inactivated, even after prolonged incubation with MCPA-CoA. Inactivation of SCADH, MCADH and IVDH was effectively prevented by the addition of substrate. This mode of inactivation by MCPA-CoA explains the urinary metabolite profile in hypoglycin treated-rats, which includes large amounts of metabolites from fatty acids and leucine, and relatively small amounts of those from valine and isoleucine. Spectrophotometric titration of SCADH and MCADH with MCPA-CoA, together with the protective effects of substrate, indicates that MCPA-CoA is acted upon by, and exerts in turn irreversible inactivation of, SCADH and MCADH, confirming that MCPA-CoA is a suicide inhibitor (Wenz et al. (1981) J. Biol. Chem. 256, 9809-9812). Spectrophotometric titration data of LCADH and MCPA-CoA is typical of non-reacting CoA ester.     

Inhibition by sporidesmin of hepatocyte bile acid transport.

Exposure of isolated rat hepatocytes (approx. 2 x 10(7)--5 x 10(7) cells/10ml of incubation mixture) to 0.5 mg of the mycotoxin sporidesmin for 30--60 min at 37 degrees C produced loss of plasma-membrane microvilli with some disruption of organelle distribution in the sub-surface region. There was accompanying inhibition of [14C]cholate and [14C]taurocholate transport, but bile acid conjugation was not altered. Inhibition of cholate uptake was maximal after exposure of hepatocytes to sporidesmin for 1 min, and was not reversed by washing cells free of extracellular sporidesmin. N-Ethylmaleimide (0.1 mM) or dithiothreitol (1 mM) partially protected hepatocytes from sporidesmin inhibition of bile acid uptake. Significant protection was not given by other thiols or by zinc sulphate, cholesterol, ascorbate or alpha-tocopherol. The results are discussed in terms of sporidesmin action on cell membranes and the toxin's effect on bile secretion.     

Studies on the mechanism of toxicity of the mycotoxin, sporidesmin. I. Generation of superoxide radical by sporidesmin

Sporidesmin (SDMS2), the mycotoxin responsible for 'facial eczema' in ruminants, contains a disulphide group which appears to be intimately involved in its toxic action. The reduced (dithiol) form of sporidesmin has been shown readily to undergo autoxidation in vitro in a reaction which generates superoxide radical (O2-). The autoxidation reaction, which takes place over a wide pH range, is strongly catalysed by trace amounts of copper, although the reaction was inhibited at high concentrations of this metal. Inhibition of the autooxidation of reduced sporidesmin (SDM(SH)2) was also observed in the presence of nickel, cobalt and manganese. Superoxide radical is also generated from SDMS2 itself in a cyclic reduction/autoxidation reaction with glutathione and other thiols; in view of the known toxicity of superoxide and its derivatives, it is suggested that oxygen-free-radicals may be involved in the initiation of the deleterious effects of the mycotoxin.     

Selective inactivation of lactate dehydrogenase of rat tissues by sodium deoxycholate.

Soluble lactate dehydrogenase (EC 1.1.1.27) extracted from brain, skeletal and cardiac muscle and liver of rats, and purified isoenzymes LDH-1 and LDH-5, were incubated with sodium deoxycholate. Deoxycholate almost totally inactivated isoenzyme LDH-5 (A4), whereas it left isoenzyme LDH-1 (B4) unaffected. Tissue lactate dehydrogenase was inactivated to different degrees depending on the origin of the enzyme. Electrophoretic isoenzyme studies of tissue lactate dehydrogenase showed the loss of activity to be quantitatively related to the overall percentage of subunit A distributed among the homotetramer LDH-5 and the heterotetramers LDH-2, LDH-3 and LDH-4. It was concluded that subunit A of lactate dehydrogenase interacts selectively with deoxycholate, irrespective of its association with subunit B. Distinct changes in electrophoretic mobilities of deoxycholate-treated isoenzymes strongly indicated an indiscriminate binding of deoxycholate by all LDH isoenzymes, probably through hydrophobic interactions. The results suggest that the inactivation of the enzyme is non-competitive, but the basis of the selectivity of deoxycholate towards subunit A is not known at present.     

Selective fluorophore-assisted light inactivation of voltage-gated calcium channels

Fluorophore-assisted light inactivation (FALI) is an investigative tool to inactivate fluorescently labeled proteins by a mechanism of in situ photodestruction. We found that Cav 1.2 (L-type) and Cav 3.1 (T-type) calcium channels, labeled by genetic fusion with GFP derivatives, show differential sensitivity to FALI. Specifically, FALI silences Cav 1.2 calcium channels containing EYFP-labeled α 1C subunits but does not affect the EYFP-α 1G Cav 3.1 calcium channels or Cav 1.2 channels containing EYFP-labeled β subunits. Our findings limit the applicability of acceptor photobleaching for the measurements of FRET but open an opportunity to combine the fluorescent imaging of the live cell expressing labeled calcium channels with selective functional inactivation of their specific subsets.