Effects of detergents and organic solvents on rat liver microsomal UDP-glucuronosyltransferase activity towards phenolic substrates

1. The effects of several detergents (Brij 58, deoxycholate and Lubrol 12A9) and ether on the initial rate of UDP-glucuronosyltransferase activity towards fixed concentrations of five phenolic acceptor substrates of widely different octanol-buffer (pH 7.4) partition coefficient have been compared with those observed in non-activated and Triton X-100-and n-pentane-activated rat liver microsomes.

2. Enzymic activity was dependent on the lipid-solubility of acceptor substrate. Each activator, except Triton X-100, enhanced enzymic activity towards all substrates by a similar factor, which was independent of the octanol-buffer partition coefficient. For Triton X-100 microsomes, the activation was also partition-dependent.

3. The highest activation factor was seen with ether. Pre-incubation of ether-activated microsomes for 30?min at 37°C before assay resulted in inactivation of the enzyme towards more water-soluble substrates. Tryptic digestion (30?min at 37°C) of the ether-activated microsomes resulted in marked reduction of enzyme activity towards all substrates.

4. Ether, and the two detergents, Brij 58 and Lubrol 12A9, released small amounts of protein (5-12% total present); both detergents also released some (8-12%) phospholipid.

5. The K^app_m towards acceptor substrate also depended on the octanol-buffer partition coefficient, and was largely unchanged on activation by n-pentane. V_mak was not dependent on partition coefficient and was significantly increased on activation.     

Inhibition of the ATP-sensitive potassium channel from mouse pancreatic β-cells by surfactants

1. We have used patch-clamp methods to study the effects of the detergents, Cremophor, Tween 80 and Triton X100 on the K_ATP channel in the pancreatic β-cell from mouse.
2. All three detergents blocked K_ATP channel activity with the following order of potency: Tween 80 (K_i<∼83 nM)>Triton X100 (K_i=350 nM)>Cremophor. In all cases the block was poorly reversible.
3. Single-channel studies suggested that at low doses, the detergents act as slow blockers of the K_ATP channel.
4. Unlike the block produced by tolbutamide, that produced by detergent was not affected by intracellular Mg²⁺-nucleotide, diazoxide or trypsin treatment, nor did it involve an acceleration of rundown or increase in ATP sensitivity of the chanel.
5. The detergents could block the pore-forming subunit, Kir6.2ΔC26, which can be expressed independently of SUR₁ (the regulatory subunit of the K_ATP channel). These data suggest that the detergents act on Kir6.2 and not SUR₁.
6. The detergents had no effect on another member of the inward rectifier family: Kir1.1a (ROMK1).
7. Voltage-dependent K-currents in the β-cell were reversibly blocked by the detergents with a far lower potency than that found for the K_ATP channel.
8. Like other insulin secretagogues that act by blocking the K_ATP channel, Cremophor elevated intracellular Ca²⁺ in single β-cells to levels that would be expected to elicit insulin secretion.
9. Given the role of the K_ATP channel in many physiological processes, we conclude that plasma borne detergent may have pharmacological actions mediated through blockage of the K_ATP channel

     

DISSOCIATION AND DENATURATION BEHAVIOUR OF SESAME α-GLOBULIN IN SODIUM DODECYL SULPHATE SOLUTION*

The effect of anionic detergent, sodium dodecyl sulphate, on the major protein, α-globulin of sesame seed (Sesamum indicum L.) has been investigated by gel filtration, sedimentation velocity, viscosity, optical rotation, difference spectra and fluorescence measurements. The detergent causes dissociation of the protein first and then denaturation. In the detergent concentration range of 1.75–4.0 times 10^-3M four components are observed in the ultracentrifuge. The specific rotation of the protein increases with the detergent concentration above 2.5 times 10^-3M and shows a cooperative transition between 3–8 times 10^-3M detergent suggesting conformational change; above 8 times 10^-3M detergent the value of –[α] does not change. The reduced viscosity η_red however, increases above 2.5 times 10^-3M detergent and does not attain a plateau value. The difference spectrum of the protein indicates that both tryptophan and tyrosine groups have been affected by the detergent. The fluorescence intensity decreases and the maxima shifts towards red in the detergent solution resulting in an ‘isoemissive point’ at 355 nm. The double difference spectra in sucrose-detergent protein system show that below 5.0 times 10^-3M detergent, the difference absorption and fluorescence spectrum result from the binding of the detergent near the chromophoric groups and are not due to conformational change. Binding studies by equilibrium dialysis indicate the presence of 50 binding sites in the protein and a binding constant of 3.0 times 10³.     

Photodynamic effect in lysozyme: a kinetic study in different micellar media

Abstract: The influence of medium heterogeneity on the kinetics of the photodynamic effect on native protein lysozyme (Lyso), as well as the interaction of protein and the medium, anionic (SDS) micelles, neutral (Triton X‐100) micelles and reversed micelles of AOT, were investigated at pH 8. The interaction between Lyso, Triton X‐100 and SDS micelles was quantified by determining the respective associations constant (K_Lyso). Values were 37 m ^–1 for Triton X‐100 and 514 m ^–1 for SDS, indicating that the Lyso molecule binds Triton X‐100 micelles effectively and SDS micelles even more strongly. Time‐resolved phosphorescence detection (TRPD) indicates that the protein interacts with O₂ (¹Δ_g), with overall rate constants of the order of 10⁸ m ^–1/s in direct micelles and 10⁷ m ^–1/s in reverse micelles. Apparent reactive rate constants for eosin‐sensitized photo‐oxidation (singlet molecular oxygen [O₂ (¹Δ_g)]‐mediated) of the protein were determined through oxygen uptake experiments for the direct micelles, while the fade in the protein fluorescence spectrum upon sensitized irradiation was used in AOT. The results indicate that the O₂ (¹Δ_g) attack on the interior of Lyso on amino acid residues, was more effective in leading to a photo‐oxidative reaction in SDS and in Triton X‐100 at surfactant concentrations < 1 × 10^–2 M than in a homogeneous solution. However, Lyso reactivity reached a maximum when the concentration of micelles was ≈ 1 × 10^–5, the same as the protein concentration In AOT reverse micelles, the quenching rate constants decreased > 75% with respect to water. This effect can be attributed to the decrease in accessibility of the amino acid residues to O₂ (¹Δ_g).     

Optimal detergent activation of rat liver microsomal UDP-glucuronosyl transferases toward morphine and 1-naphthol: contribution to induction and latency studies.

The detergent-activation profiles of UDP-glucuronosyl transferases (UGTs, EC 2.4.1.17) toward 1-naphthol and toward morphine have been determined: three non-ionic detergents, Triton X-100, Brij 58 and Lubrol Px and one zwitterion detergent, 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonic acid (CHAPS) were studied. The results showed that marked inhibition of 1-naphthol-UGT and morphine UGT activities occurred with high concentrations of Triton X-100. Lubrol Px, at high concentrations, inhibited 1-naphthol-UGT but not morphine-UGT. It appeared that the detergent/protein ratio suitable for optimal activation of both isoenzymes was limited to 0.2 for these detergents. In contrast, Brij 58 and CHAPS displayed optimal activation of the two enzymes for a large range of detergent/microsomal protein ratios (respectively from 0.2 to 1 and from 0.4 to 1), making them the most suitable for induction and/or latency studies of both isoenzymes. The influence of maximal activation status on the effect of 3-methylcholanthrene and phenobarbital treatment on morphine-UGT and 1-naphthol-UGT activity has also been evaluated. The findings provided evidence that detergent-activation profiles and optimal detergent-activated versus "native" UGT activity determination give crucial informations about the characteristics of a given isoenzymic form of UGT, i.e. its sensitivity to specific alterations of the phospholipid environment, its latency and its inducibility.     

Effect of sodium dodecyl sulfate on the heat denaturation and aggregation of arachin at pH 3.6

The effect of an anionic detergent, sodium dodecyl sulfate, on the heat-induced denaturation and protein-protein interactions of arachin at pH 3.6 was studied by melting temperature, turbidity, electrophoresis and fluorescence spectral measurements. Sodium dodecyl sulfate induced aggregation in arachin at room temperature, in the concentration range 1 times 10^-5 to 1 times 10^-2 M, as shown by turbidity, electrophoresis and fluorescence spectral measurements. However, higher detergent concentrations (> 1 times 10^-2 M SDS) decreased the aggregation. Electrophoretic experiments at pH 3.6 showed that sodium dodecyl sulfate at a concentration of 5 times 10^-2 M changes the protein charge from positive to negative. Fluroescence spectral measurements suggested that the detergent at 5 times 10^-2 M level unfolds arachin. Heating and cooling arachin at pH 3.6 in the presence of sodium dodecyl sulfate increased the protein-protein interactions as compared to those at room temperature, in the detergent concentration range 1 times 10^-5 to 1 times 10^-2 M; at higher detergent concentrations there was a sharp decrease. The observed effects of sodium dodecyl sulfate on the structure and conformation and heat-induced protein-protein interactions of arachin at the experimental pH may arise out of the detergent binding to the protein involving both ionic and hydrophobic interactions.     

Effect of cetyl trimethyl ammonium bromide on the heat-induced denaturation and protein-protein interactions of arachin at pH 3.6

The effect of a cationic detergent, cetyl trimethyl ammonium bromide, on the heat-induced denaturation and protein-protein interactions of arachin at pH 3.6 was studied by gel melting temperature, turbidity, electrophoresis, u.v. difference and fluorescence spectral measurements. Cetyl trimethyl ammonium bromide decreased the heat-induced protein-protein interactions as shown by the melting temperature, turbidity and electrophoretic measurements. Turbidity and electrophoretic measurements also showed that the detergent induces dissociation of the protein before and after heating it at higher detergent concentration (above 1 times 10^-3 M). U.v. difference and fluorescence spectral measurements suggested that the detergent induces unfolding of arachin at room temperature and this increases markedly after heating the protein in the detergent. The promotive effect of cetyl trimethyl ammonium bromide on the heat-induced denaturation and its preventive effect on the protein-protein interactions of arachin under the experimental conditions may arise out of the binding of the detergent to protein by primarily hydrophobic interactions.     

Characterization of [3H]muscimol binding to mouse brain membranes.

The binding of [³H]muscimol {[methylene-³H(N)]-3-hydroxy-5-aminoethyl isoxazole} to a membrane fraction from mouse brain was studied as a possible model for the GABA recognition site of the γ-aminobutyric acid (GABA)-anionophore receptor complex. Kinetic studies showed two distinct association and dissociation rate constants, 2.3 × 10^?4 and 1.4 × 10^?5 sec^?1 nM^?1 for association and 1.2 × 10^?2 and 1.0 × 10^?3 sec^?1 for dissociation. Equilibrium analysis (Scatchard plot) of binding data also indicated two types of sites with K_D equal to 9 × 10^?9 and 70 × 10^?9 M. Detergents (0.1% Triton X-100, 0.1% Lubrol PX or 5% Tween 20) had no effect on the binding of [³H]muscimol or other conformationally restricted agonists such as imidazoleacetic acid, isoguvacine, THIP (4,5,6,7-tetrahydroisoxazole [5,4-c] pyridine-2-ol), and (±)-isonipecotic acid. In contrast to this, the detergents potentiated the ability of the less conformationally restricted agonists such as GABA and β-alanine in displacing [³H]muscimol. Binding of bicuculline was reduced by all of the detergent treatments.     

A Solid-State NMR Study of Protein Hydration and Stability

Purpose. The mobility of protein in powders at different hydration levels was studied in relation to aggregation and activity. Methods. Magic angle spinning ¹³C, ¹⁵N, ¹H, ²H, and ¹⁷O NMR techniques were used to determine changes in the mobility of surface residues in proteins as a function of hydration and related to changes in activity. NMR relaxation measurements of high frequency (₀, T₁) and low frequency (₁, T_1p) motions have been carried out on lyophilized DNase, insulin and lysozyme stored at different relative humidities. Moisture-induced aggregation and enzymatic activity of the lyophilized proteins was determined by high performance size exclusion chromatography and bioassays. Results. There was little change in T_1p observed with increasing humidity. The results show, however, that there is a decrease in T₁, for DNase, insulin and lysozyme at relative humidities ranging from 0–98%, and we propose that the reduction in T₁, is related to the aggregation susceptibility of proteins during storage at different humidities. The water mobility was determined directly using ¹⁷O NMR experiments. We found that as the amount of weakly-bound water increases, the protein surface mobility decreases and is coupled with increased aggregation. Aggregation measurements at different humidities were correlated with bioassays for lysozyme and found to be consistent with the hydration data. Conclusions. Mobility of protein molecules was determined by solid-state NMR over a wide range of % RH and it was found that water content leads to a change in mobility of protein molecules. The aggregation and activity of proteins were strongly correlated to change in molecular mobility.     

A spectroscopic study of the interaction of allylisothiocyanate with mustard 12S protein

The intraction of allylisothiocyanate (AIT) with mustard 12S protein resulted in a change in the absorption spectrum of the protein below 280nm without any shift in the ^γmax. The spectrophotometric titration of mustard 12S protein after interaction with AIT suggested the involvement of phenolic groups. Upon interaction with AIT, a new band at 250nm appeared in the near ultraviolet circular dichroism spectrum of the protein. However, no change occurred in the far ultraviolet circular dichroism spectrum. Circular dichroism measurements with amino acids suggest the possible involvement of tyrosine residues in the interaction of AIT with mustard 12S protein.     

Characterization of benzazepine UDP-glucuronosyl-transferases in laboratory animals and man

1. The O-glucuronidation of two dopamine D1 receptor antagonists, Odapipam and Berupipam, were studied in hepatic microsomal fractions from mouse, rat, rabbit, dog, pig, and man using ¹⁴C-UDP-glucuronic acid.

2. The influence of pH, detergent, gender, drug-metabolizing enzyme inducers, and age were examined. Detergents like the zwitterionic CHAPS and non-ionic Tween 20. Triton X-100, and Brij 35 stimulated the glucuronidation rate by up to 600% of native activity with the latter being most effective. Both apparent K_m and V_max increased following detergent treatment in rat hepatic microsomes. Less marked activation of UDP glucuronosyltransferase activity was observed with Brij 35 in mouse, rabbit, dog, and pig compared with rat. In contrast, human hepatic microsomes were not stimulated by detergent treatment.

3. Marked species-dependent UDP-glucuronosyltransferase activity were observed for the two compounds. In general, Odapipam exhibited higher V_max and K_m compared with Berupipam with the exception of rabbit where the reverse was true. Similar kinetic parameters were, however, observed in human hepatic microsomes. Highest glucuronidation rate (in general) was observed in mouse followed by dog, pig, rabbit, man, and rat.

4. UGT activity in human livers showed up to a seven-fold variation. Conjugation of each compound were highly correlated (r = 0.92; n = 20) suggesting that identical isoform(s) were involved in this reaction. A significant age-related decrease in UDP-glucuronosyltranferase activity was observed, which partly could be explained by a preponderance in elderly female donor liver samples.     

Effect of trimebutine on colonic myoelectrical activity in IBS patients

Summary The binding affinities of two steroid anaesthetics, alphaxalone (Alfx) and alphadolone acetate (Alfd), for testosterone-oestradiol-binding globulin (TeBG) and corticosteroid-binding globulin (CBG) were measured in human serum. In 8 male patients, the effect of i.v. administration of Althesin (a mixture of Alfx and Alfd) on the transport of testosterone (T) and cortisol (F) was studied. Both Alfx and Alfd bind to TeBG and CBG with a relatively high affinity (10⁶M^–1). A significant change in the percentage of unbound T was observed during Althesin infusion, with no change in total T concentration or in the TeBG binding parameters. The results suggest that by interaction with TeBG binding sites Alfx and/or Alfd displaced T bound to TeBG, and transiently increased the percentage of unbound T. A significant increase in the concentration of F was observed during althesin infusion, while the percentage of unbound F and the CBG binding parameters were unchanged. The dose of Alfx and Alfd used was not sufficient to alter the transport of F during brief althesin anaesthesia in men.     

AGGREGATION,DISSOCIATION AND DENATURATION OF SESAME (Sesamum indicum L.) α-GLOBULIN IN CETYL TRIMETHYL AMMONIUM BROMIDE SOLUTION*

The behaviour of the major protein of sesame seed (Sesamum indicum L.) α-globulin has been studied in a cationic detergent, cetyl trimethyl ammonium bromide solution. Up to a critical detergent concentration the protein is precipitated from solution, above which redissolution of the protein is observed. Sedimentation velocity patterns indicate the presence of higher aggregates in the detergent concentration range 5 times 10^-5– 1 times 10^-3 M. These are considered to be the soluble precursors of the insoluble aggregates. Fluorescence measurements show that tryptophanyl groups of the protein which are in contact with the aqueous phase are perturbed by the detergent. The difference spectra of the protein in higher concentration of detergent indicate considerable red shift in the spectrum. Spectrophotometric titration of phenolic groups in 1 times 10^-2 M CTAB indicate that a conformational change in the protein has taken place.     

Direct in vitro and in vivo demonstration of muscarinic receptor binding by the novel radioligand, [3H]5-hydroxymethyltolterodine,in the bladder and other tissues of rats

In vitro and in vivo binding sites of [³H]-labeled 5-hydroxymethyltolterodine (5-HMT), a new radioligand for labeling muscarinic receptors in rat tissues were characterized. Specific [³H]5-HMT binding in rat tissues was saturable and of high affinity in each tissue. The dissociation constant (K_d) was significantly lower in bladder and heart than in submaxillary gland. Significant levels of in vivo specific [³H]5-HMT binding by intravenous injection of the radioligand were detected in tissues, except for cerebral cortex. Thus, [³H]5-HMT was shown to specifically label muscarinic receptors in rat tissues, suggesting a useful radioligand for labeling muscarinic receptors with high affinity.     

In vivo kinetic analysis of covalent binding between N-acetyl-L-cysteine and plasma protein through the formation of mixed disulfide in rats

Purpose. This investigation was undertaken to study the relationship between plasma drug clearance and covalent protein-binding kinetics of N-acetyl-L-cysteine (NAC). Methods. NAC was intravenously administered to rats via a bolus injection or continuous infusion. Plasma concentrations of protein-unbound and total NAC were analyzed using a compartment model, taking into consideration of the protein binding process, and the apparent first-order binding and dissociation rate constants (k_on and k_off) were obtained. Results. Plasma total NAC after a bolus injection showed biphasic elimination with an inflection point at 1 hr. After 1 hr, NAC was largely present in the covalent protein-bound form. During the steady state of the infusion, approximately 30%-40% of plasma NAC bound with protein covalently. The k_on, k_off, and the elimination rate constant of protein-unbound drug (k_e) were 0.23, 0.57, and 4.3 hr^-1. The dissociation half-life of NAC from protein estimated from k_off was in agreement with the elimination half-life of plasma total NAC. This suggests that the dissociation of NAC from protein rate-limited the drug elimination in plasma (k_off < k_e). Conclusion. We demonstrated that plasma total drug clearance is kinetically limited by covalent protein binding. The compartmental model described here is useful for analyzing its kinetics in vivo.     

三种洗涤剂溶血反应的比较

洗衣粉,洗洁精广泛使用于玻璃仪器的洗涤,它们为表现活性剂,可引起红细胞表面张力改变而发生溶血反应,本文对三种洗涤剂的溶血反应进行了比较。     

Covalent and Noncovalent Protein Binding of Drugs: Implications for Hepatic Clearance,Storage, and Cell-Specific Drug Delivery

This review deals with the mechanisms by which the liver disposes of drugs that are covalently or noncovalently associated with proteins. Many drugs bind to plasma proteins such as albumin (mainly anionic compounds) and ₁-acid glycoprotein (cationic compounds). Nevertheless, the liver is able to clear such drugs efficiently from the circulation because of intrahepatic dissociation of the drug-protein complex. This clearance may involve spontaneous dissociation because of progressive removal of the unbound drug during liver passage, a process that can be rate limiting in hepatic uptake. Alternatively, the porous endothelial lining of the hepatic sinusoids may allow extensive surface interactions of the drug–protein complexes with hepatocytes, leading to facilitation of drug dissociation. Binding to plasma proteins and intracellular proteins in the cytoplasm or cell organelles is an important factor determining the hepatic storage and elimination rate of drugs. Drugs noncovalently associated with glycosylated proteins, which can be endocytosed by various liver cells, are not co-endocytosed with such proteins. However, covalently bound drugs can be internalized by receptor-mediated endocytosis, which permits specific targeting to hepatocytes, endothelial cells, Kupffer cells, and lipocytes by coupling to different glycoproteins that are recognized on the basis of their terminal sugar. The endocytosed drug–carrier complex is routed into endosomes and lysosomes, where the active drug is liberated by cleavage of acid-sensitive linkages or proteolytic degradation of peptide linkers. This concept has been applied to antineoplastic, antiparasitic, and antiviral drugs.     

[3H] sertraline binding to rat brain membranes

Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [³H] sertraline binding occurred at a single site with the following parameters:K _d 0.57 nM,B _max 821 fmol/mg protein,n _h 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (K _d 0.81 nM) agreed with that determined by saturation binding experiments. [³H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase inK _d with little change inB _max). The rank order of potency of inhibition of [³H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [³H] sertraline binding in the brain of rats 7 days after treatment withp-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [³H] sertraline labels serotonin uptake sites in rat brain.     

对曾氏推导的药物蛋白结合率公式的修正

对曾氏推导的药物蛋白结合率公式的修正娄建石（天津医科大学药理教研室，天津３０００７０）测定药物蛋白结合率是药物代谢动力学研究中的重要内容之一。曾衍霖教授根据国外文献推导出药物蛋白结合率公式（１），为这方面的研究提供了很好的计算依据。本文作者在计算药物...     

Effect of probenecid on the elimination and protein binding of ceftriaxone

Summary The kinetics and binding parameters of ceftriaxone have been characterized in eight normal subjects who received, in sequence, 1.0 g ceftriaxone and 1.0 g ceftriaxone together with 250 and 500 mg probenecid q.i.d.Probenecid increased the total systemic clearance (CL _S ^T ) from 0.244 to 0.312 ml/min/kg, whereas the terminal half-life (t _1/2() ^T ) fell from 8.1 to 6.5 h. In contrast, the renal clearance of free ceftriaxone (CL _R ^F ) was decreased from 2.09 to 1.67 ml/min/kg, confirming a small but significant contribution of tubular secretion to the renal elimination of ceftriaxone.The final value of CL _R ^F was attained with the lower dose probenecid, whereas the non-renal clearance of free ceftriaxone (CL _NR ^F ) fell progressively from 2.78 to 1.90 ml/min/kg with the increasing probenecid dose. The total decrease in the systemic clearance of free ceftriaxone (CL _S ^F ) after the higher dose of probenecid was about 30% (4.87 to 3.57 ml/min/kg).As a consequence of a decreased affinity constant (K_A), the average free fraction in plasma (f) was increased by 54% after the low dose and by 74% after the high dose of probenecid.The protein binding interaction between probenecid and ceftriaxone appears to be unique. The results are of limited clinical consequence for ceftriaxone but they emphasise the importance of evaluating the kinetics of the free drug when examining interactions involving probenecid.Abbreviations AUC^T, AUC^F plasma AUC of total and free (unbound) drug - CL _S ^T , CL _S ^F clearance: total systemic, free systemic - CL _O ^T , CL _R ^F total oral, free renal - CL _NR ^F free non-renal - V _Z ^T V _SS ^T apparent volume of distribution: terminal phase, total drug; corrected steady-state, total drug [14] - f average free fraction of drug in plasma - f_e fraction of dose excreted unchanged in urine - t _1/2() ^T terminal t_1/2: total drug - t _1/2() ^F free drug - K_A affinity constant - nP capacity constant - C _av ^ss steady-state concentration in plasma during multiple dosing - C_B bound concentration - C_F free (unbound) concentration