Kinetics of methotrexate binding to dihydrofolate reductase from Neisseria gonorrhoeae

The kinetics of methotrexate inhibition of dihydrofolate reductase from Neisseria gonorrhoeae have been investigated. Methotrexate was shown to be a tight-binding inhibitor (Kt = 13 pM) competitive with dihydrofolate. However, "stoichiometric" or "pseudoirreversible" inhibition could not be demonstrated. Progress curves of inhibited assays quickly attained steady state regardless of the order of substrate addition, indicating that methotrexate association and dissociation processes were rapid. Kinetic techniques were used to measure the rate of methotrexate dissociation from the enzyme-NADPH-methotrexate ternary complex. At 30 degrees, the first-order off-rate constant (koff) was calculated to be 0.56 min-1. This value is approximately 40-fold greater than the dissociation rate constant of methotrexate for Escherichia coli dihydrofolate reductase. At lower temperatures, progress curves of methotrexate-inhibited gonococcal enzyme assays displayed marked increases in both curvature and the time to reach steady state. At 9 degrees, the methotrexate dissociation rate was slow enough (koff = 0.04 min-1) so that initial velocities of the reaction could be measured, and under these conditions methotrexate inhibition was shown to be "stoichiometric".     

Relationship between stimulated prolactin release from GH cells and cyclic AMP degradation and formation

We have studied the relationship between the prolaction (PRL) release induced by thyroliberin (TRH) and theophylline and the formation and inactivation of adenosine 3', 5'-cyclic monophosphate (cyclic AMP) in cultured rat-pituitary cells (GH3 cells). TRH, which stimulated prolactin release, increased cyclic AMP formation and stimulated transiently both the low- and high-Km cyclic phosphodiesterases. The maximal effect on the phosphodiesterase was observed at 30 mM TRH. The stimulatory effect of TRH on the activity of the cyclic AMP phosphodiesterases was duplicated by incubation of the cells with cyclic AMP (2-10 mM). In washed particulate GH3 cell fractions, TRH increased the adenylyl cyclase activity up to 180%. Treatment of GH3 cells with theophylline stimulated the release of PRL and inhibited cyclic AMP degradation probably leading to the measured increase in cellular concentrations of the nucleotide. The effects of TRH and theophylline on cellular cyclic AMP concentrations and on PRL release were additive. There was a positive correlation between PRL release and cellular cyclic AMP concentration (r = 0.97). The elevations observed in cellular cyclic AMP concentration after TRH treatment are due to increased formation which in turn leads to phosphodiesterase activation. Therefore, cyclic AMP formation appears to be an intermediary step in the stimulus-secretion coupling caused by the tripeptide.     

Metabolism and transfer of the mycotoxin zearalenone in human intestinal Caco-2 cells

The mycotoxin zearalenone (ZEA) is found worldwide as contaminant in cereals and grains. It is implicated in reproductive disorders and hyperestrogenic syndromes in animals and humans exposed by food. We investigated metabolism and transfer of ZEA using the human Caco-2 cell line as a model of intestinal epithelial barrier. Cells exposed to 10–200 μM ZEA showed efficacious metabolism of the toxin. α-zearalenol and β-zearalenol were the measured preponderant metabolites (respectively 40.7 ± 3.1% and 31.9 ± 4.9% of total metabolites, after a 3 h exposure to 10 μM ZEA), whereas ZEA-glucuronide and α-zearalenol glucuronide were less produced (respectively 8.2 ± 0.9% and 19.1 ± 1.3% of total metabolites, after a 3 h exposure to 10 μM ZEA). Cell production of reduced metabolites was strongly inhibited by α-and β-hydroxysteroid dehydrogenase inhibitors, and Caco-2 cells exhibited α-hydroxysteroid dehydrogenase type II and β-hydroxysteroid dehydrogenase type I mRNA. After cell apical exposure to ZEA, α-zearalenol was preponderantly found at the basal side, whereas β-zearalenol and both glucuronides were preferentially excreted at the apical side. As α-zearalenol shows the strongest estrogenic activity, the preferential production and basal transfer of this metabolite suggests that intestinal cells may contribute to the manifestation of zearalenone adverse effects.     

Qualitative Änderungen in Lebermikrosomen Phenobarbital-behandelter Kaninchen

Summary Phenobarbital treatment of rabbits was found to cause significant changes in some of the apparent Michaelis (K _M) constants for p- and N-hydroxylation of aniline [reactions 1 and 2, resp.] and de-ethylation, p- and N-hydroxylation of N-ethylaniline [reactions 3,4, and 5, resp.], and in the spectral dissociation constants (K _s) for aniline, N-ethylaniline and ethylisocyanide in isolated hepatic microsomes.The critical oxygen concentrations were increased in all reactions investigated.The distribution constants (K _G) for CO, determined according to Warburg, decreased in the reactions 2, 3, and 4, and increased in reaction 1. Reaction 5 was not inhibited by CO in microsomes from untreated rabbits and was stimulated by CO after phenobarbital treatment of the animals.Depending on the reaction, theK _M-values for aniline and N-ethylaniline were increased, decreased, or remained unchanged by the phenobarbital treatment of the animals. The most striking changes inK _M-values were observed with the reactions 2 and 5 in which cytochrome P-450 is either not involved or is not a limiting factor.TheK _s-value for ethylisocyanide measured after addition of dithionite to microsomes was increased. The correspondingK _s-value measured without dithionite under aerobic conditions remained unchanged.TheK _s value for aniline was decreased by the phenobarbital treatment of the animals. Using microsomes from untreated animals theK _s value for N-ethylaniline could not be determined exactly. The order of magnitude of this value, however, was greater than that of the corresponding value from phenobarbital-treated animals.The observed alterations in affinity imply that induction by phenobarbital may be associated withqualitative as well as quantitative changes in the hepatic microsomal enzymes or membranes. The qualitative changes could be important for the accessibility of the reacting groups and/or substrate guiding.

FrÄulein Barbara Suermann danken wir für wertvolle technische Mitarbeit bei der Messung der Bindungskonstanten von Anilin und N-Äthylanilin.     

Ventricular irritability associated with the use of naloxone hydrochloride. Two case reports and laboratory assessment of the effect of the drug on cardiac excitability

Two instances of ventricular tachycardia and fibrillation have followed the injection of naloxone hydrochloride (Narcan) that had been given to reverse the effects of morphine following cardiac surgical procedures. Injection of the drug into 5 dogs that had received morphine was associated with ventricular extrasystoles in 2 animals, although no increase in ventricular excitability could be demonstrated as measured by either strength/interval curves or ventricular diastolic threshold. Naloxone hydrochloride should be used with caution in patients with cardiac irritability.     

Evidence that unsaturated fatty acids are potent inhibitors of renal UDP-glucuronosyltransferases (UGT): kinetic studies using human kidney cortical microsomes and recombinant UGT1A9 and UGT2B7

Renal ischaemia is associated with accumulation of fatty acids (FA) and mobilisation of arachidonic acid (AA). Given the capacity of UDP-glucuronosyltransferase (UGT) isoforms to metabolise both drugs and FA, we hypothesised that FA would inhibit renal drug glucuronidation. The effect of FA (C2:0-C20:5) on 4-methylumbelliferone (4-MU) glucuronidation was investigated using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7 as the enzyme sources. 4-MU glucuronidation exhibited Michaelis-Menten kinetics with HKCM (apparent K(m) (K(m)(app)) 20.3 microM), weak substrate inhibition with UGT1A9 (K(m)(app) 10.2 microM, K(si) 289.6 microM), and sigmoid kinetics with UGT2B7 (S(50)(app)440.6 microM) Similarly, biphasic UDP-glucuronic acid (UDPGA) kinetics were observed with HKCM (S(50) 354.3 microM) and UGT1A9 (S(50) 88.2 microM). In contrast, the Michaelis-Menten kinetics for UDPGA observed with UGT2B7 (K(m)(app) 493.2 microM) suggested that kinetic interactions with UGTs were specific to the xenobiotic substrate and the co-substrate (UDPGA). FA (C16:1-C20:5) significantly inhibited (25-93%) HKCM, UGT1A9 or UGT2B7 catalysed 4-MU glucuronidation. Although linoleic acid (LA) and AA were both competitive inhibitors of 4-MU glucuronidation by HKCM (K(i)(app) 6.34 and 0.15 microM, respectively), only LA was a competitive inhibitor of UGT1A9 (K(i)(app) 4.06 microM). In contrast, inhibition of UGT1A9 by AA exhibited atypical kinetics. These data indicate that LA and AA are potent inhibitors of 4-MU glucuronidation catalysed by human kidney UGTs and recombinant UGT1A9 and UGT2B7. It is conceivable therefore that during periods of renal ischaemia FA may impair renal drug glucuronidation thus compromising the protective capacity of the kidney against drug-induced nephrotoxicity.     

Identification of two arginase isoenzyme activities along the nephron of Meriones shawi

 Conflicting theories on the existence of several renal arginase isoenzymes remain in debate. Because the activity of arginase is high in two embryologically different nephron segments of the Meriones shawi kidney, namely the cortical (CPST) and medullary (OSPST) proximal straight tubule and the outer medullary collecting duct (OMCD), we postulate that these nephron segments may contain different isoforms. Isolated nephron segments were dissected from collagenase-treated kidneys. Tubules were permeabilized with Triton X-100 (0.25%) and incubated with increasing Arg concentrations to characterize the arginase activity. The results were as follows: (1) in OMCD, one arginase isoform (E₁), characterized by a high Arg affinity (1.160 mM), was present; (2) in CPST, two arginase isoforms were discovered – one, E₁, had a similar K _m (1.407 mM) to that found in OMCD whereas the other (E₂) had a low affinity for Arg (K _m =18.8 mM); and (3) in OSPST, two isoenzymes were present – E₁ which had a K _m of 1.478 mM and the second isoform that we named E₂ which had a K _m of 9.07 mM. In addition, arginase located in CPST and OMCD was strongly inhibited by Orn and Lys. The K _i value for Lys varied between 1.635 and 2.288 mM. Therefore, this work demonstrates that two arginase isoforms are present in the kidney of Meriones shawi. Isoform E₁ is present in the proximal tubule and the collecting duct whereas isoform E₂ is restricted to the proximal tubule. Received: 13 August 1998 / Accepted: 25 September 1998     

Threshold analysis of selected dose-response data for endocrine active chemicals.

Using a biologically relevant mathematical model, the Michaelis-Menten equation, we examined published data from endocrine active chemicals for evidence of no-threshold dose-response curves. Data were fit to a modified Michaelis-Menten equation which accounted for total background response. Subsequently, the data sets were analyzed using non-linear regression in order to estimate the four parameters of interest (non-hormone controlled background (Bnh), maximum response (Rmax), endogenous hormone level (D0), and the dose at which a half-maximal response was observed (ED50)) and to determine the fit to the fully modified Michaelis-Menten equation. Subsequently, response data were adjusted to account for Bnh and then normalized to Rmax, while dose data were adjusted to account for D0 and then normalized to the ED50. This data set was combined into a single, composite data set and fit to the fully modified Michaelis-Menten equation. We examined 31 data sets (24 endpoints) from studies on 9 different chemical/hormone treatments. Twenty-six of the data sets fit the modified Michaelis-Menten equation with high multiple correlation coefficients (r>0.90). The normalized data demonstrated a good fit to the modified Michaelis-Menten equation. These results indicate that a variety of biological responses fit the modified Michaelis-Menten equation, which does not have a threshold dose term.     

甘氨酰脯氨酸二肽氨基肽酶检测技术及临床应用研究

该文研究了甘氨酰辅氨酸二肽氨基肽酶（GPDAEC．3．4．14．5）的最适反应条件，9次测定米氏常数（Km）值为（1．91±0．08）mmol／L，选用158mmol／LTris、64mmol/L，双甘氨肽，10mmol／L底物，pH8．6缓冲液，同时建立了手工法和自动化分析法，酶促反应速度在70min内呈线性，酶活力用手工法在750IU／L，内成线性，自动化分析法在1300IU/L，以下成线性，三份高、中、低值酶活力标本手工法及自动化分析法批内变异系数分别为1．7％～3．1％和0．7％～1．4％，批间变其分别为3.8％～6．7％和1．2％～4．4％。132例正常人GPDA为185．2±28．6（x±s），65例原发性肝癌组为408．0±216．1（x±s）明显高于正常组（P＜0．001）且与AFP水平无相关意义（r＝0．0361）．24例胃癌和59例肺癌分别为（99．5±16．6）IU／L和（11．8±23．0）IU／L，明显伏于正常组（P＜0．001）；并发现GPDA在食道癌及有门癌病人亦有下降趋势（P＜0．01）。