Enzyme deactivation in fixed bed reactors with michaelis-menten kinetics

Rate expression for enzyme poisoning which are consistent with a Michaelis-Menten main reaction are used to analyze the performance of a fixed bed reactor containing immobilized enzyme. When enzyme deactivation results from the irreversible bonding of a product molecule to an existing substrate-enzyme complex, it is shown that minimum enzyme activity can occur in the interior of the bed, well away from the ends. This suggests that bed sectioning techniques may enable direct evaluation of fundamental poisoning mechanisms.

Enzyme immobilization in porous solid supports - penetration of immobilized enzyme

The process of enzyme immobilization under the diffusion-controlled regime (i.e., fast attachment of enzyme compared to its diffusion) is modeled and theoretically solved in this article. Simple and compact solutions for the penetration depth of immobilized enzyme and the bulk enzyme concentration versus time are presented. Furthermore, the conditions for the validity of our solutions are also given in this article so that researchers can discover when the theoretical solutions can be applied to their systems.

Functional polymorphism of the human arylamine N-acetyltransferase type 1 gene caused by (CT)-T-190 and G(560)A mutations

Human N-acetyltransferase type 1 (NAT1) catalyses the N- or O-acetylation of various arylamine and heterocyclic amine substrates and is able to bioactivate several known carcinogens. Despite wide inter-individual variability in activity, historically, NAT1 was considered to be monomorphic in nature. However, recent reports of allelic variation at the NAT1 locus suggest that it may be a polymorphically expressed enzyme. In the present study, peripheral blood mononuclear cell NAT1 activity in 85 individuals was found to be bimodally distributed with approximately 8% of the population being slow acetylators. Subsequent sequencing of the individuals having slow acetylator status showed all to have either a (CT)-T-190 or G(560)A base substitution located in the protein encoding region of the NAT1 gene. The (CT)-T-190 base substitution changed a highly conserved Arg(64), which others have shown to be essential for fully functional NAT1 protein. The (CT)-T-190 mutation has not been reported previously and we have named it NAT1*17. The G(560)A mutation is associated with the base substitutions previously observed in the NAT1*10 allele and this variant (NAT1*14) encodes for a protein with reduced acetylation capacity. A novel method using linear PCR and dideoxy terminators was developed for the detection of NAT1*14 and NAT1*17. Neither of these variants was found in the rapid acetylator population. We conclude that both the (CT)-T-190 (NAT1*17) and G(560)A (NAT1*14) NAT1 structural variants are involved in a distinct NAT1 polymorphism. Because NAT1 can bioactivate several carcinogens, this polymorphism may have implications for cancer risk in individual subjects. (C) 1998 Chapman & Hall Ltd.

Localisation of aryl sulfotransferase expression in human tissues using hybridisation histochemistry and immunohistochemistry

To date, the laboratory has cloned seven unique human sulfotransferases; five aryl sulfotransferases (HAST1, HAST2, HAST3, HAST4 and HAST4v), an estrogen sulfotransferase and a dehydroepiandrosterone sulfotransferase. The cellular distribution of human aryl sulfotransferases in human hepatic and extrahepatic tissues has been determined using the techniques of hybridization histochemistry and immunohistochemistry. Human aryl sulfotransferase expression was detected in liver, epithelial cells of the gastrointestinal mucosal layer, epithelial cells lining bronchioles and in mammary duct epithelial cells. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

The excitatory behavioral and antianalgesic pharmacology of normorphine-3-glucuronide after intracerebroventricular administration to rats

In the adult male Sprague-Dawley rat, a species commonly used to study tolerance to the antinociceptive effects of morphine, approximate to 10% of the morphine dose is metabolized to normorphine-3-glucuronide (NM3G). In contrast, NM3G is a relatively minor metabolite of morphine in human urine reportedly accounting for approximate to 1% of the morphine dose. To date, the pharmacology of NM3G has been poorly characterized. Therefore, our studies were designed to determine whether the intrinsic pharmacology of NM3G is similar to that of morphine-3-glucuronide (M3G), the major metabolite of morphine, which has been shown to be a potent central nervous system (CNS) excitant and to attenuate the intrinsic antinociceptive effects of morphine in rats. The CNS excitatory potency of NM3G was found to be approximately half that of M3G, inducing convulsions in rats at intracerebroventricular (i.c.v.) doses of greater than or equal to 16.8 nmol. When administered before morphine (70 nmol i.c.v.), NM3G (8.9 nmol i.c.v.) attenuated antinociception for up to 2 hr, but when administered after morphine, no significant attenuation of morphine antinociception was observed. Thus, after i.c.v. administration, NM3G like M3G, is a potent CNS excitant and antianalgesic in the rat. NM3G may therefore play a role in the development of tolerance to the antinociceptive effects of morphine in the rat as has been proposed previously for M3G.

A novel method for selection of chymotrypsin inhibitors from a phage peptide library

A novel screening strategy has been developed for the identification of alpha-chymotrypsin inhibitors from a phage peptide library. In this strategy, the standard affinity selection protocol was modified by adding a proteolytic cleavage period to avoid recovery of alpha-chymotrypsin substrates. After four cycles of selection and further activity assay, a group of related peptides were identified by DNA sequencing. These peptides share a consensus sequence motif as (S/T)RVPR(R/H). Then, a corresponding short peptide (Ac-ASRVPRRG-NH2) was synthesized chemically and proved to be an inhibitor of alpha-chymotrypsin. The present work provides a useful way for searching proteinase inhibitors without detailed knowledge of the molecular structure.

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: implications for human prolidase

Electron paramagnetic resonance (EPR) spectra and X-ray absorption (EXAFS and XANES) data have been recorded for the manganese enzyme aminopeptidase P (AMPP, PepP protein) from Escherichia coli. The biological function of the protein, a tetramer of 50-kDa subunits, is the hydrolysis of N-terminal Xaa-Pro peptide bonds. Activity assays confirm that the enzyme is activated by treatment with Mn2+. The EPR spectrum of Mn2+-activated AMPP at liquid-He temperature is characteristic of an exchange-coupled dinuclear Mn(II) site, the Mn-Mn separation calculated from the zero-field splitting D of the quintet state being 3.5 (+/- 0.1) Angstrom. In the X-ray absorption spectrum of Mn2+-activated AMPP at the Mn K edge, the near-edge features are consistent with octahedrally coordinated Mn atoms in oxidation state +2. EXAFS data, limited to k less than or equal to 12 Angstrom(-1) by traces of Fe in the protein, are consistent with a single coordination shell occupied predominantly by O donor atoms at an average Mn-ligand distance of 2.15 Angstrom, but the possibility of a mixture of O and N donor atoms is not excluded. The Mn-Mn interaction at 3.5 Angstrom, is not detected in the EXAFS, probably due to destructive interference from light outer-shell atoms. The biological function, amino acid sequence and metal-ion dependence of E. coli AMPP are closely related to those of human prolidase, an enzyme that specifically cleaves Xaa-Pro dipeptides. Mutations that lead to human prolidase deficiency and clinical symptoms have been identified. Several known inhibitors of prolidase also inhibit AMPP. When these inhibitors are added to Mn2+-activated AMPP, the EPR spectrum and EXAFS remain unchanged. It can be inferred that the inhibitors either do not bind directly to the Mn centres, or substitute for existing Mn ligands without a significant change in donor atoms or coordination geometry. The conclusions from the spectroscopic measurements on AMPP have been verified by, and complement, a recent crystal structure analysis.

Evaluation of an immunoassay (EMIT) for mycophenolic acid in plasma from renal transplant recipients compared with a high-performance liquid chromatography assay

Mycophenolic acid is an immunosuppressant administered as a bioavailable ester, mycophenolate mofetil. The pharmacokinetics of mycophenolic acid have been reported to be variable. Accurate measurement of concentrations of this drug could be important to adjust doses. The aim of this study was to compare the enzyme-multiplied immunoassay technique (EMIT [Dade Behring; San Jose, CA, U.S.A.]) for mycophenolic acid with a high-performance liquid chromatographic (HPLC) assay using samples collected from renal transplant recipients. The HPLC assay used solid phase extraction and a C18 stationary phase with ultraviolet (UV) detection (254 nm). The immunoassay required no manual sample preparation. Plasma samples (n = 102) from seven patients, collected at various times after a dose, were analyzed using both methods. Both assays fulfilled quality-control criteria. Higher concentrations were consistently measured in patient samples when using EMIT. The mean (+/- standard deviation [SD]) bias (EMIT-HPLC) was 1.88 +/- 0.86 mg/L. The differences in concentrations were higher in the middle of a dosage interval, suggesting that a metabolite might have been responsible for overestimation. Measurement of glucuronide concentrations by HPLC demonstrated only a weak correlation between assay differences and glucuronide concentrations. If the crossreacting substance is active, EMIT could provide a superior measure of immunosuppression; if inactive, further work is needed to improve antibody specificity. In conclusion, it was found that EMIT overestimates the concentration of mycophenolic acid in plasma samples from renal transplant recipients compared with HPLC analysis.

Spider toxins: A new group of potassium channel modulators

Spider toxins that target potassium channels constitute a new class of pharmacological tools that can be used to probe the structure and function of these channels at the molecular level. The limited studies performed to date indicate that these peptide toxins may facilitate the analysis of K+ channels that have proved insensitive to peptide inhibitors isolated from other animal sources. Thus far, two classes of K+ channel-selective spider toxins have been isolated, sequenced, and pharmacologically characterised - the hanatoxins (HaTx) from Grammastola spatulata and heteropodatoxins (HpTx) from Heteropoda venatoria. The hanatoxins block Kv2.1 and Kv4.2 voltage-gated K+ channels. In Kv2.1 K+ channels this occurs as a consequence of a depolarising shift in the voltage dependence of activation and not by occlusion of the channel pore. These toxins show minimal sequence homology with other peptide inhibitors of K+ channels, but they do share some homology with other ion channel toxins from spiders, particularly with regard to the spacing between cysteine residues. We have recently isolated three K+ channel antagonists from the venom of the Australian funnel-web spider Hadronyche versuta; at least two of these toxins are likely to constitute a new class of spider toxins active on K+ channels as they are approximately twice as large as HaTx and HpTx.

Crystallization and preliminary X-ray diffraction studies of mammalian purple acid phosphatase

The oxidized form of purple acid phosphatase from pig allantoic fluid has been crystallized in the presence of phosphate using the hanging-drop technique. The crystals belong to the space group P2(1)2(1)2(1) and have unit-cell parameters a = 66.8, b = 70.3, c = 78.7 Angstrom. Diffraction data collected from a cryocooled crystal using a conventional X-ray source extend to 1.55 Angstrom resolution. A knowledge of the three-dimensional structure of mammalian purple acid phosphatase will aid in understanding the substrate specificity of the enzyme and will be important in the rational design of inhibitors, with potential in the treatment of bone diseases.

Inhibitors of beta-amyloid formation based on the beta-secretase cleavage site

A series of inhibitors of beta-amyloid formation have been developed based on the beta-secretase cleavage site (VNL-DA) of the Swedish mutant Amyloid Precursor Protein. A simple tripeptide aldehyde was found to be the most potent (IC50 = 700 nM) in the series displaying an inhibitory profile which is different from reported inhibitors of beta-amyloid formation. (C) 2000 Academic Press.

Substrate-dependent regulation of human arylamine N-acetyltransferase-1 in cultured cells

Arylamine N-acetyltransferase-1 (NAT1) is a polymorphically expressed enzyme that is widely distributed throughout the body. In the present study, we provide evidence for substrate-dependent regulation of this enzyme. Human peripheral blood mononuclear cells cultured in medium supplemented with p-aminobenzoic acid (PABA; 6 mu M) for 24 h showed a significant decrease (50-80%) in NAT1 activity. The loss of activity was concentration-dependent (EC50 similar to 2 mu M) and selective because PABA had no effect on the activity of constitutively expressed lactate dehydrogenase or aspartate aminotransferase. PABA also induced down-regulation of NAT1 activity in several human cell lines grown at confluence. Substrate-dependent downregulation was not restricted to PABA. Addition of other NAT1 substrates, such as p-aminosalicylic acid, ethyl-p-aminobenzoate, or p-aminophenol to peripheral blood mononuclear cells in culture also resulted in significant (P < .05) decreases in NAT1 activity. However, addition of the NAT2-selective substrates sulfamethazine, dapsone, or procainamide did not alter NAT1 activity. Western blot analysis using a NAT1-specific antibody showed that the loss of NAT1 activity was associated with a parallel reduction in the amount of NAT1 protein (r(2) = 0.95). Arylamines that did not decrease NAT1 activity did not alter NAT1 protein levels. Semiquantitative reverse transcriptase polymerase chain reaction of mRNA isolated from treated and untreated cells revealed no effect of PABA on NAT1 mRNA levels. We conclude that NAT1 can be down-regulated by arylamines that are themselves NAT1 substrates. Because NAT1 is involved in the detoxification/activation of various drugs and carcinogens, substrate-dependent regulation may have important consequences with regard to drug toxicity and cancer risk.

Conformational selection of inhibitors and substrates by proteolytic enzymes: Implications for drug design and polypeptide processing

Inhibitors of proteolytic enzymes (proteases) are emerging as prospective treatments for diseases such as AIDS and viral infections, cancers, inflammatory disorders, and Alzheimer's disease. Generic approaches to the design of protease inhibitors are limited by the unpredictability of interactions between, and structural changes to, inhibitor and protease during binding. A computer analysis of superimposed crystal structures for 266 small molecule inhibitors bound to 48 proteases (16 aspartic, 17 serine, 8 cysteine, and 7 metallo) provides the first conclusive proof that inhibitors, including substrate analogues, commonly bind in an extended beta-strand conformation at the active sites of all these proteases. Representative superimposed structures are shown for (a) multiple inhibitors bound to a protease of each class, (b) single inhibitors each bound to multiple proteases, and (c) conformationally constrained inhibitors bound to proteases. Thus inhibitor/substrate conformation, rather than sequence/composition alone, influences protease recognition, and this has profound implications for inhibitor design. This conclusion is supported by NMR, CD, and binding studies for HIV-1 protease inhibitors/ substrates which, when preorganized in an extended conformation, have significantly higher protease affinity. Recognition is dependent upon conformational equilibria since helical and turn peptide conformations are not processed by proteases. Conformational selection explains the resistance of folded/structured regions of proteins to proteolytic degradation, the susceptibility of denatured proteins to processing, and the higher affinity of conformationally constrained 'extended' inhibitors/substrates for proteases. Other approaches to extended inhibitor conformations should similarly lead to high-affinity binding to a protease.

Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods, These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA, To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters, In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [H-3]N-OH-IQ to DNA, ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and similar to 50% of that seen with acetyl coenzyme A (AcCoA), In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites, With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein, The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis.