Mixed Non-competitive Type Research Articles

ACTIVIDAD INHIBITORIA in vitro DE LOS EXTRACTOS ACUOSOS DE LOS FRUTOS DE Hylocereus megalanthus Y Passiflora tripartita var. mollisima SOBRE LAS ENZIMAS α-AMILASA Y α-GLUCOSIDASA

La pitahaya amarilla (Hylocereus megalanthus) y el tumbo serrano (Passiflora tripartita var. mollisima) son frutos cultivados en Perú. El objetivo de este estudio fue evaluar la propiedad hipoglicemiante de estos frutos mediante la inhibición in vitro de las enzimas α-amilasa y α-glucosidasa. Los frutos fueron despulpados y liofilizados, y luego de preparar los respectivos extractos acuosos, se determinó el contenido de polifenoles totales (TPC) mediante el método de Folin-Ciocalteu. La actividad inhibitoria se determinó mediante el IC50 y se evaluó el tipo de inhibición utilizando 0,25-2 mg/mL y 0,02-0,28 mg/mL del extracto del tumbo serrano sobre la α-amilasa y α-glucosidasa, respectivamente. Para la pitahaya se empleó 4-64 mg/ml y 0,8-25,6 mg/mL sobre la α-amilasa y α-glucosidasa, respectivamente. Los TPC para el tumbo serrano y pitahaya amarilla fueron de 614,67 y 16,17 mg/100 g de muestra, respectivamente, expresados en equivalente de ácido gálico. El extracto acuoso de tumbo serrano inhibió a las enzimas α-amilasa y α-glucosidasa (tipo no competitiva mixta) con un IC50 de 1719,18 y 77,68 μg/Ml, respectivamente, mientras que la pitahaya amarilla solo inhibió a la enzima α-glucosidasa con un IC50 de 8692,4 μg/mL (tipo competitiva).

Biologic Propensities and Phytochemical Profile ofVangueria madagascariensisJ. F. Gmelin (Rubiaceae): An Underutilized Native Medicinal Food Plant from Africa

Vangueria madagascariensis (VM), consumed for its sweet-sour fruits, is used as a biomedicine for the management of diabetes and bacterial infections in Africa. The study aims to assess the potential of VM on α-amylase, α-glucosidase, glucose movement, and antimicrobial activity. The antioxidant properties were determined by measuring the FRAP, iron chelating activity, and abilities to scavenge DPPH, HOCl, ∙OH, and NO radicals. Leaf decoction, leaf methanol, and unripe fruit methanol extracts were observed to significantly inhibit α-amylase. Active extracts against α-glucosidase were unripe fruit methanol, unripe fruit decoction, leaf decoction, and ripe fruit methanol, which were significantly lower than acarbose. Kinetic studies revealed a mixed noncompetitive type of inhibition. Leaf methanolic extract was active against S. aureus and E. coli. Total phenolic content showed a strong significant positive correlation (r = 0.88) with FRAP. Methanolic leaf extract showed a more efficient NO scavenging potential and was significantly lower than ascorbic acid. Concerning ∙OH-mediated DNA degradation, only the methanol extracts of leaf, unripe fruit, and ripe fruit had IC50 values which were significantly lower than α-tocopherol. Given the dearth of information on the biologic propensities of VM, this study has established valuable primary information which has opened new perspectives for further pharmacological research.

(p)ppGpp Inhibits Polynucleotide Phosphorylase from Streptomyces but Not from Escherichia coli and Increases the Stability of Bulk mRNA in Streptomyces coelicolor

ppGpp regulates gene expression in a variety of bacteria and in plants. We proposed previously that ppGpp or its precursor, pppGpp [referred to collectively as (p)ppGpp], or both might regulate the activity of the enzyme polynucleotide phosphorylase in Streptomyces species. We have examined the effects of (p)ppGpp on the polymerization and phosphorolysis activities of PNPase from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli. We have shown that (p)ppGpp inhibits the activities of both Streptomyces PNPases but not the E. coli enzyme. The inhibition kinetics for polymerization using the Streptomyces enzymes are of the mixed noncompetitive type, suggesting that (p)ppGpp binds to a region other than the active site of the enzyme. ppGpp also inhibited the phosphorolysis of a model RNA substrate derived from the rpsO-pnp operon of S. coelicolor. We have shown further that the chemical stability of mRNA increases during the stationary phase in S. coelicolor and that induction of a plasmid-borne copy of relA in a relA-null mutant increases the chemical stability of bulk mRNA as well. We speculate that the observed inhibition in vitro may reflect a role of ppGpp in the regulation of antibiotic production in vivo.

α-Glucosidase inhibitory antihyperglycemic activity of substituted chromenone derivatives

Series of 3,4- and 3,6-disubstituted chromenones including new chromenone derivatives were synthesized applying various synthetic strategies including Pechmann condensation, Knoevenagel condensation, Reimer–Tiemann reaction and Suzuki coupling in very good yields. Synthesized compounds ( 4a– z) were screened for in vitro α-glucosidase inhibitory and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activities. Majority of compounds displayed varying degrees of α-glucosidase inhibitory and DPPH scavenging activity. Compound 4x emerged as the most potent α-glucosidase inhibitor in present series of compounds owing to the presence of 3-acetyl-6-(6-methoxy-3-pyridyl) group on chromenone; however, it could not display DPPH scavenging activity and was found to be mixed non-competitive type inhibitor of rat intestinal α-glucosidase. When tested in vivo for antihyperglycemic activity in starch loaded Wistar rats, it displayed significant antihyperglycemic property. This is the first report assigning rat intestinal α-glucosidase inhibitory property for this class of new chromenones and presents new family of compounds possessing α-glucosidase inhibitory activities and antihyperglycemic property. Compound 4x may serve as an interesting new compound for the development of therapeutics targeted against diet-induced hyperglycemia in diabetes.

The NADPH oxidase inhibitor diphenyleneiodonium is also a potent inhibitor of cholinesterases and the internal Ca2+ pump

Diphenyleneiodonium (DPI) is often used as an NADPH oxidase inhibitor, but is increasingly being found to have unrelated side effects. We investigated its effects on smooth muscle contractions and the related mechanisms. We studied isometric contractions in smooth muscle strips from bovine trachea. Cholinesterase activity was measured using a spectrophotometric assay; internal Ca(2+) pump activity was assessed by Ca(2+) uptake into smooth muscle microsomes. Contractions to acetylcholine were markedly enhanced by DPI (10(-4) M), whereas those to carbachol (CCh) were not, suggesting a possible inhibition of cholinesterase. DPI markedly suppressed contractions evoked by CCh, KCl and 5-HT, and also unmasked phasic activity in otherwise sustained responses. Direct biochemical assays confirmed that DPI was a potent inhibitor of acetylcholinesterase and butyrylcholinesterase (IC(50) approximately 8 x 10(-6) M and 6 x 10(-7) M, respectively), following a readily reversible, mixed non-competitive type of inhibition. The inhibitory effects of DPI on CCh contractions were not mimicked by another NADPH oxidase inhibitor (apocynin), nor the Src inhibitors PP1 or PP2, ruling out an action through the NADPH oxidase signalling pathway. Several features of the DPI-mediated suppression of agonist-evoked responses (i.e. suppression of peak magnitudes and unmasking of phasic activity) are similar to those of cyclopiazonic acid, an inhibitor of the internal Ca(2+) pump. Direct measurement of microsomal Ca(2+) uptake revealed that DPI modestly inhibits the internal Ca(2+) pump. DPI inhibits cholinesterase activity and the internal Ca(2+) pump in tracheal smooth muscle.

Aleppo tannin: structural analysis and salivary amylase inhibition

The effectiveness and specificity of a tannin inhibition on human salivary amylase (HSA) catalyzed hydrolysis was studied using 2-chloro-4-nitrophenyl 4- O-β- d-galactopyranosyl-α-maltoside (GalG 2-CNP) and amylose substrates. Aleppo tannin was isolated from the gall nut of Aleppo oak. This tannin is a gallotannin, in which glucose is esterified with gallic acids. This is the first kinetic report, which details the inhibitory effects of this compound on HSA. A mixed non-competitive type inhibition has been observed on both substrates. The extent of inhibition is markedly dependent on the substrate-type. Kinetic constants were calculated from Lineweaver–Burk secondary plots for GalG 2-CNP ( K EI 0.82 μg mL −1, K ESI 3.3 μg mL −1). This indicates a 1:1 binding ratio of inhibitor–enzyme and/or inhibitor–enzyme–substrate complex. When amylose was the substrate the binding ratio of inhibitor to enzyme–substrate complex was found to be 2:1, with the binding constants of K EI 17.4 μg mL −1, K ESI 14.9 μg mL −1, K ESI 2 9.6 μg mL −1. Presumably, the tannin inhibitor can bind not only to HSA, but to the amylose substrate, as well. Kinetic data suggest that Aleppo tannin is a more efficient amylase inhibitor than the recently studied other tannin with quinic acid core (GalG 2-CNP: K EI 9.0 μg mL −1, K ESI 47.9 μg mL −1).

Alkylammonium chlorobenzoates are a new group of ester-containing reversible inhibitors of cholinesterases of different animals

Interaction with cholinesterases (ChEs) of nine specially synthesized derivatives of dimethylaminoalkyl esters of 2-chloro-and 2,4-dichlorobenzoic acids and their iodoalkylates is studied. Used as enzyme sources were partially purified preparations of acetylcholinesterase (AChE) from human erythrocytes and butyrylcholinesterase (BChE) from horse blood serum, as well as water homogenates of the frog Rana temporaria brain and of the Pacific squid Todarodes pacificus optical ganglia. The studied benzoates failed to be hydrolyzed by the studied ChEs at the enzyme concentrations exceeding 10 times those used for determination of the acetylthiocholine hydrolysis rate. These compounds have turned out to be reversible inhibitors of ChEs of the mixed-noncompetitive type of action. Effects on the anticholinesterase activity of such structural elements of the inhibitors as the acidic part of the benzoate molecule, length of polymethylene chain in the molecule alcoholic part, and the structure of ammonium group are studied. This study has allowed revealing some peculiarities of the reaction capability of vertebrate and invertebrate ChEs.

Inhibition of mackerel ( Scomber scombrus) muscle lipoxygenase by green tea polyphenols

The high polyunsaturated fatty acid content of oily fish such as mackerel ( Scomber scombrus) makes it particularly susceptible to oxidative degradation. We have shown previously the presence of lipoxygenase (LOX), a lipid oxygenase, in mackerel muscle. In the current study, commercially available green tea polyphenols were shown to effectively inhibit the LOX activity of mackerel muscle. EGCG (epigallocatechin gallate) was the strongest inhibitor tested with an IC 50 (concentration for half maximal inhibition) value of 0.13 nM. All the tea catechins showed a mixed non-competitive type inhibition. In addition, antioxidants such as BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), esculetin, caffeic acid, ascorbic acid, and ethylene diamine tetraacetic acid (EDTA) were effective to varying degrees (IC 50 values between 0.02 and >50 μM) in the inhibition of mackerel muscle LOX. Nordihydroguaiaretic acid (NDGA), a classical LOX inhibitor and potassium cyanide (KCN), a heme protein inhibitor were assayed for their inhibitory activities for comparison. Post harvest spoilage of fish account for loss of as much as 10% of the world’s catches of cultured fish. This data indicates that the green tea polyphenols, nature’s very potent antioxidants, may be used as an effective and natural means of reducing post harvest spoilage in fish.

Kinetic investigation of a new inhibitor for human salivary α-amylase

This study is the first report on the effectiveness and specificity of α-acarviosinyl-(1 → 4)-α- d-glucopyranosyl-(1 → 6)- d-glucopyranosylidene-spiro-thiohydantoin (PTS-G-TH) inhibitor on the 2-chloro-4-nitrophenyl-4- O-β- d-galactopyranosyl-maltoside (GalG 2CNP) and amylose hydrolysis catalysed by human salivary α-amylase (HSA). Synthesis of PTS-G-TH was carried out by transglycosylation using acarbose as donor and glucopyranosylidene-spiro-thiohydantoin (G-TH) as acceptor. This new compound was found to be a much more efficient HSA inhibitor than G-TH. The inhibition is a mixed-noncompetitive type on both substrates and only one molecule of inhibitor binds to the enzyme. Kinetic constants calculated from secondary plots are in micromolar range. Values of K EI and K ESI are very similar in the presence of GalG 2CNP substrate; 0.19 and 0.24 μM, respectively. Significant difference can be found for K EI and K ESI using amylose as substrate; 8.45 and 0.5 μM, respectively. These values indicate that inhibition is rather uncompetitive than competitive related to amylose hydrolysis.

Oral Contraceptives as Substrates and Inhibitors for Human Cytosolic SULTs

Cytosolic sulfotransferases (SULTs) in mammals are involved in the biotransformation and homeostasis of various endogenous and xenobiotic compounds. The current study aimed to examine the sulfation of contraceptive compounds by various human cytosolic SULTs and to investigate the inhibitory effects and mode of action of these compounds on the sulfation of 17beta-estradiol, a major endogenous estrogen. A systematic study using all eleven known human cytosolic SULTs revealed the differential substrate specificity of these enzymes for the eight representative contraceptive compounds and two endogenous estrogens (estrone and 17beta-estradiol) tested as substrates. Activity data showed that SULT1A1 displayed the strongest activity toward 17alpha-ethynylestradiol. Kinetic studies revealed that the V (max) value of the sulfation of 17alpha-ethynylestradiol by SULT1A1 was 1.64 times that of the sulfation of 17beta-estradiol, while the K (m) values were almost equal for the two compounds. The inhibitory effects of three contraceptive compounds on the sulfation of 17beta-estradiol by SULT1A1 were examined. IC(50) values determined were 0.193, 1.84, and 2.98 mM, respectively, for 19-norethindrone acetate, ethynodiol diacetate and mifepristone. Kinetic analyses indicated that the mechanism underlying the inhibition by these contraceptives is of a mixed noncompetitive type. Metabolic labeling experiments confirmed the sulfation of contraceptive compounds and the release of their sulfated derivatives by HepG2 human hepatoma cells. Collectively, the results obtained suggest a role of sulfation in the metabolism of contraceptive compounds in vivo. Moreover, in view of their inhibitory effects on the sulfation of 17beta-estradiol, these compounds may potentially act to disrupt the homeostasis of endogenous estrogens.

Inhibitory effects of tannin on human salivary α-amylase

Here, we first report on the effectiveness and specificity of tannin inhibition of 2-chloro-4-nitrophenyl-4- O-β- d-galactopyranosylmaltoside hydrolysis that is catalyzed by human salivary α-amylase (HSA). Tannin was gallotannin in which quinic acid was esterified with 2–7 units of gallic acid. A number of studies establish that polyphenols—like tannins—may prevent oral diseases, e.g., dental caries. Kinetic analyses confirmed that the inhibition of hydrolysis is a mixed non-competitive type and only one molecule of tannin binds to the active site or the secondary site of the enzyme. Since Dixon plots were linear, product formation could be excluded from the enzyme–substrate–inhibitor complex (ESI). Kinetic constants calculated from secondary plots and non-linear regression are almost identical, thereby confirming the suggested model. Kinetic constants ( K EI=9.03 μg mL −1, K ESI=47.84 μg mL −1) show that tannin is as an effective inhibitor of HSA as acarbose and indicate a higher stability for the enzyme–inhibitor complex than ESI.

Cloning and expression of Lipomyces starkeyi α-amylase in Escherichia coli and determination of some of its properties

The Lipomyces starkeyi α-amylase (LSA) gene encoding soluble starch-degrading α-amylase was cloned and characterized from a derepressed and partially constitutive mutant for both dextranase and amylase activities. The nucleotide (nt) sequence of the cDNA fragment reveals an open reading frame of 1944 bp encoding a 619 amino acid (aa) mature protein (LSA) with a calculated molecular weight of 68.709 kDa that was estimated to be about 73 kDa, including His tag (4 kDa) based on SDS–PAGE (10% acrylamide gel), activity staining, and the Western blotting, using anti-amylase-Ab. LSA had a sequence similar to other α-amylases in four conserved regions of the α-amylase family: (I) 287DIVVNH 292, (II) 372GLRIDTVKH 380, (III) 399GEVFD 403, (IV) 462FLENQD 467. Polymerase chain reaction and sequence analysis showed one intron of 60 nucleotides in the genomic lsa at positions between 966 and 967 of cDNA. The cloned LSA amylase showed a maximum activity at pH 6 and optimum temperature of 40 oC, with greater than 90% stability between pH 5 and pH 8 for 16 h. It was inhibited by Cu 2+ and stimulated by Ca 2+ and Mg 2+. Enzyme activity was not affected by 1 mM EGTA but was inhibited by 1 mM EDTA. LSA did not hydrolyze maltodextrins of G2 to G4, yet formed G2 + G3 from G5, G2 + G4 or G3 + G3 from G6, and G3 + G4 from G7. LSA did not hydrolyze soluble starch in the present of 2% (w/v) of acarbose. Kinetics of LSA was carried out by using starch as a substrate and the inhibition type of acarbose was the mixed non-competitive type ( K i=3.4 μ M).

Inhibition of human salivary α-amylase by glucopyranosylidene-spiro-thiohydantoin

This study is the first report on the effectiveness and specificity of glucopyranosylidene-spiro-thiohydantoin (G-TH) inhibitor on the 2-chloro-4-nitrophenyl-4- O-β- d-galactopyranosyl-maltoside (GalG 2CNP) hydrolysis catalysed by human salivary α-amylase (HSA). The inhibition of hydrolysis is a mixed-noncompetitive type. In any case, only one molecule of inhibitor binds to HSA. Since our substrate and inhibitor are small molecules the long enough active site facilitates accommodating both of them simultaneously. However, the product formation can be excluded from enzyme–substrate–inhibitor complex (ESI) since Dixon plots are linear. Kinetic constants calculated from secondary plots and nonlinear regression are almost entirely equal, confirming the fidelity of the suggested model. Kinetic constants ( K 1i=7.3 mM, L 1 i =2.84 mM) show that G-TH is not such a potent inhibitor of HSA as acarbose and indicate higher stability for ESI than for enzyme–inhibitor complex.

Isolation, characterization and inhibition by acarbose of the α-amylase from Lactobacillus fermentum: comparison with Lb. manihotivorans andLb. plantarum amylases

Extracellular α-amylase from Lactobacillus fermentum (FERMENTA) was purified by glycogen precipitation and ion exchange chromatography. The purification was approximately 28-fold with a 27% yield. The FERMENTA molecular mass (106 000 Da) is in the same range as the ones determined for L. amylovorus (AMYLOA), L. plantarum (PLANTAA) and L. manihotivorans (MANIHOA) α-amylases. The amino acid composition of FERMENTA differs from the other lactobacilli considered here, but however, indicates that the peptidic sequence contains two equal parts: the N-terminal catalytic part; and the C-terminal repeats. The isoelectric point of FERMENTA, PLANTAA, MANIHOA are approximately the same (3.6). The FERMENTA optimum pH (5.0) is slightly more acidic and the optimum temperature is lower (40 °C). Raw starch hydrolysis catalyzed by all three amylases liberates maltotriose and maltotretaose. Maltose is also produced by FERMENTA and MANIHOA. Maltohexaose FERMENTA catalyzed hydrolysis produces maltose and maltotriose. Finally, kinetics of FERMENTA, PLANTAA and MANIHOA using amylose as a substrate and acarbose as an inhibitor, were carried out. Statistical analysis of kinetic data, expressed using a general velocity equation and assuming rapid equilibrium, showed that: (1) in the absence of inhibitor k cat/ Km are, respectively, 1×10 9, 12.6×10 9 and 3.2×10 9 s −1 M −1; and (2) the inhibition of FERMENTA is of the mixed non-competitive type ( K 1i=5.27 μM; L 1i=1.73 μM) while the inhibition of PLANTAA and MANIHOA is of the uncompetitive type ( L 1i=1.93 μM and 1.52 μM, respectively). Whatever the inhibition type, acarbose is a strong inhibitor of these Lactobacillus amylases. These results indicate that, as found in porcine and barley amylases, Lactobacillus amylases contain in addition to the active site, a soluble carbohydrate (substrate or product) binding site.

Starch digestion in tropical fishes: isolation, structural studies and inhibition kinetics of α-amylases from two tilapias Oreochromis niloticus and Sarotherodon melanotheron

α-Amylases from the intestinal cavity of two tilapia species, Oreochromis niloticus (ONI-AMY) and Sarotherodon melanotheron (SME-AMY), were purified using ammonium sulfate precipitation, affinity chromatography and chromatofocusing procedures. The purification was approximately 100-fold. The amylolytic activity, specific activity, product distribution, pH and temperature profile of ONI-AMY and SME-AMY are quite similar. The molecular mass differs slightly: 56 600 (ONI-AMY) vs. 55 500 (SME-AMY). As shown by isoelectric focusing analysis, both amylases contain two isoforms A and B with distinct p I: 7.2 (A) and 7.8 (B), vs. 8.3 (A) and 8.8 (B), respectively. It was not possible to isolate B, since B converts into A with time. The kinetics of the inhibition of ONI-AMY and SME-AMY activity by α-, β- and γ-cyclodextrin (α-, β- and γ-CD) were investigated using amylose as the substrate. Statistical analysis of the kinetic data expressed using a general velocity equation and assuming rapid equilibrium showed that the inhibition is of the mixed noncompetitive type. Similar results were obtained with ONI-AMY and SME-AMY. β- and γ-CD are stronger inhibitors than α-CD. ONI-AMY and SME-AMY are then closely related and show the general features common to the members of the α-amylase class (family 13). They enable ONI and SME tilapias to digest starch in food.

Mechanism of porcine pancreatic alpha-amylase. Inhibition of amylose and maltopentaose hydrolysis by alpha-, beta- and gamma-cyclodextrins.

The effects of alpha-, beta- and gamma-cyclodextrins on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic alpha-amylase (PPA) were investigated. The results of the statistical analysis performed on the kinetic data using the general initial velocity equation of a one-substrate reaction in the presence of one inhibitor indicate that the type of inhibition involved depends on the substrate used: the inhibition of amylose hydrolysis by alpha-, beta- and gamma-cyclodextrin is of the competitive type, while the inhibition of maltopentaose hydrolysis is of the mixed noncompetitive type. Consistently, the Lineweaver-Burk plots intersect on the vertical axis when amylose is used as the substrate, while in the case of maltopentaose, the intersection occurs at a point located in the second quadrant. The inhibition of the hydrolysis therefore involves only one abortive complex, PPA-cyclodextrin, when amylose is used as the substrate, while two abortive complexes, PPA-cyclodextrin and PPA-maltopentaose-cyclodextrin, are involved with maltopentaose. The mixed noncompetitive inhibition thus shows the existence of one accessory binding site. In any case, only one molecule of inhibitor binds to PPA. In line with these findings, the difference spectra of PPA produced by alpha-, beta- and gamma-cyclodextrin indicate that binding occurs at a tryptophan and a tyrosine residue. The corresponding dissociation constants and the inhibition constants obtained using the kinetic approach are in the same range (1.2-7 mM). The results obtained here on the inhibition of maltopentaose hydrolysis by cyclodextrin are similar to those previously obtained with acarbose as the inhibitor [Alkazaz, M., Desseaux, V., Marchis-Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100-107], but differ from those obtained with amylose as the substrate and acarbose as inhibitor [Alkazaz, M., Desseaux, V., Marchis-Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787-796]. It is concluded that the hydrolysis of both long and short chain substrates requires at least one secondary binding site, including a tryptophan residue.

The human pancreatic α-amylase isoforms: isolation, structural studies and kinetics of inhibition by acarbose

A rapid method is proposed for isolating the two main components of human pancreatic α-amylase (HPA I and HPA II). The isoelectric point of HPA I (7.2), the main component, was determined using an isoelectrofocusing method and found to differ from that of HPA II (6.6). The molecular mass of HPA I (55 862±5 Da) and that of HPA II (55 786±5 Da) were determined by performing mass spectrometry and found to be quite similar to that of the protein moiety calculated from the amino acid sequence (55 788 Da), which indicates that the human amylase is not glycosylated. The structure of both HPA I and HPA II was further investigated by performing limited proteolysis. Two fragments with an apparent molecular mass of 41 kDa and 14 kDa were obtained by digesting the isoforms with proteinase K and subtilisin, whereas digestion with papain yielded two cleaved fragments with molecular masses of 38 kDa and 17 kDa. Proteinase K and subtilisin susceptible bonds are located in the L8 loop (A domain), while the papain cut which occurs in the presence of the calcium chelator EDTA is in the L3 loop (B domain). The kinetics of the inhibition of HPA I and HPA II by acarbose, a drug used to treat diabetes and obesity, were studied using an amylose substrate. The Lineweaver-Burk primary plots of HPA I and HPA II, which did not differ significantly, indicated that the inhibition was of the mixed non-competitive type. The secondary plots gave parabolic curves. All in all, these data provide evidence that two acarbose molecules bind to HPA. In conclusion, apart from the p I, no significant differences were observed between HPA I and HPA II as regards either their molecular mass and limited proteolysis or their kinetic behavior. As was to be expected in view of the high degree of structural identity previously found to exist between human and porcine pancreatic amylases, the present data show that the inhibitory effects of acarbose on the kinetic behavior of these two amylases are quite comparable. In particular, the process of amylose hydrolysis catalyzed by HPA as well as by PPA in both cases requires two carbohydrate binding sites in addition to the catalytic site.

The mechanism of porcine pancreatic alpha-amylase. Inhibition of maltopentaose hydrolysis by acarbose, maltose and maltotriose.

A kinetic study was carried out on the inhibitory effects of acarbose, maltose, and maltotriose on porcine pancreatic alpha-amylase (PPA), using maltopentaose as the substrate. Lineweaver-Burk plots showed that the inhibitory action of acarbose is of the mixed non-competitive type. The secondary plots gave straight lines. A model involving abortive complexes accounts for these results. Dixon plot analysis led to the same conclusion. According to the proposed model, one molecule of acarbose per amylase molecule binds either directly to free enzyme at the active site or to the enzyme-substrate complex at a secondary carbohydrate-binding site, which becomes functional after the substrate has bound to the enzyme molecule at the active site. Kinetic analysis of the inhibition exerted by either the maltose or maltotriose reaction products of maltopentaose hydrolysis were then performed. The inhibitory effect of maltose was found to be of the non-competitive type, while that of maltotriose was competitive. It can therefore be concluded that the first reaction product to be released upon maltopentaose hydrolysis is maltose, and that the second product is maltotriose. This indicates that after hydrolysis of the maltopentaose chain, the reducing side fragment is released first.

Methadone: a potent inhibitor of rat liver aldehyde oxidase

Several drugs with structural similarities to SKF-525A were tested for their ability to inhibit rat liver aldehyde oxidase using the experimental antitumour agent N-[(2'-dimethylamino)ethyl]acridine-4-carboxamide (AC; NSC 601316; acridine carboxamide) as substrate. The antihistamine d-chlorpheniramine, and the antiarrhythmics disopyramide, procainamide and lignocaine were ineffective in inhibiting this reaction. The antihistamines diphenhydramine, pheniramine, doxylamine, orphenadrine, methapyrilene and pyrilamine, gave ic 50 values of 100–500 μM. The narcotic analgesics d-propoxyphene and, in particular, methadone were potent inhibitors of acridone formation with ic 50 values of 15.5 and 0.31 μM, respectively. Further analysis indicates mixed non-competitive type inhibition by methadone with inhibition constants ( K is and K ii , respectively) of 0.03 ± 0.01 (SE) and 0.57 ± 0.12μM.

Inhibition by SKF-525A of the aldehyde oxidase-mediated metabolism of the experimental antitumour agent acridine carboxamide

Oxidation of the experimental anti-tumour agent N-[(2'-dimethylamino) ethyl]acridine-4-carboxamide (AC; NSC 601316; acridine carboxamide) to the 9(10 H)acridone, followed by ring hydroxylation and glucuronidation, appears to be the main pathway of detoxication of AC in the rat and mouse. The acridone formation has been further characterized in vitro using an enzyme-enriched fraction where activity per milligram protein is increased approximately 10-fold compared with the cytosolic fraction. Inhibition by amsacrine [4'-(9-acridinylamino)methanesulphon- m-anisidide; NSC 249992] and menadione (50% inhibition at 6.4 and 1.8 μM, respectively) but not allopurinol (to 30μM) indicates that the activity is due to aldehyde oxidase, without the involvement of xanthine oxidase. Interestingly, acridone formation in both the cytosolic and enzyme-enriched fractions is highly sensitive to the classical cytochrome P450 inhibitor SKF-525A [proadifen hydrochloride; 2'-(diethylamino)ethyl 2,2-diphenylpentenoate] (50% inhibition at 9.2 and 1.9 μM, respectively). Further analysis indicates mixed non-competitive type inhibition by SKF-525A ( K is , 0.3 μM; K ii , 4.9 μM). Little or no inhibition was seen with cimetidine, metyrapone or methimazole. No NADPH-dependent acridone formation was observed with the microsomal fraction. These data indicate that acridone formation previously observed in isolated rat hepatocytes and in vivo is most likely due to aldehyde oxidase rather than cytochrome P450.