枯草芽孢杆菌SA-22 β-甘露聚糖酶的纯化及其特性

利用硫酸铵沉淀、羟基磷灰石柱层析、Sephadex G-75凝胶过滤和DEAE-52离子交换柱层析的方法,将枯草芽孢杆菌SA-22 β-甘露聚糖酶纯化了30.75倍,同时,该酶比活达到3478056 u/mg,收率达到23.43%。利用SDS-PAGE凝胶电泳和Sephadex G-75凝胶过滤的方法测得枯草芽孢杆菌SA-22 β-甘露聚糖酶的分子量分别为38 kD和34 kD。实验发现该酶的最适pH为6.5,在pH 5～10的范围内稳定;该酶最适温度为70℃,在50℃保温4h后其活力不变,在60℃保温4 h后剩余酶活为74.2%,70℃的酶活半衰期为3h。实验还发现Hg²⁺对酶活力有明显抑制作用。该酶对槐豆胶和魔芋胶的K_m和V_max值分别为11.30mg/mL, 4.76mg/mL和188.68(μmol·mL^-1·min^-1), 114.94(μmol·mL^-1·min^-1)。     

Purification of carbonic anhydrase-II from sheep liver and inhibitory effects of some heavy metals on enzyme activity

In this study; sheep carbonic anhydrase-II (SCA-II) (E.C: 4.2.1.1) was purified from sheep liver and in vitro effects of heavy metals on the enzyme was examined. SCA-II isozyme was purified with about 203 purification fold, a specific activity of 2320 EU/mg-protein and a yield of 72 by using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. Purity of the SCA-II enzyme was verified by SDS-PAGE technique and subunit molecular mass of the enzyme was found as 29?kDa. In addition to this, inhibitory effects of some metal ions on the enzyme were examined. In this study, sheep liver tissue was chosen; because the liver is an organ in which metal wastes of air, water and food are collected and it is easy to obtain the liver tissue. Because of the very important duties of CA enzyme on living beings, the effect of metals on the CA enzyme was investigated.     

Inhibition of aspartate aminotransferase by hydrazinosuccinate

DL-Hydrazinosuccinic acid was synthesized by the reaction of DL-bromosuccinic acid with hydrazine. The compound strongly inhibited aspartate aminotransferase and gave 50% inhibition at 1.3 μM when added simultaneously with L-aspartate to an assay mixture containing enzyme. Incubation of the enzyme with the compound prior to assay resulted in a much stronger inhibition, which proceeded time-dependently. The inhibition was protectable with L-aspartate and was substantially reversed by dialysis.     

Steroidal fraction of Carissa carandas L. inhibits microbial hyaluronidase activity by mixed inhibition mechanism

Abstract

Hyaluronidase (hyase) is a hyaluronic acid (HA) depolymerizing enzyme produced by many pathogenic bacteria as a virulence factor to establish and spread infections. Present studies established that a steroidal fraction (SF) isolated from leaves of Carissa carandas act as a strong hyase inhibitor. The kinetic parameters involved in the inhibition of hyase by purified SF were studied and compared with standard hyase inhibitor quercetin. The purified SF showed the highest inhibition with an IC50 of 5.19 mM in comparison with a standard inhibitor, quercetin (IC50 8.63 mM). The inhibition constant (Ki) of purified SF determined by Dixon plot was 8.32 mM, which was significantly lower than that of quercetin standard. The kinetic behavior of enzyme hyase revealed to be more complex than classical competitive and uncompetitive inhibition where inhibitor affects both Km and Vmax. The inhibitor (I) favored the binding to the enzyme–substrate (ES) complex where Km value appeared to decrease (Kmapp < Km). The inhibitor also leads to decrease in the apparent maximum velocity of the enzyme–substrate reaction (Vmaxapp < Vmax). These results signpost toward mixed nature of inhibition of enzyme hyase by purified SF. Anti-hyaluronidase activity by a bioactive metabolite from C. carandas has not been reported so far and has high therapeutic potential against spread of pathogen and its toxins in the host.     

Inhibition of mammalian aspartate transcarbamylase by quinazolinone derivatives

Quinazolinone derivatives have been studied as both in vitro and in vivo inhibitors of aspartate transcarbamylase (ATCase). In vitro treatment of mammalian ATCase with four compounds revealed that they inhibited enzyme activity and that 2-phenyl-1,3-4(H)benzothiazin-4-thione was the most potent one. This compound acts as a noncompetitive inhibitor towards both aspartate and carbamoyl phosphate. The values of the inhibition constant (K_i) indicate that this compound exerts a potent inhibitory effect upon ATCase activity. Moreover, in vivo treatment with different doses of these derivatives showed also an inhibitory effect upon ATCase, the relative activity being decreased by 40%–58% with a 1 mg dose. These data support the inhibition of ATCase by quinazolinone derivatives as a new type of inhibitor for the enzyme.     

Inhibition of octopus glutathione transferase by Meisenheimer complex analog, S-(2,4,6-trinitrophenyl) glutathione

The tight binding of Meisenheimer intermediate with octopus digestive gland glutathione transferase was analyzed with 1,3,5-trinitrobenzene, which forms a trapped Meisenheimer complex with glutathione because there is no leaving group at the ipso carbon. By steady-state enzyme kinetic analysis, an inhibition constant of 1.89 ± 0.17 M was found for the transient formed, S-(2,4,6-trinitrophenyl) glutathione. The above inhibition constant is 407-fold smaller than the K _m value for the substrate (2,4-dinitrochlorobenzene). Thus, S-(2,4,6-trinitrophenyl) glutathione is considered to be a transition-state analog. The tight binding of this inhibitor to the enzyme provides an explanation for the involvement of the biological binding effect on the rate enhancement in the glutathione transferase-catalyzed S_NAr mechanism.     

Basic models for differential inhibition of enzymes

The possible preferential action exerted by an inhibitor on the transformation of one of two agonist substrates catalyzed by the same enzyme has recently been reported in studies on aldose reductase inhibition. This event was defined as “intra-site differential inhibition” and the molecules able to exert this action as “differential inhibitors”. This work presents some basic kinetic models describing differential inhibition. Using a simple analytic approach, the results show that differential inhibition can occur through either competitive or mixed type inhibition in which the inhibitor prevalently targets the free enzyme. The results may help in selecting molecules whose differential inhibitory action could be advantageous in controlling the activity of enzymes acting on more than one substrate.     

Peptidyl cyclopropenones: Reversible inhibitors,irreversible inhibitors,or substrates of cysteine proteases?

Peptidyl cyclopropenones were previously introduced as selective cysteine protease reversible inhibitors. In the present study we synthesized one such peptidyl cyclopropenone and investigated its interaction with papain, a prototype cysteine protease. A set of kinetics, biochemical, HPLC, MS, and ¹³C‐NMR experiments revealed that the peptidyl cyclopropenone was an irreversible inhibitor of the enzyme, alkylating the catalytic cysteine. In parallel, this cyclopropenone also behaved as an alternative substrate of the enzyme, providing a product that was tentatively suggested to be either a spiroepoxy cyclopropanone or a gamma‐lactone. Thus, a single family of compounds exhibits an unusual variety of activities, being reversible inhibitors, irreversible inhibitors and alternative substrates towards enzymes of the same family.     

Inhibition of jack bean urease by p-benzoquinone: elucidation of the role of thiols and reversibility of the process

p-Benzoquinone (pBQ) was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 °C. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. The influence of the inhibitor concentration and the preincubation time on the enzyme activity was elucidated. It was found that increase in pBQ concentration resulted in a linear decrease of urease activity. The dependence of the enzyme activity on the preincubation time showed that the rate of inhibition rapidly decreased at the beginning of the process in order to achieve the constant value. The inhibition became time independent in the studied time range. This observation is characteristic of a slow binding mechanism of inhibition. The protective experiment proved that the urease active site is involved in the binding of pBQ. High effectiveness of thiol protectors against pBQ inhibition indicates the strategic role of the active site sulfhydryl group in the blocking process. There were two methods used for reactivation of pBQ-inhibited urease. The dilution of the urease-pBQ complex in urea solution did not result in a regain of enzyme activity. Alternatively, the addition of dithiothreitol into the urease-pBQ mixture caused the instant and efficient reactivation of the enzyme. The experiments showed that the nature of the urease-pBQ complex is irreversible but the application of a specific thiol reagent can release the active enzyme from the complex.     

Inactivation of acetylcholinesterase by various fluorophores

The inhibition of recombinant mouse acetylcholinesterase (rMAChE) and electric eel acetylcholinesterase (EEAChE) by seven, structurally different chromophore-based (dansyl, pyrene, dabsyl, diethylamino- and methoxycoumarin, Lissamine rhodamine B, and Texas Red) propargyl carboxamides or sulfonamides was studied. Diethylaminocoumarin, Lissamine, and Texas Red amides inhibited rMAChE with IC₅₀ values of 1.00 μM, 0.05 μM, and 0.70 μM, respectively. Lissamine and Texas Red amides inhibited EEAChE with IC₅₀ values of 3.57 and 10.4 μM, respectively. The other chromophore amides did not inhibit either AChE. The surprising inhibitory potency of Lissamine was examined in further detail against EEAChE and revealed a mixed-type inhibition with K_i?=?11.7 μM (competitive) and K_i′?=?24.9 μM (noncompetitive), suggesting that Lissamine binds to free enzyme and enzyme–substrate complex.     

Purification and Properties of Sarcosine Oxidase from Cylindrocarpon didymum M–1

Sarcosine oxidase was purified to homogeneity from the cell extract of Cylindrocarpon didymum M–1, aerobically grown in medium containing choline as the carbon source. The molecular weight of the enzyme was estimated to be 45,000 by gel filtration method and 48,000 by the sodium dodecylsulfate disc gel electrophoresis method. The enzyme exhibited an absorption spectrum with maxima at 277 and 450 run and shoulders at 370 and 470 nm. The anaerobic addition of sarcosine to the enzyme resulted in the disappearance of the peak at 450 nm. The enzyme contained one mol of covalently bound FAD per mol of enzyme. Enzyme activity was inhibited by Ag⁺, Cu²⁺, Hg²⁺, p-chloromercuribenzoate and iodoacetate. The enzyme oxidized sarcosine but was inert toward choline, betaine, dimethylglycine and N-methyl amino acids. Km and V_max values for sarcosine were 1.8 ihm and 26.2 μmol/min/mg, respectively. The enzyme catalyzed the following reaction: Sarcosine+O₂+H₂O→glycine +formaldehyde+H₂O₂.     

Product inhibition of the enzymatic hydrolysis of lactose

Kinetic experiments have been conducted with acetone-dried cells of Kluyveromyces fragilis to study product inhibition of the enzymatic hydrolysis of lactose. Both hydrolytic products, d-glucose and d-galactose, showed efficient inhibition effect on enzyme activity. The fact that d-glucose and d-galactose are mutually exclusive for the inhibition was verified by Dixon plots. The kinetic constants were also estimated using the experimental data. The rate equation was derived based on a multiple inhibition model of competitive inhibition of d-galactose and non-competitive inhibition of d-glucose. The good agreement between experiment and prediction indicated the validity of the established model.     

Expression of human β-N-acetylhexosaminidase B in yeast eases the search for selective inhibitors

Human lysosomal β-N-acetylhexosaminidases from the family 20 of glycoside hydrolases are dimeric enzymes catalysing the cleavage of terminal β-N-acetylglucosamine and β-N-acetylgalactosamine residues from a broad spectrum of glycoconjugates. Here, we present a facile, robust, and cost-effective extracellular expression of human β-N-acetylhexosaminidase B in Pichia pastoris KM71H strain. The prepared Hex B was purified in a single step with 33% yield obtaining 10 mg of the pure enzyme per 1 L of the culture media. The enzyme was used in the inhibition assays with the known mechanism-based inhibitor NAG-thiazoline and a wide variety of its derivatives in the search for specific inhibitors of the human GH20 β-N-acetylhexosaminidases over the human GH84 β-N-acetylglucosaminidase, which was expressed, purified and used in the inhibition experiments as well. Moreover, enzyme-inhibitor complexes were analysed employing computational tools in order to reveal the structural basis of the results of the inhibition assays, showing the importance of water-mediated interactions between the enzyme and respective ligands. The presented method for the heterologous expression of human Hex B is robust, it significantly reduces the costs and equipment demands in comparison to the expression in mammalian cell lines. This will enhance accessibility of this human enzyme to the broad scientific community and may speed up the research of specific inhibitors of this physiologically important glycosidase family.     

In vitro effects of some drugs on human erythrocyte glutathione reductase

The effects of ketotifen, meloxicam, phenyramidol–HCl and gadopentetic acid on the enzyme activity of GR were studied using human erythrocyte glutathione reductase (GR) enzymes in vitro. The enzyme was purified 209-fold from human erythrocytes in a yield of 19% with 0.31?U/mg. The purification procedure involved the preparation of haemolysate, ammonium sulphate precipitation, 2′′,5′-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies. In the in vitro studies, IC₅₀ values and K_i constants were 0.012?mM and 0.0008?±?0.00021?mM for ketotifen; 0.029?mM and 0.0061?±?0.00127?mM for meloxicam; 0.99?mM and 0.4340?±?0.0890?mM for phenyramidol–HCl; 138?mM and 28.84?±?4.69?mM for gadopentetic acid, respectively, showing the inhibition effects on the purified enzyme. Phenyramidol–HCl showed competitive inhibition, whereas the others showed non-competitive inhibition.     

Urease activity in the crystalline state.

Crystalline Klebsiella aerogenes urease was found to have less than 0.05% of the activity observed for the soluble enzyme under standard assay conditions. Li2SO4, present in the crystal storage buffer at 2 M concentration, was shown to inhibit soluble urease by a mixed inhibition mechanism (Ki's of 0.38 +/- 0.05 M for the free enzyme and 0.13 +/- 0.02 M for the enzyme-urea complex). However, the activity of crystals was less than 0.5% of the expected value, suggesting that salt inhibition does not account for the near absence of crystalline activity. Dissolution of crystals resulted in approximately 43% recovery of the soluble enzyme activity, demonstrating that protein denaturation during crystal growth does not cause the dramatic diminishment in the catalytic rate. Finally, crushed crystals exhibited only a three-fold increase in activity over that of intact crystals, indicating that the rate of substrate diffusion into the crystals does not significantly limit the enzyme activity. We conclude that urease is effectively inactive in this crystal form, possibly due to conformational restrictions associated with a lid covering the active site, and propose that the small amounts of activity observed arise from limited enzyme activity at the crystal surfaces or trace levels of enzyme dissolution into the crystal storage buffer.     

Aluminum: a pH-dependent inhibitor of NADP-isocitrate dehydrogenase from porcine heart

Aluminum showed a pH-dependent inhibitory effect on NADP-isocitrate dehydrogenase from porcine heart. Aluminum ions (Al³⁺) acted as a partial competitive inhibitor of the enzyme with respect to the substratethreo-Ds-isocitrate and inhibited the enzyme non-competitively with respect to NADP at pH 6.85. Fractional velocity plot analysis showed theK _i of the enzyme for aluminum ions to be 0.88m. When pH was elevated to 8.0, aluminum ions, which occur as a form of the Al(OH)₄ ^– anion, acted as partial uncompetitive and non-competitive inhibitors of the enzyme with respect to the substrates isocitrate and NADP, respectively. TheK _i of the enzyme was determined to be 5.64 m at pH 8.0 by fractional velocity plot analysis. The inhibition of NADP-isocitrate dehydrogenase by two forms of aluminum ions may explain aluminum toxicity in various tissues and organs.     

Inhibition of choline acetyltransferase by excitatory amino acids as a possible mechanism for cholinergic dysfunction in the central nervous system

Choline acetyltransferase (ChAT) activity was reduced by more than 85% in cultured retina cells after 16 h treatment with 150 microM kainate (T(1/2) : 3.5 h). Glutamate, AMPA and quisqualate also inhibited the enzyme in equivalent proportion. Cell lesion measured by lactate dehydrogenase (LDH) release, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - thiazolyl blue (MTT) reduction and microscopic observation was not detected even after 48 h with kainate. Other retina neurochemical markers were not affected by kainate and full recovery of the enzyme was achieved 9 days after kainate removal. Moreover, hemicolinium-3 sensitive choline uptake and hemicolinium-3 binding sites were maintained intact after kainate treatment. The immunoblot and immunohistochemical analysis of the enzyme revealed that ChAT molecules were maintained in cholinergic neurons. The use of antagonists showed that ionotropic and group 1 metabotropic receptors mediated the effect of glutamate on ChAT inhibition, in a calcium dependent manner. The quisqualate mediated ChAT inhibition and part of the kainate effect (30%) was prevented by 5 mM N(G)-nitro-L-arginine methyl ester (L-NAME). Veratridine (3 microM) also reduced ChAT by a Ca(2+) dependent, but glutamate independent mechanism and was prevented by 1 microM tetrodotoxin.     

Kinetics of Inhibition of Green Crab (Scylla serrata) Alkaline Phosphatase by L-Cysteine

The inhibition of alkaline phosphatase from green crab (Scylla serrata) by L-cysteine has been studied. The results show that L-cysteine gives a mixed-type inhibition. The progress-of-substrate-reaction method previously described by Tsou [(1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 391–436] was used to study the inactivation kinetics of the enzyme by L-cysteine. The microscopic rate constants were determined for reaction of the inhibitor with the free enzyme and the enzyme–substrate complex (ES) The results show that inactivation of the enzyme by L-cysteine is a slow, reversible reaction. Comparison of the inactivation rate constants of free enzyme and ES suggests that the presence of the substrate offers marked protection of this enzyme against inactivation by L-cysteine.     

Aggregation and inhibition of rat intestinal alkaline phosphatase by high concentrations of calcium. Reversibility of the processes

Intestinal alkaline phosphatase (IAP) is an enzyme of the brush border of the enterocyte. The activity of IAP biphasically depends on calcium. Although calcium increases IAP activity, when calcium is higher than 20 mmole/L, IAP activity decreases and the amount of an aggregated form increases. The reversibility of the effect of calcium and the aggregation process are unknown. The isoelectric point of the enzyme was higher in the presence of calcium, but was the same for the enzyme and the aggregated form. The treatment with EGTA after calcium addition did not restore the enzymatic activity but produced desaggregation of the enzyme and increase in the monomeric subunits of IAP. It is concluded that the binding of calcium does not produce important modifications on the structure of the protein, that the inhibitory effect is not reversible and that calcium could be involved in the stability of the structure of the enzyme.     

The inhibition effect of some n-alkyl dithiocarbamates on mushroom tyrosinase

Three new n-alkyl dithiocarbamate compounds, as sodium salts, C₄H₉NHCS₂Na (I), C₆H₁₃NHCS₂Na (II) and C₈H₁₇NHCS₂Na (III), were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agaricus bisporus in 10 mM phosphate buffer pH 6.8, at 293K using UV spectrophotometry. Caffeic acid and p-coumaric acid were used as natural substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver–Burk plots showed different patterns of mixed and competitive inhibition for catecholase and cresolase reactions, respectively. These new synthetic compounds can be classified as potent inhibitors of MT due to K_i values of 0.8, 1.0 and 1.8 μM for cresolase inhibitory activity, and also 9.4, 14.5 and 28.1 μM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater potency in the inhibitory effect towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (α>1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds. The inhibition mechanism is presumably related to the chelating of the binuclear coppers at the active site and the different K_i values may be related to different interaction of the aliphatic chains of I, II and III with the hydrophobic pocket in the active site of the enzyme.