MUHAMMAD ATHAR ABBASI et al., Kinetics Studies on Symplocomoside: A Urease Inhibitor (original) (raw)
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CYCLIC UREA ANALOGUES AS INHIBITORS OF JACK BEAN UREASE.
Based on the structural homology with urea, the substrate of urease, a variety of cyclic urea analogues viz. barbiturate, imidazolidinone and imidazolone derivatives were tested for their effect on jack bean urease in 50mM phosphate buffer, pH=7.0. These compounds were found to inhibit jack bean urease in a concentration dependent manner IC50 varies from 0.6 mM to 3.5mM with no further time dependent changes in the reaction velocities. Evaluation of the kinetic data from Lineweaver-Burk plots and Dixon plots revealed that the analyzed compounds act as reversibile competitive inhibitors of the plant enzyme.
Purification and biochemical characteristics of urease from Proteus rettgeri
Biochimica Et Biophysica Acta (bba) - Enzymology, 1971
The β-D-glucosidase produced by Thermomyces lanuginosus-SSBP was purified to apparent homogeneity. The purified enzyme consisted of two identical subunits with a native molecular mass of 200 kDa. The purified β-Dglucosidase only hydrolysed the glucoside substrates containing a terminal, non-reducing β-D-glucose residue and was active on both aryl-β-glucoside and cellobiose. This enzyme also exhibited less, but significant α-Dglucosidase activity and was capable of hydrolysing β-1,6-linked diglucosides and gentiobiose. The K app m , V max and k cat values for p-nitrophenyl-β-D-glucopyranoside were calculated to be 0.075 mM, 12.12 units/mg of protein and 44.44 glucose molecules released/s respectively. The β-D-glucosidase retained its full activity after a 30 min incubation at 50 mC but was inactive after the same treatment at 70 mC. The enzyme appeared to be stable when the pH of the storage buffer was above 5.0. Maximal β-D-glucosidase activity occurred at 65 mC and pH 6.0. This enzyme was competitively inhibited by glucose, cellobiose and salicin with K i values of 0.55, 0.52 and 0.81 mM respectively. The presence of Hg 2 + and Nbromosuccinimide inhibited the enzyme activity completely at 2 mM, while cysteine enhanced β-D-glucosidase activity.
ANAR-8 Formal Lab Report-- Jack Bean urease kinematics and mode of inhibition of AHA.docx
This paper was produced by Ali and I as a comprehensive laboratory report for a 5 week long biochemistry experiment in Biochem lab. This experiment set out to determine the kinematics of Jack Bean urease and to determine the mode of inhibition of acetohydroxamide (AHA). This was accomplished by comparing the results from urease activity assay data collected when introducing a 1 mM and 2 mM inhibitor to the urease to the results of the urease activity assay with no inhibitor present. A spectrometer was used to determine the activity of the enzyme with the use of an indophenol standard curve. Km was determined to be 2.04 mM, Vmax was calculated to be 0.03922 mM NH3 /min, Kcat was determined to be 2.51 s-1, and Ki was averaged and found to be 1.6 mM. These results indicated that acetohydroxamide acted as a mixed inhibitor in the presence of Jack Bean urease; however, previous literature determined that AHA acted as a competitive inhibitor in the presence of Jack Bean urease.
Significance of sulfhydryl groups in the activity of urease from pigeonpea (Cajanus cajan L.) seeds
Plant Science, 2000
Titration of urease from pigeonpea (Cajanus cajan L.), a hexameric protein (mol. wt. 480 000; subunit mol. wt. 80 000), with 5,5%-dithiobis-(2-nitrobenzoate) (DTNB) reveals the presence of 5.82 9 0.13 'accessible' sulfhydryl groups per molecule of the enzyme protein (i.e. about one 'accessible' SH group per subunit). Denatured enzyme was found to titrate for 12.1 90.1 SH groups per molecule (i.e. about two SH groups per subunit). Half of the 'accessible' groups react faster than the remaining at pH 8.5 as well as pH 7.5. However, the reaction was slower at pH 7.5 than 8.5. Time-dependent loss of enzyme activity with DTNB was also found to be biphasic. The enzyme was inactivated at low concentration of p-chloromercuribenzoate (p-CMB), N-ethyl maleimide (NEM) and iodoacetamide. The inactivation reactions were biphasic, with half of the activity lost more rapidly than the remaining half. The loss of activity with p-CMB was linearly related to the blocking of accessible SH groups. Inactivation by p-CMB is largely reversible by addition of excess of cysteine. Fluoride ion strongly protects the enzyme against NEM inactivation, however, substrate urea provides much weaker protection against SH group reagents. The significance of these results is discussed.
Iranian Journal of Pharmaceutical Research, 2012
Helicobacter pylori (H. pylori) infection leads to different clinical and pathological outcomes in humans, including chronic gastritis, peptic ulcer disease and gastric neoplasia and even gastric cancer and its eradiation dependst upon multi-drug therapy. The most effective therapy is still unknown and prompts people to make great efforts to find better and more modern natural or synthetic anti-H. pylori agents. In this report 21 randomly selected herbal methanolic extracts were evaluated for their effect on inhibition of Jack-bean urease using the indophenol method as described by Weatherburn. The inhibition potency was measured by UV spectroscopy technique at 630 nm which attributes to released ammonium. Among these extracts, five showed potent inhibitory activities with IC 50 ranges of 18-35 µg/ mL. These plants are Matricaria disciforme (IC 50 :35 µg/mL), Nasturtium officinale (IC 50 :18 µg/mL), Punica granatum (IC 50 :30 µg/mL), Camelia sinensis (IC 50 :35 µg/mL), Citrus aurantifolia (IC 50 :28 µg/mL).
A New α-Glucosidase Inhibiting Dithiadiazetidin Derivative from Symplocos racemosa
HETEROCYCLES, 2005
The phytochemical investigation of the n-butanol soluble fraction of Symplocos racemosa Roxb. resulted in the isolation of a new dithiadiazetidin derivative; symploate (1). Its structure was established through various 1D and 2D NMR techniques together with high-resolution mass spectrometric techniques and spectral evidences. The symploate (1) showed moderate inhibitory activity against α-glucosidase in a concentration-dependent fashion with an IC 50 value of 691.1 ± 3.29 µM.
Inhibition of jack bean urease by amphiphilic peptides
Medicinal Chemistry Research
In the current study, amphiphilic peptides were designed and screened against Jak bean urease by using computer aided drug discovery approach. The result showed that out of thirty eight amphiphilic peptides 1, 3, 12, 18, 30, and exhibit stronger binding a nity with the active site of the enzyme and establish hydrogen bonds with the nickel ions i.e. Ni 841 and Ni 842 as well as make non-polar and hydrophobic contacts with the nonpolar residues in the active site. The selected amphiphilic peptides were synthesized by solid-phase peptide synthesis strategy, characterized by Fast atomic bombardment mass spectroscopy (FAB-MS) and Nuclear magnetic resonance spectroscopy (1 H and 13 C-NMR) and performed in vitro urease inhibitory activity. Amphiphilic peptides 12 and showed excellent urease inhibitory activity, (p < 0.001) with IC 50 values 20.5 ± 0.01 µM, and 28.1 ± 0.03 µM respectively, which was considerably better than thiourea used as positive control. We suggest that these amphiphilic could be potent urease inhibitors and sever as the starting point for effective interventions.
Jackbean, soybean and Bacillus pasteurii ureases: Biological effects unrelated to ureolytic activity
European Journal of Biochemistry, 2004
In this work we compared two plant ureases, jackbean urease (JBU) and embryo-specific soybean urease (SBU) and a bacterial (Bacillus pasteurii) urease, for kinetic parameters and other biological properties described recently for ureases that are independent of the ureolytic activity. The insecticidal effect of ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Contrasting with B. pasteurii urease (PBU), both plant ureases presented potent insecticidal activity, with LD50 values of 0.017% (w/w) and 0.052% (w/w) for JBU and SBU, respectively. The insecticidal property of JBU or SBU was not affected by treatment with p-hydroxymercuribenzoate, an irreversible inhibitor of ureolytic activity of both proteins. Also, contrasting with canatoxin – a urease isoform from jackbean seeds that displays a toxic effect in mice (LD50 = 2 mg·kg−1) – no lethality was seen in mice injected intraperitoneally with JBU or SBU (20 mg·kg−1). Similarly to canatoxin, the three enzymes promoted aggregation of blood platelets (EC50 = 400.0 µg·mL−1, 22.2 µg·mL−1, 15.8 µg·mL−1 for BPU, SBU and JBU, respectively). This platelet activating property was also independent of urease activity. Comparison of the kinetic properties indicated that SBU is fivefold less susceptible than JBU to inhibition by acetohydroxamic acid, a chelator of Ni+2 and Zn+2 ions. The ureases also showed different susceptibility to agents that modify cysteine residues, such as p-hydroxymercuribenzoate and p-benzoquinone. Altogether, these data emphasize that biological properties that are independent of ureolytic activity are not restricted to jackbean ureases and that these proteins may have a role in plant defense against insect predators.
Jackbean, soybean and Bacillus pasteurii ureases
European Journal of Biochemistry, 2004
In this work we compared two plant ureases, jackbean urease (JBU) and embryo-specific soybean urease (SBU) and a bacterial (Bacillus pasteurii) urease, for kinetic parameters and other biological properties described recently for ureases that are independent of the ureolytic activity. The insecticidal effect of ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Contrasting with B. pasteurii urease (PBU), both plant ureases presented potent insecticidal activity, with LD 50 values of 0.017% (w/w) and 0.052% (w/w) for JBU and SBU, respectively. The insecticidal property of JBU or SBU was not affected by treatment with p-hydroxymercuribenzoate, an irreversible inhibitor of ureolytic activity of both proteins. Also, contrasting with canatoxin -a urease isoform from jackbean seeds that displays a toxic effect in mice (LD 50 ¼ 2 mgAEkg )1 ) -no lethality was seen in mice injected intraperitoneally with JBU or SBU (20 mgAEkg )1 ). Similarly to canatoxin, the three enzymes promoted aggregation of blood platelets (EC 50 ¼ 400.0 lgAEmL )1 , 22.2 lgAEmL )1 , 15.8 lgAEmL )1 for BPU, SBU and JBU, respectively). This platelet activating property was also independent of urease activity. Comparison of the kinetic properties indicated that SBU is fivefold less susceptible than JBU to inhibition by acetohydroxamic acid, a chelator of Ni +2 and Zn +2 ions. The ureases also showed different susceptibility to agents that modify cysteine residues, such as p-hydroxymercuribenzoate and p-benzoquinone. Altogether, these data emphasize that biological properties that are independent of ureolytic activity are not restricted to jackbean ureases and that these proteins may have a role in plant defense against insect predators.
Anti-urease Secondary Metabolites from Seriphidium quettense
2016
Ethyl acetate layer of the methanolic extract of Seriphidium quettense was subjected to silica gel column chromatography to isolate one new; seriphiloid (1), and four known compounds; ilicic acid (2), 6hydroxy-8(10)-oplopen-14-one (3), 2-(4-hydroxyphenyl)-5,6,7-trimethoxy-4H-chromen-4-one (4) and 2-(3,4dihydroxyphenyl)-5,6,7-trimethoxy-4H-chromen-4-one (5). The chemical structure of the new isolate was established with the help of 1D, 2D NMR techniques and high resolution mass spectrometry. Known compounds were identified because of 1D NMR and mass spectrometric analysis and in comparison with the literature values. Compounds 1-5 were evaluated for their acetylcholinesterase, butyrylcholinesterase, -glucosidase and urease inhibitory activities. Most of the metabolites were found inactive; however, compounds 2 and 3 showed good antiurease activity with IC50 value 21.5±0.1 and 20.8±0.1 μg/mL, respectively.