Fungicidal, Corrosive, and Mutational Effects of Polyhexamethylene Biguanide Combined with 1-Bromo-3-chloro-5,5-dimethylimidazolidine-2,4-dione.

BACKGROUND: The disinfectants polyhexamethylene biguanide (PHMB) and 1-bromo-3-chloro-5,5-dimethylimidazolidine-2,4-dione (BCDMH) each have limitations. So far, their combined usage has not been examined. In this study, the fungicidal activity of combined disinfectant using PHMB and BCDMH, named PB, against Candida albicans was evaluated. METHODS: Suspension quantitative fungicidal test and viable fungi count were used to test fungicidal effects against C. albicans. Coupon corrosion testing was used to evaluate disinfectants' corrosive effects on stainless steel, copper, and aluminum. The mouse lymphoma assay was used to detect mutations induced by PB. RESULTS AND DISCUSSION: Fungicidal activity of the combination of 40 mg/L PHMB and 40 mg/L BCDMH was comparable to, or even better than, those of 600 mg/L PHMB or 640 mg/L BCDMH alone. The combination of 400 mg/L PHMB and 400 mg/L BCDMH exhibited good fungicidal effects in field applications. The combination of 100 mg/L PHMB and 100 mg/L BCDMH did not have corrosive effects on stainless steel and no mutagenic effect was observed under the test conditions. CONCLUSIONS: The combination of PHMB and BCDMH has strong fungicidal effects and little metal corrosive and mutagenic effect and can be used as one suitable fungicide for wide household and industrial applications, including shipping containers.

Nf-GH, a glycosidase secreted by Naegleria fowleri, causes mucin degradation: an in vitro and in vivo study.

AIM: The aim of this work was to identify, characterize and evaluate the pathogenic role of mucinolytic activity released by Naegleria fowleri. MATERIALS & METHODS: Zymograms, protease inhibitors, anion exchange chromatography, MALDI-TOF-MS, enzymatic assays, Western blot, and confocal microscopy were used to identify and characterize a secreted mucinase; inhibition assays using antibodies, dot-blots and mouse survival tests were used to evaluate the mucinase as a virulence factor. RESULTS: A 94-kDa protein with mucinolytic activity was inducible and abolished by p-hydroxymercuribenzoate. MALDI-TOF-MS identified a glycoside hydrolase. Specific antibodies against N. fowleri-glycoside hydrolase inhibit cellular damage and MUC5AC degradation, and delay mouse mortality. CONCLUSION: Our findings suggest that secretory products from N. fowleri play an important role in mucus degradation during the invasion process.

D-Amino acid oxidase and presence of D-proline in Xenopus laevis.

We purified D-amino acid oxidase (EC 1.4.3.3, DAO) from Xenopus laevis tadpoles. The optimal temperature and pH for enzyme activity were 35-40 °C and 8.3-9.0, respectively, depending on the substrate amino acids available to the enzyme; the highest activity was observed with D-proline followed by D-phenylalanine. Activity was significantly inhibited by p-hydroxymercuribenzoate, but only moderately by p-chloromercuribenzoate or benzoate. Enzyme activity was increased until the final tadpole stage, but was reduced to one-third in the adult and was localized primarily in the kidney. The tadpoles contained high concentrations of D-proline close to the final developmental stage and nearly no D-amino acids were detected in the adult frog, indicating that D-amino acid oxidase functions in metamorphosis.

Pitfalls in the determination of human acylated ghrelin plasma concentrations using a double antibody enzyme immunometric assay.

OBJECTIVES: To investigate the effect of hemolysis and protease inhibitors on acylated ghrelin (AG) concentrations measured with a double antibody enzyme immunometric assay that uses an acetylcholinesterase (AChE)-Fab' conjugate. DESIGN AND METHODS: Samples were hemolysed or treated with PHMB (p-hydroxymercuribenzoate), PMSF (phenylmethanesulfonylfluoride) or AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride) to prevent AG degradation. RESULTS: Hemolysis decreased AG concentrations. PHMB or PMSF did not affect the assay. The standard curve was abolished by AEBSF but rescued by addition of a washing step prior to the AChE-Fab' conjugate. CONCLUSIONS: Hemolysis and AEBSF may affect AG determination.

Efficacy of commercial soft contact lens disinfectant solutions against Acanthamoeba.

PURPOSE: To investigate the relative efficacy of Japanese commercial soft contact lens disinfectant solutions against Acanthamoeba trophozoites and cysts. MATERIALS AND METHODS: Eight types of multipurpose solution (MPS), two types of hydrogen peroxide solution, and one povidone-iodine solution were evaluated to determine their effect against Acanthamoeba trophozoites and cysts (ATCC 50514). Acanthamoeba cysts were cultured in encystment medium for either 1 or 2 weeks (1 and 2-week-old cysts). The trophozoites and cysts were treated with each disinfectant solution for 0, 2, 4, 8, or 24 h. After performing four tenfold serial dilutions of each test solution, dilutions were cultured for 10 days. The number of surviving organisms was calculated using the trimmed Spearman-Karber method. RESULTS: Among the MPS tested, only four were effective against trophozoites after treatment for 4 h, and none was effective against 2-week-old cysts. Hydrogen peroxide had a significant effect on trophozoites and 1-week-old cysts, but not on 2-week-old cysts. In contrast, povidone-iodine caused a 2.6 log reduction in 2-week-old cysts. CONCLUSIONS: MPS were found to have limited efficacy against trophozoites and no efficacy against 2-week-old cysts. Only povidone-iodine had any efficacy against 2-week-old cysts.

A cysteine proteinase in the penetration glands of the cercariae of Cotylurus cornutus (Trematoda, Strigeidae).

A cysteine proteinase from the penetration glands of Cotylurus cornutus cercariae was examined with histochemical and biochemical methods. The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-L-arginine-4-methoxy-2-naphthylamide, and N-blocked-p-nitroanilide, but did not degrade elastin. The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking compounds p-hydroxymercuribenzoate and N-ethylmaleimide (NEM) inhibited it. The enzyme was also sensitive to leupeptin but insensitive to soybean trypsin inhibitor. An electrophoretic separation of extract proteins from the cercariae under acidic, non-denaturing conditions and in the presence of 0.1% gelatin in a polyacrylamide gel revealed the presence of two distinct and three weak transparent bands in the gel resulting from a gelatinolytic activity at pH 6.8. The distinct bands apparently resulted from the activity of the glandular enzyme and lysosomal cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure. No gelatinolysis occurred following treatment of an extract sample with 0.1 mM NEM.

Interactions of lens care with silicone hydrogel lenses and effect on comfort.

PURPOSE: The purpose of this study was to investigate the effect of lens care products on short-term subjective and physiological performance silicone hydrogel lenses. METHODS: Ten subjects wore either lotrafilcon B or galyfilcon A silicone hydrogel contact lenses soaked in a lens care product containing either Polyquad/Aldox or PHMB or control lenses inserted directly from the pack. Subjects wore the lenses for 6 h. Ocular comfort (graded on a 1 to 10 scale) and ocular physiology were assessed. Unworn but soaked lenses were analyzed for metrological changes, release of excipients into phosphate buffered saline, and changes to their surface chemical composition. RESULTS: None of the lens metrology measures or clinically observed conjunctival or limbal redness changed. Corneal staining was significantly (p < 0.008) raised, albeit to low levels, after 6 h wear for either lens type when soaked in the PHMB solution compared with the control lens (lotrafilcon B 0.4 to 0.9 ± 0.7 to 0.4 vs. 0.1 to 0.4 ± 0.3 to 0.5; galyfilcon A 0.2 to 0.3 ± 0.2 to 0.4 vs. 0.0 ± 0.0). For lotrafilcon B lenses, there were decreases in comfort (p = 0.002), increases in burning/stinging (p = 0.002) after 1 h of wear, and increases in lens awareness on lens insertion (p = 0.0001) when soaked in PHMB. However, lotrafilcon B lenses soaked in Polyquad/Aldox showed increases in burning/stinging after 1 and 6 h (p < 0.008) of lens wear. For galyfilcon A lenses, most significant (p ≤ 0.002) changes to symptomatology occurred after soaking in Polyquad/Aldox solution. More PHMB was released from lotrafilcon B lenses, and more MPDS material was released from galyfilcon A lenses. The surface of galyfilcon A lenses changed but irrespective of lens solution type, whereas the changes to the lens surface was dependent on solution type for lotrafilcon B lenses. CONCLUSIONS: Lens care products can change corneal staining and comfort responses during wear. These changes may be associated with release of material soaked into lenses or changes to the lens surface composition.

Bicarbonate-dependent and bicarbonate-independent mechanisms contribute to nondiffusive uptake of acetate in the ruminal epithelium of sheep.

The present study investigated the significance of apical transport proteins for ruminal acetate absorption and their interaction with different anions. In anion competition experiments in the washed reticulorumen, chloride disappearance rate (initial concentration, 28 mM) was inhibited by the presence of a short-chain fatty acid mixture (15 or 30 mM of each acetate, propionate, and butyrate). Disappearance rates of acetate and propionate, but not butyrate (initial concentration, 25 mM each) were diminished by 40 or 80 mM chloride. In isolated ovine ruminal epithelia mounted in Ussing chambers, an increase in chloride concentration from 4.5 to 90 mM led to a decrease of apical acetate uptake at a concentration of 0.5 mM. Mucosal nitrate inhibited acetate uptake most potently whereas sulfate had no effect. Decreasing mucosal pH from 7.4 to 6.1 approximately doubled uptake of acetate both at 0.5 and 10 mM, but this doubling was almost abolished when HCO(3)(-) was absent. The stimulated uptake at mucosal pH 6.1 consisted of a bicarbonate-dependent, nitrate-inhibitable part (K(m) = 54 mM) and a bicarbonate-independent component (K(m) = 12 mM) that was also sensitive to nitrate inhibition. Maximal uptake was three times larger for bicarbonate-dependent vs. bicarbonate-independent uptake. Mucosal addition of 200 microM DIDS, 400 microM p-chloromercuribenzene sulfonic acid, 800 microM p-hydroxymercuribenzoic acid, or 100 microM phloretin had no effects on acetate uptake although the latter two inhibited l-lactate uptake. Our data conclusively show a dominant involvement of proteins in apical acetate uptake. Previously described pH effects on acetate absorption originate mainly from modulation of acetate/bicarbonate exchange. Additionally, there is bicarbonate-independent uptake of acetate anions that is protein coupled but not via monocarboxylate cotransporter.

The interplay of processivity, substrate inhibition and a secondary substrate binding site of an insect exo-beta-1,3-glucanase.

Abracris flavolineata midgut contains a processive exo-beta-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are beta-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUbetaGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUbetaGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUbetaGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity.

[Effects of hand hygiene on feline calicivirus inactivation and removal as norovirus surrogate treated with antiseptic hand rubbing, wet wipes, and functional water].

As a preventive action plan against gastroenteritis caused by the Norovirus (NV), we studied hand hygiene effects using with three hand rubbing products, four wet wipe products, and two functional water types using Feline Calicivirus as a Norovirus surrogate. After treatment using antiseptic hand rubbing products containing chlorhexidine, quaternary ammonium, and povidone-iodine, high inactivation detected by TCID50 was observed compared to products containing povidone-iodine, although no difference was seen in viral removal measured by the amount of viral genome copies in real-time-PCR. Among wet wipes soaked in chlorhexidine, quaternary ammonium, benzoic acid and PHMB, two groups showed viral inactivation and removal. Two products were more effective for functional water, viral decrease was seen in rinsing in running electrolyzed acid water and handwashing by soap. Results underscore the importance of selection in hand washing metheds (alternative soap and also) in preventing viral gastroenteritis.

gamma-Glutamyl hydrolase, not glutamate carboxypeptidase II, hydrolyzes dietary folate in rat small intestine.

Dietary polyglutamyl folates are hydrolyzed to monoglutamyl folate derivatives prior to intestinal transport. In humans and pigs, the reaction occurs at pH 6.5 at the jejunal brush border membrane by folate hydrolase and is encoded by the glutamate carboxypeptidase II (GCPII) gene. Intracellular folate hydrolase with an optimal pH of 4.5 is encoded by the gamma-glutamyl hydrolase (gamma-GH) gene and predominates in rats. We determined the respective roles of GCPII and gamma-GH in dietary folate hydrolysis in rat small intestine. Duodenal, jejunal, and ileal mucosa, pancreas, and duodenal luminal fluid were collected from 10 Sprague-Dawley rats that had not been food deprived. Folate hydrolase was assayed at pH 4.5 and 6.5 with and without parahydroxymercuribenzoate (pHMB), an inhibitor of intracellular folate hydrolase. Folate hydrolase activity occurred at pH 4.5 in all tissues, was significantly inhibited by the addition of pHMB at both pH 4.5 and 6.5, and was virtually absent from brush border fractions at pH 6.5. The highest activity was in the postprandial duodenal luminal fluid at pH 4.5. Rat-specific primers for GCPII and gamma-GH were used to detect mRNA expression in pancreas, jejunal mucosa, and liver. GCPII expression was detected only in the liver, whereas gamma-GH was expressed in all 3 tissues. These results suggest that the hydrolysis of polyglutamyl folates in rats requires the intracellular folate hydrolase that is expressed by pancreatic gamma-GH, in contrast to GCPII that is expressed in the jejunal mucosal brush border in pigs and humans. gamma-GH folate hydrolase is abundant in rat postprandial pancreatic secretions and appears to hydrolyze dietary folates in the intestinal lumen prior to intestinal absorption.

Effect of metal ions and calcium on purified PON1 and PON3 from rat liver.

The effect of several metal ions and calcium on purified paraoxonases (PON1 and PON3) from rat liver was studied. PON1 and PON3 were also inhibited by EDTA and both enzyme activities were restored by the addition of free calcium. The reactivation by calcium was a time-dependent effect for PON1; however, this was not the case for PON3. We also studied the response of PON1 and PON3 to several inhibitors: Co, Cu, Mn, Hg and p-hydroxymercurybenzoate (pOHMB), and determined the type of inhibition and the inhibition constants. Among all the compounds tested, mercurials (Hg and pOHMB) were the most potent inhibitors of PON1. For PON3 mercurials and copper showed the highest inhibitory potency. Purified PON3 also showed different inhibition patterns as compared to PON1. A comparison of PON1 and PON3 shows qualitative and quantitative differences in the sensitivity against the inhibitors tested, showing major differences in the case of cobalt, copper and pOHMB, which may be related to structural differences of both PONs. These results increase our knowledge of the biochemical properties of PON1 and PON3 and may help in the understanding of their physiological role as a potential detoxification mechanism against environmental metal ions.

Purification, characterization and cloning of aldehyde dehydrogenase from Rhodococcus erythropolis UPV-1.

The enzyme responsible for formaldehyde removal in industrial wastewaters by cells of Rhodococcus erythropolis UPV-1 was identified as a broad-specific aldehyde dehydrogenase (EC 1.2.1.3). The enzyme was purified to electrophoretic homogeneity from ethanol-grown cells with a specific activity of 19.5 U mg-1 protein and an activity recovery of 56%. The enzyme showed an isoelectric point (pI) of 5.3 and was a trimer of 162 kDa consisting of three identical 54-kDa subunits. It was specific for NAD+ and showed hyperbolic kinetics for this coenzyme (Km=90 microM), but sigmoidal kinetics for the aliphatic aldehydes used as substrates. The enzyme affinity for aldehydes increased with their hydrocarbon chain length, ranging from 333 microM for formaldehyde to 85 nM for n-octanal. The corresponding calculated Hill coefficients were in the 1.55-2.77 range. With n-propanal as substrate, the optimum pH and temperature for activity were 9.5-10.0 and 47.5 degrees C, respectively, with an Ea for catalysis of 28.6 kJ mol-1. NAD+ protected the enzyme against thermal inactivation, but aldehydes were ineffective. The activity was severely inhibited by p-hydroxymercuribenzoate, indicating that a thiol was essential for catalysis. The 1,524-bp aldhR gene encoding a 507-amino-acid protein was expressed in cells of Escherichia coli M15 as a hexahistidine-tagged protein.

Differential effects of peroxynitrite on the function of arginine vasopressin V(1a) receptors and alpha(1)-adrenoceptors in vivo.

OBJECTIVE: The aim of this study was to provide evidence that peroxynitrite may differentially affect the function of arginine vasopressin (AVP) V(1a) receptors and alpha(1)-adrenoceptors in vascular smooth muscle of the rat METHODS: The vasoconstrictor responses elicited by AVP, or the alpha(1)-adrenoceptor agonist, phenylephrine, were determined in anesthetized rats before and after injections of (i) peroxynitrite, the thiol chelator, para-hydroxymercurobenzoic acid (PHMBA), or the electron acceptor, nitroblue tetrazolium (NBT). The ability of the reducing agent, glutathione, to reverse the loss of response to phenylephrine and AVP in peroxynitrite-treated rats was also examined. RESULTS: The AVP-induced responses were suppressed 10-20 min but not 60-70 min after the administration of peroxynitrite. Glutathione reversed the above loss of response to AVP at 10-20 min. The responses elicited by phenylephrine were suppressed 10-20 min and 60-70 min after administration of peroxynitrite. Glutathione did not reverse the above losses of response to phenylephrine. In addition, the vasoconstrictor actions of AVP and phenylephrine were markedly suppressed after administration of PHMBA or nitroblue tetrazolium. CONCLUSIONS: The above findings provide evidence that exogenously administered peroxynitrite may differentially affect the function of AVP V(1a) receptors and alpha(1)-adrenoceptors in vascular smooth muscle of the rat. The possibility that peroxynitrite impairs AVP V(1a) receptor function by transient oxidation events whereas peroxynitrite impairs alpha(1)-adrenoceptor function by transient oxidation and permanent nitration events will be discussed.

Conformation and microenvironment of the active site of a low molecular weight 1,4-beta-D-glucan glucanohydrolase from an alkalothermophilic Thermomonospora sp.: involvement of lysine and cysteine residues.

Conformation and microenvironment at the active site of 1,4-beta-D-glucan glucanohydrolase was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde. OPTA has been known to form a fluorescent isoindole derivative by cross-linking the proximal thiol and amino groups of cysteine and lysine. Modification of lysine of the enzyme by TNBS and of cysteine residue by PHMB abolished the ability of the enzyme to form an isoindole derivative with OPTA. Kinetic analysis of the TNBS and PHMB-modified enzyme suggested the presence of essential lysine and cysteine residues, respectively, at the active site of the enzyme. The substrate protection of the enzyme with carboxymethylcellulose (CMC) confirmed the involvement of lysine and cysteine residues in the active site of the enzyme. Multiple sequence alignment of peptides obtained by tryptic digestion of the enzyme showed cysteine is one of the conserved amino acids corroborating the chemical modification studies.

Effects of thiol chelation on alpha1-adrenoceptor-induced vasoconstriction in vivo.

The aims of this study were to determine whether systemic injections of the lipophobic thiol chelator, para-hydroxymercurobenzoic acid (PHMBA) would reduce the vasoconstrictor responses elicited by the alpha1-adrenoceptor agonist, phenylephrine, in urethane-anesthetized rats by chelation of thiol residues in alpha1-adrenoceptors in vascular smooth muscle rather than voltage-sensitive Ca(2+)-channels (Ca(2+)VERSUS-channels). The magnitudes and durations of the vasoconstrictor responses elicited by phenylephrine were markedly reduced after the injections of PHMBA. In contrast, the maximal phenylephrine-induced responses were not affected whereas the durations of these responses were markedly attenuated after injection of the Ca(2+)VERSUS-channel blocker, nifedipine. Nifedipine elicited pronounced and sustained falls in mean arterial blood pressure and vascular resistances in PHMBA-treated rats. Moreover, the vasodilator actions of the nitric oxide-donor, sodium nitroprusside were minimally attenuated by PHMBA whereas they were markedly attenuated by nifedipine. These findings support evidence that the vasoconstrictor responses due to activation of alpha1-adrenoceptors are initiated by mobilization of intracellular pools of Ca(2+) whereas they are sustained by opening of Ca(2+)VERSUS-channels. These findings also suggest that PHMBA diminishes the vasoconstrictor effects of phenylephrine by chelation of thiol residues in alpha1-adrenoceptors rather than by blockade of Ca(2+)VERSUS-channels, and that chelation of these thiol residues prevents agonist occupation and/or activation of these receptors and subsequent mobilization of intracellular pools of Ca(2+).

Potential role of nitration and oxidation reactions in the effects of peroxynitrite on the function of beta-adrenoceptor sub-types in the rat.

This study examined the hemodynamic responses elicited by the beta-adrenoceptor agonist, isoproterenol (1 and 10 microg/kg, i.v.) before and after administration of (i) peroxynitrite (10 x 10 micromol/kg, i.v.), (ii) the thiol chelator, para-hydroxymercurobenzoic acid (pHMBA, 75 micromol/kg, i.v.), and (iii) the electron acceptor, nitroblue tetrazolium (NBT, 10 micromol/kg, i.v.) in pentobarbital-anesthetized rats. The tachycardia elicited by the lower dose of isoproterenol was diminished whereas the tachycardia elicited by the higher dose was not attenuated after administration of peroxynitrite. The falls in hindquarter and renal vascular resistances elicited by both doses of isoproterenol were substantially diminished whereas the isoproterenol-induced falls in mesenteric vascular resistance were not changed after administration of peroxynitrite. All of the isoproterenol-induced responses were markedly attenuated after administration of pHMBA or NBT. These findings suggest that the oxidation and/or nitration of beta-adrenoceptors impair the ability of isoproterenol to bind to and/or activate these G protein-coupled receptors. beta1-, beta2- and beta3-adrenoceptors contain extracellular cysteine residues susceptible to oxidation (i.e., disulfide-bridge formation) whereas only the beta1- and beta2-adrenoceptors contain extracellular tyrosine residues susceptible to nitration. These findings also suggest that sustained impairment of beta1- and beta2-adrenoceptor function by peroxynitrite is due to nitration of extracellular tyrosine residues in these receptors. By analogy, beta3-adrenoceptors may not be permanently affected by peroxynitrite because these receptors are devoid of extracellular tyrosine residues.

A novel soluble protein factor with non-opioid dynorphin A-binding activity.

A novel soluble non-opioid dynorphin A-binding factor (DABF) was identified and characterized in neuronal cell lines, rat spinal cord, and brain. DABF binds dynorphin A(1-17), dynorphin A(2-17), and the 32 amino acid prodynorphin fragment big dynorphin consisting of dynorphin A and B, but not other opioid and non-opioid peptides, opiates, and benzomorphans. The IC50 for dynorphin A(1-17), dynorphin A(2-17), and big dynorphin is in the 5-10 nM range. Using dynorphin A and big dynorphin fragments a binding epitope was mapped to dynorphin A(6-13). DABF has a molecular mass of about 70 kDa. SH-groups are apparently involved in the binding of dynorphin A since p-hydroxy-mercuribenzoic acid inhibited this process. Upon interaction with DABF dynorphin A was converted into Leu-enkephalin, which remained bound to the protein. These data suggest that DABF functions as an oligopeptidase that forms stable and specific complexes with dynorphin A. The presence of DABF in brain structures and other tissues with low level of prodynorphin expression suggests that DABF as an oligopeptidase may degrade other peptides. Dynorphin A at the sites of its release in the CNS may attenuate this degradation as a competitor when it specifically binds to the enzyme.

3alpha/beta,20beta-hydroxysteroid dehydrogenase (porcine testicular carbonyl reductase) also has a cysteine residue that is involved in binding of cofactor NADPH.

Besides residue of the catalytic triad that is conserved in the short-chain dehydrogenase/reductase (SDR) superfamily, a Cys side chain reportedly plays functional roles in NADP-dependent 15-hydroxyprostaglandin dehydrogenase and human carbonyl reductase (CR). The three-dimensional structure of porcine 3alpha/beta,20beta-hydroxysteroid dehydrogenase, also known as porcine testicular carbonyl reductase, demonstrates the proximity of the Cys 226 side chain to the bound NADP. However, no clear explanation with respect to the basis of the catalytic function of the Cys residue is yet available. By chemical modification, point mutation, and kinetic analysis, we determine that two Cys residues, Cys 149 and Cys 226, are involved in the enzyme activity. Furthermore, we found that pretreatment with NADP markedly protects the enzyme from inactivation by 4-(hydroxyl mercury) benzoic acid (4-HMB), thereby confirming that Cys 226 is involved in binding of the cofactor. On the basis of the tertiary structure of 3alpha/beta,20beta-HSD, the possible roles of Cys residues, especially that of Cys 226, in enzyme action and in the binding of cofactor NADPH are discussed.

Jackbean, soybean and Bacillus pasteurii ureases: biological effects unrelated to ureolytic activity.

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) = 2 mg x kg(-1)) - no lethality was seen in mice injected intraperitoneally with JBU or SBU (20 mg x kg(-1)). Similarly to canatoxin, the three enzymes promoted aggregation of blood platelets (EC(50) = 400.0 micro g x mL(-1), 22.2 micro g x mL(-1), 15.8 micro g x 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.