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.

Folate conjugase: two separate activities in human jejunum.

Previous perfusion studies of the human jejunum suggested that conjugated folate is hydrolyzed on the mucosal surface. The techniques of cell fractionation and DEAE and gel chromatography led to the identification of two separate folate conjugase activities in human jejunal mucosa: one membrane-bound and concentrated in the brush border, the other soluble and intracellular. These enzyme activities exhibit different pH optima, molecular weights, and inhibition characteristics. Folate conjugase in the brush border may accomplish the initial digestion of dietary pteroylpolyglutamates.

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.

Structural and functional studies on hemoglobin Bethesda (alpha2beta2 145His), a varient associated with compensatory erythrocytosis.

Studies have been performed on a 12-yr-old Chinese girl with compensatory erythrocytosis due to the presence of hemoglobin Bethesda comprising about 45% of the red cell hemoglobin. Her parents and three siblings were normal. The oxygen affinity of her blood was markedly increased: under physiological conditions (pH 7.40, 37 degrees C). P(50) was 12.8 mm Hg (normal = 26.5 mm Hg). The red cell 2,3-diphosphoglycerate (2.3-DPG) level was normal. The abnormal hemoglobin could not be separated from hemoglobin A by zone electrophoresis at pH 8.6 or isoelectric focusing on polyacrylamide gel. However, after the hemoglobin was split into free alpha and beta chains by treatment with p-hydroxymercuribenzoate (PMB) or 6 M urea, an abnormal beta chain was readily demonstrated having a higher isoelectric point (more positive net charge) than normal beta(A). Structural analysis of the variant beta chain demonstrated the substitution of histidine for tyrosine at position 145: hemoglobin Bethesda (alpha(2)beta(2) (145His)). From earlier chemical and crystallographic studies, it has been postulated that this residue is a critical determinant of hemoglobin function. Hemoglobin Bethesda was separated from hemoglobin A by column chromatography. Oxygen equilibria of purified hemoglobin Bethesda revealed an extremely high oxygen affinity (exceeding that of isolated alpha and beta chains), and markedly reduced cooperativity. The Bohr effect of hemoglobin Bethesda was 1/3 that of hemoglobin A. However, hemoglobin Bethesda showed a significant interaction with 2.3-DPG and inositol hexaphosphate.

The responses of rat intestinal brush border and cytosol peptide hydrolase activities to variation in dietary protein content: dietary regulation of intestinal peptide hydrolases.

The effects of variation in dietary protein content on small intestinal brush border and cytosol peptide hydrolase activities have been investigated. One group of rats was fed a high protein diet (55% casein) and another group was fed a low protein diet (10% casein). After 1 wk, brush border peptide hydrolase activity (L-leucyl-beta-naphthylamide as substrate) and cytosol peptide hydrolase activity (L-prolyl-L-leucine as substrate) were determined in mucosae taken from the proximal, middle, and distal small intestine. As judged by several parameters, brush border peptide hydrolase activity was significantly greater in rats fed the high protein diet when data for corresponding segments were compared. In contrast, no significant difference was seen in cytosol peptide hydrolase activity. IN A SECOND STUDY, BRUSH BORDER AND CYTOSOL PEPTIDE HYDROLASE ACTIVITIES WERE DETERMINED IN THE PROXIMAL INTESTINE BY UTILIZING AN ADDITIONAL THREE PEPTIDE SUBSTRATES: L-leucyl-L-alanine, L-phenylalanylglycine, and glycyl-L-phenylalanine. Sucrase, maltase, and alkaline phosphatase activities were also determined. As before, brush border peptide hydrolase activities were significantly greater in rats fed the high protein diet. However, activities of the nonproteolytic brush border enzymes did not vary significantly with diet. In contrast to the results obtained with L-prolyl-L-leucine as substrate for the cytosol enzymes, cytosol activity against the three additional peptide substrates was greater in rats fed the high protein diet. It is suggested that the brush border peptide hydrolase response to variation in dietary protein content represents a functional adaptation analogous to the regulation of intestinal disaccharidases by dietary carbohydrates. The implication of the differential responses of the cytosol peptide hydrolases is uncertain, since little is known of the functional role of these nonorgan-specific enzymes.

The interaction of bovine factor VIII with human platelets.

Treatment of human platelets with purified bovine Factor VIII caused three types of aggregation: (a) primary agglutination; (b) secondary aggregation involving the platelet release reaction; and (c) super-aggregation, in which the platelets were gathered into only a few large clumps. Removal of calcium ions or treatment with p-hydroxymercuiriphenyl sulfonate blocked the release reaction, but not primary agglutination or super-aggregation. Platelets treated with formalin were not aggregated by ADP, thrombin, or collagen, but were agglutinated by bovine Factor VIII, although they did not show super-aggregation. For malin-treated platelets were agglutinated by phytohemagglutinin P less extensively and less rapidly than by bovine Factor VIII. Treatment of platelets and Factor VIII with neuraminidase released 60 and 53%, respectively, of the sialic acid residues without affecting the agglutination reaction or the procoagulant activity of the Factor VIII. Agglutination was inhibited by high salt concentrations, dextran sulfate, and heparin. During agglutination, both the procoagulant and platelet-agglutinating activities of Factor VIII became bound to the platelet surface.

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.

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.

[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.

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.

Differential scanning calorimetry of the thermal denaturation of lactate dehydrogenase.

1. Differential scanning calorimetry has been used to study the thermal denaturation of lactate dehydrogenase. At pH 7.0 in 0.1 M potassium phosphate buffer, only one transition was observed. Both the enthalpy of denaturation and the melting temperature are linear function of heating rate. The enthalpy is 430 kcal/mol and the melting temperature 61 degrees C at 0 degrees C/min heating rate. The ratio of the calorimetric heat to the effective enthalpy indicated that the denaturation is highly cooperative. Subunit association does not appear to significantly contribute to the enthalpy of denaturation. 2. Both cofactor and sucrose addition stabilized the protein against thermal denaturation. Pyruvate addition produced no changes. Only a small time-dependent destabilization was observed at low concentrations of urea. Large effects were observed in concentrated NaCl solutions and with sulfhydryl-modified lactate dehydrogenase.

Effects of thiol inhibitors on hepatic guanylate cylase activity.

Several thiol blocking agents inhibit basal guanylate cyclase activity of 100 000 X g hepatic supernatant fractions and the stimulation of enzyme activity by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), NaN3, NaNO2 and nitroprusside. The relative potency of the thiol blockers as inhibitors was CdCl2 greater than p-hydroxymercuribenzoate greater than N-ethylmaleimide greater than arsenite greater than iodoacetamide. Inhibition of basal and MNNG-responsive soluble guanylate cyclase activities by arsenite was markedly potentiated by an equimolar concentration of 2,3-dimercaprol, but not by mercaptoethanol. Inhibition of soluble guanylate cyclase by either arsenite or CdCl2 was completely reversed by excess 2,3-dimercaprol. Qualitatively similar effects were observed with DE-52 cellulose purified soluble hepatic guanylate cyclase, and suggested an involvement of closely juxtaposed thiol groups in the regulation of enzyme activity. For several reasons inhibition by thiol blockers appeared to be mediated through multiple mechanisms and/or sites of interaction: (1) Concentrations of the thiol inhibitors which had no effect on basal activity strikingly inhibited the responsiveness of the enzyme to a submaximal concentration of MNNG. (2) CdCl2 abolished the action of excess MnCl2 to stimulate purified guanylate cyclase, but was a relatively ineffective inhibitor when MnCl2 and GTP were present in equimolar concentrations. By contrast, arsenite-2,3-dimercaprol was uniformly effective in inhibiting guanylate cyclase activity in the presence or absence of excess MnCl2. (3) Arsenite-2,3-dimercaprol increased the Km for MnGTP (control, 0.13 +/- 0.02 mM; 0.2 mM arsenite-2,3-dimercaprol, 0.31 +/- 0.03 mM), whereas CdCl2 had no effect on this parameter. (4) Hepatic particulate guanylate cyclase activity was significantly inhibited by arsenite 2,3-dimercaprol but not by CdCl2. Thus, the data not only indicate that vicinal dithiol groups are required for expression of basal guanylate cyclase activity and enzyme responses to agonists, but strongly suggest the involvement of more than one interacting site containing free thiol residues.

Novel biological transformations of 15-Ls-hydroperoxy-5,8,11,13-eicosatetraenoic acid.

[1-14C]Arachidonic acid was incubated with homogenates of the fungus, Saprolegnia parasitica. The products consisted of comparable amounts of two epoxy alcohols, 15-Ls-hydroxy-11,12-epoxy-5cis,8cis,13trans- eicosatrienoic acid and 15-hydroxy-13,14-epoxy-5cis,8cis,11cis-eicosatrienoic acid. Results of incubations carried out in the presence of nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid, p-hydroxymercuribenzoate as well as glutathione peroxidase plus reduced glutathione demonstrated that transformation of arachidonic acid into epoxy alcohols occurred with the formation of 15-Ls-hydroperoxy-5cis,8cis,11cis,13trans- eicosatetraenoic acid (15-HPETE) as an intermediate. The pathway involved a lipoxygenase catalyzing the oxygenation of arachidonic acid at the 15L position to produce 15-HPETE, and a hydroperoxide isomerase activity which catalyzed conversion of 15-HPETE into the two epoxy alcohols. Studies with 15-[18O2]HPETE demonstrated that both oxygens of 15-HPETE were retained in the epoxy alcohols. Furthermore, experiments with mixtures of 15-[18O2]-and 15-[16O2]HPETE showed that conversion of 15-HPETE into epoxy alcohols occurred by an intramolecular transfer of hydroperoxide oxygen.

Purification and properties of a peptidase acting on a synthetic substrate for collagenase from monkey kidney.

A peptidase cleaving a synthetic substrate for collagenase, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (designated as PZ-peptide) has been purified extensively (about 5200-fold) from a soluble extract of monkey kidney with a view of carrying out studies on its possible physiological role. The purified PZ-peptidase appeared essentially free of collagenase, nonspecific protease and di- and tri-peptidase activities. The properties of the purified PZ-peptidase resemble very much the granuloma enzyme. It is optimally active around pH 7.0. Its apparent Km value for PZ-peptide is 0.72 mM and V is 10.1 mumol/mg protein/min. It is reversibly inhibited by p-hydroxymercuribenzoate and HgCl2, whereas iodoactetamide does not affect the enzyme activity. N-Ethylmaleimide inhibited the enzyme partially (50%). Heavy metals like Cu-2+, Cd-2+, Ag+, Pb-2+, Ni-2+, and Zn-2+ completely inhibited the enzyme activity, while the inhibition by Co-2+ was only partial. Fe-2+ did not exert any effect on the activity. The enzyme activity was completely inhibited by EDTA and was restored almost to the original value by metal ions like Mn-2+, Mg-2+, Ca-2+ and Ba-2+. The approximate molecular weight of the purified enzyme was estimated to be 56 000.

Differential sensitivity of low molecular weight DNA polymerase to sulfhydryl-blocking reagents.

Activity of a 2.5 S mouse myeloma DNA polymerase (termed DNA polymerase II) measured with either poly(rA) or poly(dA) as template did not require sulfhydryl-reducing reagents, but was sensitive to inhibition by p-hydroxymercuribenzoate and the sulfhydryl-alkylating reagent, N-ethylmaleimide; however, the activity was much more sensitive to inhibition by p-hydroxymercuribenzoate than by the sulfhydryl-alkylating reagent. The p-hydroxymercuribenzoate inhibition appeared to involve the mercurial portion of the p-hydroxymercuribenzoate molecule because HgCl2 was an equally effective inhibitor, while p-hydroxybenzoate had little effect upon enzyme activity. The p-hydroxymercuribenzoate inhibition was reversed by an equal concentration of the sulfhydryl-reducing reagent, dithiothreitol.

The multiple forms and kinetic differences of rat colonic beta-N-acetylhexosaminidases.

Rat colonic beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) has been separated into three forms by DEAE-cellulose chromatography with an increasing salt gradient. It was not possible to separate the glucosaminidase activity from the galactosaminidase activity by a variety of chromatographic procedues, but the ratio of the two specific activities varied during purification. The pH optima were however identical, for both activities and all three forms. Kinetic measurements including inhibition by substrate analogues showed differences between the two activities as well as among the three forms. A common active site model was inconsistent with the results. Data from mixed substrate experiments were consistent with a model wherein the two activities reside in seperate active sites, each able to be inhibited by the substrate for the other site. The effect of acetate and SH reagents confirmed the two-site model. Treatment with neuraminidase, thimerosal, p-hydroxymercuribenzoate, HgCl2 and AgNO3 or heating at 50 degrees C did not produce any effect on the A form that could be identified as a conversion to the B form. Measurement of the effects on both activities supported the two-site model. It is concluded that the relationship between the A and B forms in the rat colonic mucosa hexosaminidases must be different from that reported for such enzymes from other sources.

Effects of pH and sulfhydryl specific reagents on 4-fumarylacetoacetate fumarylhydrolase.

The pH-dependence of fumarylacetoacetase (4-fumarylacetoacetate fumaryl-hydrolase, EC 3.7.1.2) activity was studied in the pH range 6.25-8.50. After correction of the substrate concentration for enolate formation, the Michealis constant was found to be pH independent in this range. Likewise, the Ki values for the competitive inhibitors chloride and fluoride were found to be independent of pH between 6.25-8.50. A bell-shaped curve described the log V vs. pH dependence, and ionization constants of 6.5 and 8.2 were calculated. Tentatively an imidazole group and a sulfhydryl group were assigned to the constants 6.5 and 8.2, respectively. Both p-hydroxymercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid) react with both sulfhydryl groups per subunit in the native protein and three sulfhydryl groups per subunit in the denatured protein. Substrate protects one sulfhydryl group in the native protein from reaction with 5,5'-dithiobis(2-nitrobenzoid acid). Substrate or the competitive inhibitor, fluoride, protect the enzyme from inactivation by p-hydroxymercuribenzoate. In addition p-hydroxymercuribenzoate shows saturation kinetics. Neither sulfhydryl inhibitor completely inactivates the enzyme. The enzyme is described as having three sulfhydryl groups per subunit, one of which is inaccessible to the sulfhydryl specific reagents when the protein is in the native state. One of the two accessible sulfhydryl groups is either near the active site or essential in maintaining the structure of the protein.

Hybrid, immobilised dimers of human liver arginase.

The activity of matrix-bound monomers of arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was not changed by incubation with p-hydroxymercuribenzoate. When the chemically modified, matrix-bound monomers were incubated with soluble subunits in the presence of Mn2+, dimers were obtained. These dimers were hybrids between modified and native monomers. The results obtained are in accord with a D2-symmetry where two dimers meet to form the tetrameric enzyme. From kinetic studies it is concluded that the structure of the active sites of arginase is not affected by the chemical modification with p-hydroxymercuribenzoate.