Methyl Methanethiosulfonate Research Articles

Factors that control the reactivity of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi.

The amino acid sequences and X-ray structures of homodimeric triosephosphate isomerase from the pathogenic parasites Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) are markedly similar. In the two TIMs, the side chain of the only interface cysteine (Cys14) of one subunit docks into loop 3 of the other subunit. This portion of the interface is also markedly similar in the two enzymes. Nonetheless, Cys14 of TcTIM is nearly 2 orders of magnitude more susceptible to the thiol reagent methylmethane thiosulfonate (MMTS) than Cys14 of TbTIM. The causes of this difference were explored by measuring the second-order rate constant of inactivation by MMTS (k(2)) under various conditions. At pH 7.4, k(2) in TcTIM is 70 times higher than in TbTIM. The difference decreases to 30 when the amino acid sequence of loop 3 and adjoining residues of TbTIM are conferred to TcTIM (triple mutant). The pK(a) values of the thiol group of the interface cysteine of TcTIM and the triple mutant were 0.7 pH unit lower than in TbTIM. Because this difference could account for the different sensitivity of the enzymes to thiol reagents, we determined the k(2) of inactivation at equal levels of ionization of their interface cysteines. Under these conditions, the difference in k(2) between TcTIM and TbTIM became 8-fold, whereas that of the triple mutant to TbTIM was 1.5 times. The substrate analogue phosphoglycolate did not modify the pK(a) of the thiol group of the interface, albeit it diminished the rate of its derivatization by MMTS. In the presence of phosphoglycolate, under conditions in which the interface cysteines of the enzymes had equal levels of protonation, the difference in k(2) of TcTIM and TbTIM became smaller, whereas k(2) of the triple mutant was almost equal to that of TbTIM. Thus, from measurements of the reactivity of the interface cysteine in various conditions, it was possible to obtain information on the factors that control the dynamics of a portion of the dimer interface.

Novel Quantitative Assays for Estimating the Antimicrobial Activity of Fresh Garlic Juice

Novel agar diffusion and broth dilution assays were developed for quantitatively estimating the antimicrobial activity of fresh garlic juice. Bacteria found to be inhibited by garlic juice in agar diffusion assay included two gram-positive and five gram-negative species. Leuconostoc mesenteroides was not inhibited. Escherichia coli B-103 (HB101, with pJH101, ampicillin resistant, 100 μg ml−1) was inhibited and chosen as the standard culture for quantitative assays. The agar diffusion assay was based on the slope ratio method, where the slope of dose response for garlic juice was divided by the slope of dose response for methylmethane thiosulfonate (MMTSO2). Juice from fresh garlic varied in activity between 1.76 and 2.31 μg of MMTSO2 per mg of garlic juice. The activity of juice decreased during 11 months of storage of garlic cloves at 5°C from 2.31 to less than 0.1 μg of MMTSO2 per mg of juice. The broth dilution assay also used the E. coli B-103 culture, which permitted selective enumeration of this bacterium when 100 μg ml−1 of ampicillin was incorporated into the enumerating agar. Selective enumeration was essential since the garlic juice was not sterile and, thus, contained natural flora. Growth of E. coli was unaffected by 0.1%, delayed by 0.25%, and completely inhibited at 0.5 and 2% garlic juice in broth during 24 h of incubation at 37°C. The minimum inhibition concentration of garlic juice by broth dilution assay was, thus, estimated to be 0.5%, which is equivalent to 3.46 μg of MMTSO2 per mg of garlic juice by the agar diffusion assay.

Oxidants Depress the Synthesis of Phosphatidylinositol 4,5-Bisphosphate in Heart Sarcolemma

Phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) is the substrate for phosphoinositide–phospholipase C (PLC) and is required for the function of several cardiac cell plasma membrane (sarcolemma, SL) proteins. PtdIns 4,5-P2 is synthesized in the SL membrane by coordinated and successive actions of PtdIns 4-kinase and PtdIns 4-phosphate 5-kinase. These kinases and the generation of PtdIns 4,5-P2 may be a factor in the cardiac dysfunction during pathophysiological conditions of oxidative stress. Therefore, we examined the effects of different reactive oxygen species (ROS) on the kinases' activities and subsequent generation of PtdIns 4,5-P2. Exposure to the xanthine–xanthine oxidase–ROS generating system significantly reduced both SL kinase activities. Superoxide dismutase did not prevent this inhibition; however, catalase significantly prevented the xanthine–xanthine oxidase induced inhibition. Treatment of SL with hydrogen peroxide (H2O2) resulted in inhibition of both the kinases, which was prevented by catalase and dithiothreitol (DTT). Hypochlorous acid also inhibited both the kinases, which was prevented by DTT. Deferoxamine (an iron chelator) and mannitol (an •OH scavenger) did not modify the H2O2-induced depression of the kinases, eliminating any role of •OH. Furthermore, the IC50 of H2O2 on PtdIns 4-kinase and PtdIns 4-P 5-kinase was 27 and 81 μM, respectively. In addition, inclusion of reduced glutathione in the assay of the kinases in the absence of H2O2 did not affect the activities of the kinases; however, oxidized glutathione induced a significant depression. Also, a significant decline of the PtdIns 4-kinase and PtdIns 4-P 5-kinase activities due to changing of the redox ratio was observed. Thiol modifiers (N-ethylmaleimide, methyl methanethiosulfonate, or p-chloromercuriphenylsulfonic acid) were detected to depress the kinases' activities, which were substantially prevented by DTT. The results suggest that functionally critical thiol groups may be associated with PtdIns 4-kinase and PtdIns 4-P 5-kinase and that changes of their redox state by ROS can impair their activities, which may be an important factor in the oxidant-induced cardiac dysfunction.

Cysteine residues and the structure of the rat renal proximal tubular type II sodium phosphate cotransporter (rat NaPi IIa).

The rat renal Na/P(i) cotransporter type IIa (rat NaP(i) IIa) is a 637 amino acid protein containing 12 cysteine residues. We examined the effect of different cysteine modifying methanethiosulfonate (MTS)-reagents and the disulfide bond reducing agent tris(2-carboxyethyl)phosphine (TCEP) on the transport activity of wild-type and 12 single cysteine substitution mutants of rat NaPi IIa expressed in Xenopus laevis oocytes. The transport activity of the wild-type protein was resistant to three membrane impermeant MTS-reagents (MTSEA, MTSET and MTSES). In contrast, membrane permeant methyl methanethiosulfonate (MMTS) and TCEP inhibited the transport activity of both the wild-type, as well as all the single mutant proteins. This indicated the existence of more than one functionally important cysteine residue, not accessible extracellularly, and at least 2 disulfide bridges. To identify the disulfide bridges, three double mutants lacking 2 of the 3 cysteine residues predicted to be extracellular in different combinations were examined. This led to the identification of one disulfide bridge between C306 and C334; reconsideration of the topological model predictions suggested a second disulfide bridge between C225 and C520. Evaluation of a fourth double mutant indicated that at least one of two disulfide bridges (C306 and C334; C225 and C520) has to be formed to allow the surface expression of a functional cotransporter. A revised secondary structure is proposed which includes two partially repeated motifs that are connected by disulfide bridges formed between cysteine pairs C306-C334 and C225-C520.

Irreversible extrusion of the first loop facing the matrix of the bovine heart mitochondrial ADP/ATP carrier by labeling the Cys(56) residue with the SH-reagent methyl methanethiosulfonate.

The effect of the SH-reagent methyl methanethiosulfonate (MMTS) on the ADP/ATP carrier of bovine heart mitochondria was studied under various conditions. MMTS labeled predominately Cys(56) in the first loop facing the matrix (loop M1), and the labeling inhibited ADP transport via the carrier. The transport inhibition was found to be due to fixation of the carrier in the m-state conformation. MMTS labeling was suggested not to affect ADP binding to its major binding site. These features were the same as those of another commonly used SH-reagent, N-ethylmaleimide (NEM). Although the van der Waals volume of the non-hydrogen-bondable methylthio group of MMTS is much smaller than that of the ethylsuccinimide group of NEM, modification of Cys(56) inhibited the interconversion between the m- and c-state conformation. The mechanism by which MMTS inhibited the transport activity is discussed in terms of stabilization of conformation of the loop M1.

Steroidal affinity labels of the estrogen receptor alpha. 4. Electrophilic 11beta-aryl derivatives of estradiol.

Ten electrophilic estradiol 11beta-aryl derivatives were synthesized, with three different types of 11beta-substituent: (i) pOO(CH(2))(2)X (compounds: 6, X = OSO(2)CH(3); 7, X = I; 13, X = NHCOCH(2)Cl; 15, X = N(CH(3))COCH(2)Br; and 16, X = N(CH(3))COCH(2)Cl); (ii) pOO(CH(2))(5)X (compounds: 17, X = I; 20, X = NHCOCH(2)Br; and 22, X = N(CH(3))COCH(2)Br); and (iii) pOC(triple bond)CCH(2)X (compounds: 27, X = NHCOCH(2)Cl; and 29, X = N(CH(3))COCH(2)Cl). The range of their apparent affinity constants for binding the lamb uterine estrogen receptor alpha (ERalpha) was 3-40% that of estradiol. Six electrophiles, chloroacetamides 13, 16, 27, and 29, iodide 17, and bromoacetamide 20 (whose arm linking the electrophilic carbon to the 11beta-phenyl group includes at least six bonds), were able to irreversibly inhibit the binding of [(3)H]estradiol to ER (25-60% decrease in binding sites), with the following compound effectiveness order: 17 < 13 < 16 approximately 20 approximately 27 approximately 29. Mesylate 6, iodide 7 (whose linking arm includes only three bonds), and bromoacetamides 15 and 22 (which differ from 16 by the Cl to Br change and from 20 by the NH to NCH(3) change, respectively) were much less effective (<10% decrease in binding sites, if any). The fact that the inactivation of estradiol-binding sites by the six electrophiles was totally prevented by estradiol indicated that they were ER affinity labeling agents. When ER was modified by methyl methanethiosulfonate, an SH-specific reagent, the different compounds led to very contrasting results in ER affinity labeling. With modified ER, iodide 17 and chloroacetamides 27 and 29 were practically inactive, chloroacetamides 13 and 16 and bromoacetamide 20 were still active but less effective than on the native ER, whereas tertiary bromoacetamides 15 and 22, found to be practically inactive on native ER, became the most effective electrophiles ( approximately 45% and approximately 65% binding sites inactivated, respectively). The results indicate that in the steroid-filled hormone-binding pocket: (i) nucleophilic residues are localized on the beta-side but relatively remote from the steroid nucleus (distance from C-11 > "seven bonds"); (ii) relatively discrete changes in the electrophilic functionality, such as Cl to Br or NH to NCH(3) of haloacetamido compounds, can markedly modify the positioning of the electrophilic center which could no longer react with the nucleophilic residues; and (iii) cysteine residues (probably homologues of human ERalpha cysteine 381 and/or cysteine 530) are, at least partly, the covalent attachment sites of the electrophiles. Moreover, modification of cysteine residues by methyl methanethiosulfonate changes the structure of the hormone-binding pocket, whose labeling by the various electrophiles is profoundly altered.

Cysteine 530 of the human estrogen receptor alpha is the main covalent attachment site of 11beta-(aziridinylalkoxyphenyl)estradiols.

The efficiency of 11beta-[p(aziridinylethoxy)phenyl]estradiol 1 and 11beta-[p(aziridinylpentoxy)phenyl]estradiol 2 affinity labeling of the estrogen receptor alpha (ERalpha) was evaluated on the basis of their capacity to inhibit [(3)H]estradiol binding to lamb and human ERalphas. Relative to RU 39 411 (11beta-[p(dimethylaminoethoxy)phenyl]estradiol), the most closely related and chemically inert analogue of 1, the two electrophiles irreversibly inhibited [(3)H]estradiol binding to the lamb ERalpha. The fact that the compound effects were prevented (i) when the ERalpha hormone-binding site was occupied by estradiol and (ii) when the ERalpha-containing extracts were pretreated with methyl methanethiosulfonate (an SH-specific reagent) suggested that the compounds specifically alkylated ERalpha at cysteine residues. Wild-type human ERalpha was alkylated as efficiently as lamb ER, whereas the quadruple cysteine --> alanine mutant, in which all cysteines of the hormone-binding domain (residues 381, 417, 447, and 530) were changed to alanines, showed no significant electrophile labeling. The single C530A mutant was much less sensitive to the action of the electrophiles than the three other single mutants (C381A, C417A, and C447A). Moreover, analysis of the three double mutants (C381A/C530A, C417A/C530A, and C447A/C530A) showed that only the C381A/C530A mutant was less susceptible to electrophile labeling than the single C530A mutant. We concluded that in the hormone-binding pocket C530 was the main covalent attachment site of aziridines 1 and 2, whereas C381 could be a secondary site. These results agreed with the crystal structure of the hormone-binding domain of the human ERalpha bound to estrogen or antiestrogen, since C381 and C530 appeared to be (i) located in structural elements involved in delineating the hormone-binding pocket and (ii) in spatial proximity to each other, which was closer in the crystal structure of the ER:antiestrogen complex than in that of the ER:estrogen complex. Since C530 and C381 were also the main and secondary covalent attachment sites of tamoxifen aziridine (a nonsteroidal affinity-labeling agent), we propose a selective mode of superimposition of tamoxifen-class antiestrogens with RU 39 411-class antiestrogens, which could account for the relative positioning of the two types of ligands in the ERalpha hormone-binding pocket.

Modification of Leukotriene A4 Hydrolase/Aminopeptidase by Sulfhydryl-Blocking Reagents: Differential Effects on Dual Enzyme Activities by Methyl-Methane Thiosulfonate

The presence of a cysteine residue at or near the active site of leukotriene A4 hydrolase (EC 3.3.2.6) was suggested by inactivation of the enzyme with sulfhydryl-blocking reagents and by protection against inactivation afforded by substrates and competitive inhibitors. The aminopeptidase activity was more susceptible to inactivation than the epoxide hydrolase activity. The sulfhydryl-modifying reagent methyl-methane thiosulfonate reacted with one thiol as judged by kinetic data and titration with 5,5′-dithiobis-2-nitrobenzoate. Inactivation was a time- and dose-dependent process of apparent pseudo-first-order and maximal at 80–85%. The inactivation rate was nonsaturable and strongly influenced by ion strength. The second-order rate constant increased from 0.9 to 4.3 M−1 s−1 in the presence of 0.2 M NaCl. Albumin, a stimulator of the aminopeptidase activity, increased apparent inactivation rates by shifting pKa for the modification from 8.2 to 7.8. The inactivated enzyme partially regained activity upon treatment with β-mercaptoethanol. Peptide substrates and competitive inhibitors protected against inactivation. Bestatin, a competitive inhibitor, afforded complete protection with a KD = 0.15 μM, similar to Ki = 0.17 μM for inhibition of peptidase activity. Treated enzyme had an unchanged Km but a reduced Vmax. The epoxide hydrolase activity was only weakly affected by methyl-methane thiosulfonate with a maximal inactivation of 15–20% after prolonged treatment. Pretreatment of leukotriene A4 hydrolase with the reagent did not protect against mechanism-based inactivation by its lipid substrate, leukotriene A4. On the other hand, leukotriene B4 was a competitive inhibitor of aminopeptidase activity and protected against modification by methyl-methane thiosulfonate. Our results suggest the presence of a cysteine at or close to subsite S1′ of the active site of leukotriene A4 hydrolase and that modification of this residue interferes with the function of the aminopeptidase activity, but not the epoxide hydrolase activity. This is the first report to distinguish the two catalytic activities of leukotriene A4 hydrolase by chemical means.

Natural microflora of prepeeled garlic and their resistance to garlic antimicrobial activity

Prepeeled garlic carried 106-108cfu g−1micro-organisms; 103-105times more than freshly peeled garlic. Scanning electron micrographs revealed the presence of various types of micro-organisms on the surface of the garlic. Fifty-one lactic acid bacteria, 40 non-lactic acid bacteria and 15 yeasts were isolated from prepeeled garlic with heterofermentative lactic acid bacteria predominating among the lactic acid bacteria, andLeuconostococcurring most frequently. Twenty-six isolates of lactic acid bacteria grown in MRS broth with 20% garlic juice or higher were extremely resistant to garlic. Gram negative rod-shaped bacteria were the most frequently occurring non-lactic acid bacteria, with a predominance of the generalPseudomonasandEnterobacter. Cryptococcus neoformanswas the only species of yeast isolated. The inhibitory action of methyl methanethiosulfonate, but not of iodoacetate, appeared similar to that of garlic.

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface.

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.

Inhibition of estradiol binding to its receptor by the cupric ion

Treatment of human recombinant estrogen receptor (hER) expressed in yeast with very low concentrations of the cupric ion decreased its ability to bind [3H]estradiol ([3H]E2) and [125I]tamoxifen aziridine (minimal Cu2+ concentration: 1 nM). This decrease was reflected in a loss of immunoreactivity for monoclonal antibodies raised against the hormone binding domain (HBD). An ER recombinant expressing solely the HBD confirmed that the ion operated at this level. Cysteines located within this domain contributed to the inhibitory action of the Cu2+ in view of a partial restoration of the [3H]E2 binding activity with β-mercaptoethanol. Histidines were also implicated since the influence of Cu2+ on the [3H]E2 binding parameters (Scatchard plot analysis) was maintained after oxidation of thiol groups by methyl methanethiosulfonate, and partly reversed by imidazole.

Human Betaine–Homocysteine Methyltransferase Is a Zinc Metalloenzyme

We have overexpressed recombinant human liver betaine–homocysteine methyltransferase (BHMT; EC 2.1.1.5) inEscherichia coliand have purified the enzyme to homogeneity. The Michaelis constants for betaine andl-homocysteine are 2.2 mM and 4 μM, respectively. Analysis of the pure protein for metals by inductively coupled plasma emission spectrometry indicate that the recombinant enzyme contains zinc. Extensive dialysis in buffer containing high levels of EDTA could not strip the protein of zinc. However, dialysis against buffer containing EDTA and methyl methanethiosulfonate, followed by buffer containing EDTA and dithiothreitol, could remove zinc from the enzyme with concomitant loss of activity. Dialyzing the zinc-depleted enzyme against buffer containing 1 M urea and 2 mM zinc, followed by dialysis with buffer alone, completely restored BHMT activity and zinc content. BHMT was also partially purified from human liver. The purest BHMT-containing fractions also contained zinc and the enzyme was kinetically indistinguishable from the recombinant enzyme. As with the recombinant enzyme, the partially purified human liver enzyme could be inactivated by treatment with methyl methanethiosulfonate, EDTA, and dithiothreitol. Reconstitution of the zinc-depleted enzyme completely restored activity. We conclude that BHMT is a major zinc metalloenzyme in liver and that cysteineresidues are likely involved in zinc binding.

Changes in side chain packing during apomyoglobin folding characterized by pulsed thiol-disulfide exchange.

It is clear that close-packed side chain interactions play a dominant role in stabilizing native proteins, but the extent to which they stabilize kinetic intermediates and shape the energetic landscape of folding is not known. A method for characterizing structural changes at the level of individual side chains is presented and applied to study the refolding of apomyoglobin mutants containing engineered cysteine residues at key helical packing interfaces. The formation of buried side chain structure at the probe sites is followed by the extent of thiol-disulfide exchange during a pulse of thiol labeling reagent (either methyl methanethiosulfonate or 5,5'-dithiobis (2-nitrobenzoic acid)) applied at various stages of folding. The results suggest that the eight helices pack in at least three distinct stages, involving formation of two intermediates with time constants of <2 ms and 50 ms. In some parts of the refolding protein, stable side chain structure can be attained very rapidly, possibly in advance of backbone hydrogen bond formation as detected by previous pulsed amide hydrogen exchange experiments.

Identification of oligosulfide odorous compounds and their source in the Lake of Galilee

The chemical source of objectionable odors that are emitted from the Lake of Galilee (Lake Kinneret) and the relationship to the lake algae were elucidated. The lake serves for recreation and as an operational water reservoir, supplying a quarter of the water consumption of Israel. Analysis by closed loop stripping apparatus (CLSA), coupled with GC/MSD, olfactory GC and other detectors, revealed that the odorous compounds belong to the methylsulfide family, including, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and methyl methanethiosulfonate. The odor episodes coincide with a bloom of Peridinium gatunense algae. Peridinium gatunense rich samples, taken from the lake during the bloom season, emitted the same sulfur compounds. Depth profiles of Peridinium algae, sulfur compounds, dissolved oxygen, pH and temperature revealed strong correlation between the algal photo-induced motion in the water column and the distribution of dimethyldisulfide and dimethyltrisulfide, while the concentrations of dimethylsulfide and methyl methanethiosulfonate did not follow the same trend. In all cases, the dimethyloligosulfides were produced in the oxic region and were independent of the anoxic conditions in the hypolimniumon or in the sediment.

Sulfhydryl reagent susceptibility in proteins with high sequence similarity--triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi and Leishmania mexicana.

The amino acid sequence of triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68%. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei triosephosphate isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100-fold more sensitive. The sensitivity of wild type triosephosphate isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana triosephosphate isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accessibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more susceptible for perturbation.

Allosteric Regulation of Pyruvate Kinase M2 Isozyme Involves a Cysteine Residue in the Intersubunit Contact

Pyruvate kinase M2 isozyme mutants with amino acid substitutions in the subunit interface were prepared and characterized. The substitutions were made in the allosteric M2 isozyme by the corresponding residues of the nonallosteric M1 isozyme to identify the residue involved in the allosteric effects. The replacement of Cys-423 by Leu led to substantial loss of both homotropic and heterotropic allosteric effects while the substitutions at Phe-389, Arg-398, Ala-401, Pro-402, Thr-408, and Ile-427 did not. The altered kinetic properties of the Cys-423-substituted mutant resulted from the shift of the allosteric transition toward the active R-state since the mutant exhibits the allosteric properties in the presence of an allosteric inhibitor, L-phenylalanine. The inverse correlation between the hydrophobicity of residue 423 and the extent of stabilization of the R-state was found by analysis of mutants with un-ionizable amino acids at position 423. Furthermore, the modification of Cys-423 with methyl methanethiosulfonate led to a shift of the allosteric transition toward the R-state, probably the result of increased hydrophobicity of the residue. These results suggest that Cys-423 is involved in the allosteric regulation of the enzyme through hydrophobic interactions.

Effect of Disulfide Bonds of Transcobalamin II Receptor on Its Activity and Basolateral Targeting in Human Intestinal Epithelial Caco-2 Cells

Transcobalamin II-receptor (TC II-R) contains 10 half-cysteines, of which 8 are involved in intramolecular disulfide bonding. Reduction followed by alkylation with N-ethylmaleimide (NEM) of the 62-kDa TC II-R monomer in vitro or treatment of human intestinal epithelial Caco-2 cells with low concentrations (10(-6) M) of NEM resulted in TC II-R exhibiting a loss of ligand binding and an increase in its apparent molecular mass by 10 kDa to 72 kDa. Domain-specific biotinylation studies using NEM-treated filter-grown cells revealed loss of TC II-R but not cation-independent mannose 6-phosphate receptor protein at the basolateral cell surface. Pulse-chase labeling of NEM-treated cells with [35S]methionine revealed that the modified 72-kDa TC II-R, like the native 62-kDa TC II-R in untreated cells, turned over rapidly with a t1/2 of 7.5 h and was sensitive to treatment with peptide N-glycosidase F, sialidase alone, or sialidase and O-glycanase but not to treatment with endoglycosidase H. Labeled 72-kDa TC II-R, which was retained intracellularly following treatment of Caco-2 cells with methyl methanethiosulfonate, returned to the basolateral cell surface following withdrawal of cells from methyl methanethiosulfonate treatment and exposure to dithiothreitol. Based on these results, we suggest that formation and maintenance of intramolecular disulfide bonds of TC II-R is important for its acquisition of ligand binding and post-trans-Golgi trafficking to basolateral surface membranes but not for its turnover and exit from the endoplasmic reticulum or trafficking through the Golgi.

Two modes of ligand binding in maltose-binding protein of Escherichia coli. Electron paramagnetic resonance study of ligand-induced global conformational changes by site-directed spin labeling.

Binding of ligands to the maltose-binding protein (MBP) of Escherichia coli often causes a global conformational change involving the closure of its two lobes. We have introduced a cysteine residue onto each of these lobes by site-directed mutagenesis and modified these residues with spin labels. Using EPR spectroscopy, we examined the changes, caused by the ligand binding, in distance between the two spin labels, hence between the two lobes. The binding of both maltose and maltotetraose induced a considerable closure of the N- and C-terminal lobes of MBP. Little closure occurred upon the binding of maltotetraitol or beta-cyclodextrin. Previous study by fluorescence and UV differential absorbance spectroscopy (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609) showed that maltose and a large portion of maltotetraose bound to MBP via one mode (R mode or "end-on" mode), which is physiologically active and leads to the subsequent transport of the ligands across the cytoplasmic membrane. In contrast, maltotetraitol and beta-cyclodextrin bound to MBP via a different mode (B mode or "middle" mode), which is physiologically inactive. The present work suggests that the B mode is nonproductive because ligands binding in this manner prevent the closure of the two domains of MBP, and, as a result, the resulting ligand-MBP complex is incapable of interacting properly with the inner membrane-associated transporter complex.

Mercurochrom can be used for the histochemical demonstration and microphotometric quantitation of both protein thiols and protein (mixed) disulfides.

Mercurochrom [2,7-dibromo-4-(hydroxymercuri)-fluorescein disodium salt] used for staining of protein thiols in addition binds to other groups of proteins. Experimental evidence is provided that mercurochrom bound to non-thiol groups forms a 1:1 adduct with protein (mixed) disulfides. The disulfide contents of three different types of cells determined biochemically correlated with the corresponding mean integrated optical densities determined microphotometrically after mercurochrom staining of groups other than thiols. Intracellular disulfide exchange has been studied, leading to a transformation of protein mixed disulfides to protein disulfides and an equimolar loss of protein thiols. Protein mixed disulfides were generated from protein thiols using both methyl methanethiosulfonate (MMTS) and 2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD). Loss of thiols as well as the equimolar increase of protein mixed disulfides were followed using both mercurochrom staining for thiols and for disulfides. Generation of protein mixed disulfides due to the DDD reaction was also followed by azocoupling with Fast blue B. On the basis of the observed stoichiometry between the loss of protein thiols and the quantity, increase or conversion of protein disulfides determined microphotometrically using both mercurochrom staining and DDD Fast blue B staining, we conclude that: (1) 1 mol of mercurochrom is bound per mol of protein (mixed) disulfide; and (2) the molar absorptivity of mercurochrom bound to disulfides is epsilon 520 = 34940. This study demonstrates that mercurochrom can be used for the quantitative determination of the oxidative status of protein thiols in cells.

Antibacterial Activity of Heated Cabbage Juice, S‐Methyl‐L‐Cysteine Sulfoxide and Methyl Methanethiosulfonate

ABSTRACTAutoclaved cabbage juice was inhibitory to growth of Staphylococcus aureus. S‐Methyl‐L‐cysteine sulfoxide (SMCSO), autoclaved either together with or separately from nutrient broth, also inhibited the growth of S. aureus, but inhibition was greater when SMCSO was autoclaved separately. Methyl methanethiosulfonate (MMTSO2), a thermal breakdown product of SMCSO, completely inhibited growth of S. aureus at 10 ppm. MMTSO2 was formed in both autoclaved samples of cabbage juice and aqueous solution of SMCSO. Thus, evidence indicates that the bacterial inhibitory activity in autoclaved cabbage juice was due to heatinduced formation of MMTSO2 from SMCSO.