Sulfhydryl Localization and Tetrazolium Reduction. 1. Reversible Inhibition of its Reduction by N-Ethyl Maleimide

The sulfhydryl inhibitor N-ethyl maleimide completely inhibited the reduction of 2,3,5-triphenyltetrazolium chloride in meristematic and embryonic vascular tissues of Coleus sp. stems, Ricinus communis root tips, ungerminated Tea mays embryos, and epicotyls and coleoptiles of germinated Tea mays embryos, in a concentration of 200 mg/lit. Inhibition was reversed by the addition of cysteine or reduced glutathione (200 mg/lit) to the inhibitor medium. N-ethyl maleimide was effective also in blocking the nitro-prusside and 1-(4-chIoromercuriphenylazo)-naphthol-2 sulfhydryl staining reactions, but other substituted maleimides were ineffective in inhibiting the tetrazolium reaction in these tissues. Experiments were conducted to determine the histological pattern of sulfhydryl groups as indicated by a modification of the Bennett 1-(4-chloro-mercuriphenylazo)-naphthol-2 test and a modification of the Rap-kine nitroprusside test in certain plant tissues. A positive correlation was observed between tissues reducing the tetrazolium indicator and tissues exhibiting sulfhydryl localization as indicated by the nitroprusside reagent (trichloroacetic acid pretreated) and 1—(4— chloromercuriphenylazo)—naphthol—2.     

Mitochondrial K + transport: effect of N-ethyl maleimide on 42 K flux

The energy-linked flux of K⁺ into rat liver mitochondria is found to be stimulated by the sulfhydryl reagent, N-ethyl maleimide. The stimulation of K⁺ influx by N-ethyl maleimide is observed only at alkaline external pH. N-ethyl maleimide also stimulates efflux of K⁺ from the mitochondria. The stimulation by N-ethyl maleimide of K⁺ influx, but not K⁺ efflux, is dependent on the availability of metabolic energy. It is suggested that the effect of N-ethyl maleimide on K⁺ influx may be secondarily the result of an inhibition of phosphate-hydroxyl exchange. The dependence of energy-linked K⁺ influx on the external pH may be interpreted as evidence for a role of OH^? as a counterion accompanying K⁺ through the mitochondrial pump mechanism.     

N-ethyl maleimide as a probe for the study of functional sites and conformations of 30 S ribosomal subunits

Escherichia coli 30 S ribosomal subunits are inactive in a number of specific functions when Mg²⁺ concentration is reduced to 1 mM, and activity is recovered on heating under appropriate ionic conditions. When active and inactive forms were treated with N-ethyl maleimide, both forms reacted to a similar extent, but the reagent attached mostly to different proteins. Moreover, it caused irreversible inactivation only when reacting with the inactive form of the subunit. Though the activating treatment failed to restore activity to these subunits it did expose the same sulfhydryl groups as are available in the active state for reaction with the maleimide.Different ribosomal activities were eliminated at different maleimide concentrations, permitting the assignment of specific functions to sulfhydryl groups of specific ribosomal proteins. Protein S18 appears to be involved in subunit association, binding of fMet-tRNA and of aminoacyl-tRNA to the P-site. Proteins S1, S14 and S21 are all or in part involved in the binding of aminoacyl-tRNA to the A-site and in the binding of the antibiotic dihydrostreptomycin.The reaction with N-ethyl maleimide thus provides a criterion other than biological activity for characterizing different ribosomal forms and a tool for mapping the 30 S subunit for specific functional sites.     

Evidence for Sulfhydryl Involvement in Regulation of Phytoalexin Accumulation in Trifolium repens Callus Tissue Cultures

White clover (Trifolium repens L.) callus tissue cultures accumulated the phytoalexin medicarpin after treatment with sulfhydryl reagents. After 24-hour exposures to sulfhydryl reagents, maximum obtainable levels of medicarpin, determined by high performance liquid chromatography analysis, were found with 50 millimolar N-ethyl maleimide, 25 millimolar HgCl₂, 2 millimolar p-chloromercuribenzoic acid, and 0.5 millimolar iodoacetamide. Increased medicarpin levels were also observed in callus treated with p-chloromercuribenzene sulfonic acid, but the highest concentration tested (11.8 millimolar) did not produce the maximum response. After sulfhydryl treatment, medicarpin levels were unchanged for 4 to 6 hours, but steadily increased thereafter with maximum accumulation occurring by 48 to 50 hours for p-chloromercuribenzoic acid, p-chloromercuribenzene sulfonic acid, and HgCl₂ treated callus. Medicarpin levels did not increase in iodoacetamide-treated callus until 8 hours after sulfhydryl exposure, and medicarpin levels were still increasing linearly after 50 hours. Three other metabolic inhibitors, KCN, NaF, and Na₃AsO₄, did not exhibit elicitor activity, indicating cell death was not a factor in the response. Pretreatment of callus with 20 millimolar dithiothreitol followed by 40 millimolar N-ethyl maleimide did not produce the phytoalexin response. Preincubation with dithiothreitol also prevented elicitor activity of HgCl₂ and p-chloromercuribenzene sulfonic acid. These results suggested that dithiothreitol pretreatment somehow prevented sulfhydryl groups within the cell from reacting with the test compounds. These experiments established that the integrity of sulfhydryl groups is important in regulating phytoalexin accumulation in callus cells.     

Cytochemistry of the chromatin replication band in hypotrichous ciliated protozoa staining with silver and thiol-specific coumarin maleimide

A modification of the silver-staining techniques for nucleolar organizing regions (NORs) was used to stain selectively the macronuclear replication bands (RBs) and nucleoli in hypotrichous ciliated protozoa (Euplotes, Stylonychia, and Oxytricha). Silver staining of both types of structures was trypsin-sensitive and DNase I-insensitive, suggesting the involvement of proteins. Silver-staining proteins in the RB were differentially extracted with acid, without any decrease in nucleolar staining. Triton-acid-urea gel electrophoresis of an acid extract of Euplotes macronuclei revealed enhanced silver reaction with a single protein upon selective silver staining. An abundance of thiol groups was also demonstrated in the RBs and nucleoli by the fluorochrome 3-(4-maleimidylphenyl)-7-diethylamino-4-methyl coumarin (coumarin maleimide). Histochemical studies, including blocking thiols with N-ethyl maleimide (NEM), indicated that thiols were not necessary for silver staining, and that proteins in the RBs and nucleoli reacting with coumarin maleimide were not acid extractable.     

Effects on Mitochondrial K Flux of pH,K Concentration,and N-Ethyl Maleimide

Based on published evidence that cation transport in mitochondria is not significantly dependent on a membrane potential, it is suggested that the process of mitochondrial cation transport may be nonelectrogenic. These experiments focused on the possibility that K⁺ flux into rat liver mitochondria may be directly coupled, via an energy-linked carrier mechanism, to OH^? influx or H⁺ efflux. The dependence of the unidirectional K⁺ influx on the external K⁺ concentration indicates involvement of a saturable mechanism. Increasing the external pH from 7.0 to 8.0 increases the apparent V_max of the K⁺ influx without significantly altering the apparent K_m for K⁺. The pH dependence is greater in the presence of N-ethyl maleimide, a known inhibitor of the mitochondrial P_i/OH^? exchange mechanism. N-Ethyl maleimide decreases the apparent V_max at pH 7.0 and increases it at pH 8.0. Evidence indicates that both N-ethyl maleimide and a high external P_i concentration may stimulate the K⁺ influx at alkaline external pH (8.0) by preventing net exchanges between endogenous P_i and external OH^?. An apparent first-order dependence of the K⁺ influx on the external OH^? concentration is observed in the presence of N-ethyl maleimide. These results are consistent with a possible role of external OH^? as a cosubstrate of the K⁺ transport mechanism.     

Electron microscopic demonstration of two types of mitochondria with different affinities for lead

Synopsis Incubation of glutaraldehyde-fixed, non-frozen tissue slices of mouse seminal vesicle, ventral prostate and small intestine in the media described by Hugon & Borgers (1966) and by Mayaharaet al. (1967) for the cytochemical demonstration of alkaline phosphatase resulted in the deposition of lead along smooth muscle mitochondrial membranes but not along epithelial mitochondrial membranes. Control studies using boiled tissues; media lacking substrate; inhibitors such as L-cysteine, EDTA,N-ethyl maleimide andp-chloromercuribenzoate; and tissues incubated in full media after fixation, embedding and sectioning, showed that the reaction in muscle mitochondria was non-enzymatic. It was concluded that the phospholipid component of muscle mitochondrial membranes differed from that of epithelial mitochondrial membranes.     

Lipid-protein interactions of erythrocyte membranes: comparison of normal O, Rh (D) positive with the rare O, Rh null

Phospholipase A₂ modification of lipid-protein interactions of normal O,Rh(D) positive erythrocyte membranes increased the fluorescence intensity of the membrane bound probe, 1-anilinonaphthalene-8-sulfonate (ANS) and increased the N-1-[¹⁴C]-ethyl maleimide ([¹⁴C]-NEM) labeling of sulfhydryl groups in two proteins of molecular weight >200,000. In marked contrast, phospholipase A₂ modification of the rare phenotype O,Rh_null membranes resulted in no significant increase in ANS fluorescence or labeling of sulfhydryl groups by [¹⁴C] NEM. Since the O,Rh_null erythrocytes demonstrated an increased osmotic fragility and decreased survival time, the fluorescence and sulfhydryl labeling data support the conclusion that hydrophobic bonding between β-fatty acid side chains and non-polar regions of asymmetric proteins is necessary for maintaining the native structure of the O,Rh(D) positive membrane. Comparative studies with phospholipase C or D implied that ionic bonding played a similar though less important structural role in both membranes.     

Inactivation of rat ovarian 20α-hydroxysteroid dehydrogenase by N-alkylmaleimides

A series of N-alkylmaleimides, varying in chain length from N-ethylmaleimide and N-butyl to N-octyl, inclusive, was shown to effectively inactivate rat ovarian 20α-hydroxysteroid dehydrogenase at pH 7.7, 25 °C. The apparent second-order rate constants for inactivation were observed to increase with increasing chain length of the N-alkylmaleimide used. Positive chain length effects were also indicated by the K_d values for N-alkylmaleimides calculated from double-reciprocal plots resulting from the saturation kinetics observed in the inactivation reactions. The maximum rate constant for inactivation at enzyme saturation was 0.3 min^?1 for each maleimide studied. NADP-and coenzyme-competitive inhibitors such as 3-aminopyridine adenine dinucleotide phosphate and various adenosine derivatives protected the enzyme against maleimide inactivation, whereas no protection was observed with the steroid substrate, 20α-hydroxypregn-4-en-3-one. The pH profile for maleimide inactivation indicated the involvement of an enzyme functional group with a pK_a near 8.0. Sulfhydryl modification was also indicated by fluorescein mercuric acetate inactivation and titration experiments. Inactivation of the enzyme by a lysine-modifying reagent exhibited a pH profile differing from that observed in the maleimide inactivation process. It is proposed that N-alkylmaleimides inactivate the enzyme through covalent modification of sulfhydryl groups located in a nonpolar region of the enzyme.     

Milk-clotting Enzyme from Microorganisms

The milk-clotting activity of Mucor-rennin obtained from Mucor pusillus Lindt, was not changed by the addition of DFP in the reaction mixture. This finding suggested the probable absence of a serine residue at the active center of the enzyme. Sulfhydryl reagents such as Nekelgon, N-ethyl maleimide, PCMB failed to influence the milk-clotting reaction, indicating that a. reactive sulfhydryl group is not required for the enzymatic activity. The activity was inhibited when Mucor-rennin was treated with iodine at pH higher than 5.0. It was shown that ¹³¹I₂ was incorporated into the enzyme. When Mucor-rennin was photooxidized in the presence of methylene blue, the milk-clotting activity was inactivated. In this case, tyrosine, tryptophan, and histidine residues in the enzyme were oxidized. Among these amino acids, the histidine residue was more rapidly oxidized than other amino acids. A parallel relation was observed between the decrease of the amount of histidine residue and the inactivation of the enzyme. From these results, it is concluded that the histidine residue present in Mucor-rennin has a relation to the active center of this enzyme.     

Sulfhydryl reagents and energy-linked reactions in monocot thylakoids

Monofunctional maleimides have been used to covalently modify the coupling factor protein of monocot thylakoid membranes. As with dicot thylakoids, incubation of the monocot thylakoids with maleimides in the light but not in the dark results in inhibition of both ATP synthesis and hydrolysis. In the dark, sites on the γ and ε subunits of maize Zea mays coupling factor 1 are modified after incubation of maize mesophyll thylakoids with the fluorescent maleimide N-(anilinonaphthyl-4) maleimide. A light accessible site localized solely to the γ subunit has also been demonstrated. In contrast to the case with dicot thylakoids (spinach [Spinacia oleracea] and pea [Pisum sativum]) treatment of monocot thylakoids (maize, barley [Hordeum vulgare], crabgrass [Digitaria sanguinalis]) with bifunctional maleimides or thiol oxidants in the light does not result in functional uncoupling, i.e the bifunctional reagents act more like energy transfer inhibitors. The lack of functional uncoupling could be due either to a failure of the reagents to cross-link key sulfhydryl residues in the γ subunit or to the continued ability of the γ subunit to gate proton movements through the chloroplast coupling factor complex even though its conformation has been altered by sulfhydryl reagents.     

Modification of the erythrocyte membrane by sulfhydryl group reagents

Summary In this study, the consequences of modification of human erythrocyte membrane sulfhydryl groups by N-ethyl maleimide (NEM), 5,5dithiobis-(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuriphenyl sulfonate (PHMPS) were investigated. These reagents differ in chemical reactivity, membrane penetrability and charge characteristics.Results of sulfhydryl modification were analyzed in terms of inhibitory effects on activities of five membrane enzymes; Mg⁺⁺- and Na⁺, K⁺-ATPase, K⁺-dependent and independentp-nitrophenyl phosphatase (NPPase) and DPNase. Structural considerations involved in the sulfhydryl-mediated inhibition were evaluated by studying the changes in susceptibility to sulfhydryl alteration produced by shearing membranes into microvesicles and by the addition of the membrane modifiers, Mg⁺⁺ and ATP.Conclusions from the data suggest that the effects of NEM appeared to result from modification of a single class of sulfhydryls; DTNB interacted with two different sulfhydryl classes. Increasing concentrations of PHMPS resulted in the sequential modification of many types of sulfhydryls, presumably as a result of increasing membrane structural disruption. DTNB and PHMPS caused solubilization of about 15% of membrane protein at concentrations giving maximal enzyme inhibition.In contrast to the usually observed parallels between Na⁺, K⁺-ATPase and K⁺-dependent NPPase, activities of Mg⁺⁺-ATPase, Na⁺, K⁺-ATPase and K⁺-dependent NPPase varied independently as a result of sulfhydryl modification. We suggest complex structural and functional relationships exist among these components of the membrane ATP-hydrolyzing system.Our studies indicate that the effects of sulfhydryl group reagents on these membrane systems should not be ascribed to sulfhydryl modificationper se, but rather to the resulting structural perturbations. These effects depend upon the structural characteristics of the particular membrane preparation studied and on the chemical characteristics of the sulfhydryl group reagent used.     

Helix VIII of NhaA Na(+)/H(+) antiporter participates in the periplasmic cation passage and pH regulation of the antiporter

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na⁺/H⁺ antiporter. However, because NhaA is active at physiological pH (pH 6.5-8.5), many questions related to the active state of NhaA have remained unanswered. Our Cys scanning of the highly conserved transmembrane VIII at physiological pH reveals that (1) the Cys replacement G230C significantly increases the apparent K_m of the antiporter to both Na⁺ (10-fold) and Li⁺ (6-fold). (2) Variants G223C and G230C cause a drastic alkaline shift of the pH profile of NhaA by 1 pH unit. (3) Residues Gly223-Ala226 line a periplasmic funnel at physiological pH as they do at pH 4. Both were modified by membrane-impermeant negatively charged 2-sulfonatoethyl methanethiosulfonate and positively charged 2-(trimethyl ammonium)-ethylmethanethiosulfonate sulfhydryl reagents that could reach Cys replacements from the periplasm via water-filled funnels only, whereas other Cys replacements on helix VIII were not accessible/reactive to the reagents. (4) Remarkably, the modification of variant V224C by 2-sulfonatoethyl methanethiosulfonate or 2-(trimethyl ammonium)-ethylmethanethiosulfonate totally inhibited antiporter activity, while N-ethyl maleimide modification had a very small effect on NhaA activity. Hence, the size—rather than the chemical modification or the charge—of the larger reagents interferes with the passage of ions through the periplasmic funnel. Taken together, our results at physiological pH reveal that amino acid residues in transmembrane VIII contribute to the cation passage of NhaA and its pH regulation.     

meso-α,ɛ-Diaminopimelate d-Dehydrogenase: Sulfhydryl Group Modification

meso-α,?-Diaminopimelate D-dehydrogenase was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and HgCl₂. Two sulfhydryl groups were titrated per molecule in the presence and absence of 6 M guanidine hydrochloride: the enzyme contained one sulfhydryl group per subunit. Modification of the sulfhydryl groups with p-chloromercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 4,4'-dithiopyridine, N-ethylmaleimide, and iodoacetic acid was accompanied by a loss of enzyme activity. However, modification of sulfhydryl groups of the enzyme with cyanide did not affect the activity. Thus, the introduction of bulky or charged substituents to sulfhydryl groups decreased the catalytic activity of the enzyme, but modification of the groups with the small and uncharged group, a cyano group, did not. The sulfhydryl groups did not play an essential role in catalysis.     

Cadmium and zinc-mediated oxidative burst in tobacco BY-2 cell suspension cultures

Generation of reactive oxygen species (ROS) constitutes an important first reaction under many stress conditions in plants. We demonstrate that Nicotiana tabacum L. cv. Bright Yellow 2 (TBY-2) cells in suspension cultures, generate superoxide radical and hydrogen peroxide upon treatment with cadmium and zinc. Addition of catalase and N,N-diethyldithiocarbamate (DDC) decreased the level of H₂O₂, whereas superoxide dismutase (SOD) induced a slight increase of the H₂O₂ production. The effects of catalase, DDC and SOD on the heavy metal-induced ROS production indicate that it occurs outside of the cells, and that at least part of the hydrogen peroxide is produced by dismutation of the superoxide radical (O₂ ^·−). The effect of pretreatment of the cell cultures with commonly used mammalian NADPH oxidase inhibitors was also tested. Strong inhibitions of cadmium and zinc-mediated ROS production were obtained with the flavoprotein inhibitors—diphenylene iodonium (DPI) and quinacrine and with an inhibitor of b-type cytochromes—imidazol. Membrane permeable-N-ethyl maleimide (NEM) and iodoacetate, and membrane non-permeable thiol reagents—para-chloromercuribenzoic acid (pCMBS) also inhibited the ROS production. These results suggested that the enzyme responsible for cadmium and zinc-induced ROS production in tobacco cells contains a flavocytochrome. They also show the importance of intra- and extracellular thiol groups in the observed stress reaction. The induction of ROS production with heavy metals showed properties comparable to the elicitor-induced oxidative burst in other plant cells.     

Substrate-mediated increased reactivity of a critical sulfhydryl group of crystals of cytoplasmic aspartate transaminase

The extramitochondrial isozyme of aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase EC 2.6.1.1) contains a cysteinyl residue (cysteine-390) which, in the presence of substrate, displays enhanced reactivity toward sulfhydryl reagents. To gain insight into the structural similarity of the enzyme in solution compared to its crystalline state and into the type of structural change induced by substrates, the reactivity of Cys-390 in the crystalline enzyme has been studied. The flat yellow plates, crystallized from polyethylene glycol, form spectroscopically detectable enzyme-substrate complexes (C. M. Metzler, D. E. Metzler, D. S. Martin, R. Newman, A. Arnone, and P. Rodgers, 1978, J. Biol. Chem. 253, 5251–5254). The crystals, both in the presence and absence of the substrate pair, glutamate and α-ketoglutarate, were treated with N-ethylmaleimide or N-ethyl[1-¹⁴C]maleimide and the extent of the reaction was monitored by the colorimetric sulfhydryl reaction with 5,5′-dithiobis-2-nitrobenzoic acid, by amino acid analysis, by radioactivity incorporated, and by the measurement of enzyme activity. A cysteine residue was modified only in the presence of substrate; the crystals remained undamaged. Since, any large conformational change in the enzyme would either be prevented by the crystalline lattice or would disrupt its integrity, it is concluded that the enhanced reactivity of cysteine-390 in the presence of substrates must be due to only a small local conformational change in the substrate binding region.     

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. The inactivation by mono and bifunctional maleimides was partially prevented by Mg²⁺ and l-malate, and NADP prevented the inactivation almost totally. Determination of the number of reactive sulfhydryl groups of the native enzyme with [³H]NEM in the absence or presence of NADP showed that inactivation occurred concomitantly with the modification of two cysteinyl residues per enzyme monomer. The presence of these two essential residues was confirmed by titration of sulfhydryl groups with [³H]NEM in the enzyme previously modified by o-phenylenebismaleimide in the absence or presence of NADP.     

Evaluation of the cytotoxicity of a two photon absorbing fluorescence compound on human HepG2 cells and its application to tracking human hepatic cancer cells in mice

Abstract

Small organic dyes have been applied widely in fluorescence imaging techniques for biomedical research. We investigated the cytotoxicity of a novel fluorescent dye, trans-4-(N-2-hydroxyethyl-N-ethyl amino)-4′-(dimethyl amino) stilbene (DMAHAS), on human hepatocellular carcinoma (HepG2) cells using methyl thiazolyl tetrazolium(MTT), a neutral red assay, a Coomassie brilliant blue assay, and flow cytometric analysis. Our results showed that DMAHAS had live cell permeability, stable cytosolic localization and no significant cytotoxicity to HepG2 cells. We explored its application further for tumor cell tracking in a human liver tumor xenograft mouse model. Tumor xenografts were examined by fluorescence imaging and conventional histological methods. In addition, a method based on DMAHAS release was developed for tumor-specific cytotoxicity analysis. Our study indicated that DMAHAS is a reliable probe for tumor tracking and fluorescence imaging.     

SOME FACTORS AFFECTING THE UPTAKE, BINDING AND RETENTION OF [3H]CORTISOL BY THE CHICK EMBRYO RETINA AS RELATED TO ENZYME INDUCTION

—The accumulation of [³H]cortisol by the embryonic chick neural retina was studied at a time of rapid biochemical differentiation. In retinal cultures, uptake of steroid was decreased by inhibitors of energy metabolism and of sulphhydryl groups but not by inhibitors of macromolecular synthesis. The retention of steroid was inhibited by dinitrophenol and N-ethyl maleimide (NEM) over a 3 h incubation period. This inhibition may be due, in part, to the effect of NEM on the binding of steroid to intraretinal receptor as can be demonstrated by gel filtration. Neuraminidase had no effect on [³H]cortisol uptake or retention but markedly inhibited the induction of glutamine synthetase by the steroid. Uptake and retention of steroid at 37° was greater in the retinal nuclear pellet than in the cytosol fraction; the reverse was true at 4°. After treatment with NEM, [³H]cortisol accumulation in the nuclear pellet was drastically decreased, with approximately the same level of uptake as that seen at 4°. A temperature-dependent, sulphhydryl-sensitive process of steroid translocation from cytoplasm to nucleus may thus be indicated.     

PROTEIN-BOUND SULFHYDRYL GROUPS IN COLEUS WOUND MERISTEMS

Roberts , Lorin W. (U. Idaho, Moscow.) Protein-bound sulfhydryl groups in Coleus wound meristems. Amer. Jour. Bot. 47(2): 110—114. Illus. 1960.–A hisiochemical examination was conducted of the development of the stem wound-meristem of Coleus blumei Benth. with 2,2‘-dihydroxy-6,6‘-dinaphthyl disulfide (DDD) and 1-(4-chloromercuriphenylazo)-naphthol-2 (Mercury Orange) to test for protein-bound sulfhydryl groups. Specificity for the histochemical reactions was indicated by complete blocking of staining in sections pretreated with 0.001 M iodine in npropanol and 0.1 M aqueous solutions of iodoaceiamide. Both histochemical methods yielded comparable results in protein-SH localization (zone of cell division and differentiating xylem elemments) and staining intensity. The zone of cell division did not exhibit staining at the time of initial cell division (3 days). However, the cytoplasm of the dividing cells of the wound-meristem demonstrated protein-SH 4—5 days following wounding. A high concentration of protein-SH was observed in the cytoplasm of the dividing cells 7 days after wounding. Redifferentiating parenchyma cells, destined to become wound-xylem elements, first exhibited wall staining at the time of protoplast contraction. The subsequent walled xylem stage exhibited strong staining in the reticulated striations of the secondary walls.