Parachloromercuribenzoate Research Articles

A protease-resistant α-galactosidase from Pleurotus djamor with broad pH stability and good hydrolytic activity toward raffinose family oligosaccharides

An acidic α-galactosidase designated as PDGI (Pleurotus djamor α-galactosidase) was purified to homogeneity with 290-fold purification and a specific activity of 52.18 units/mg by means of ion exchange chromatography and gel filtration chromatography. PDGI is a monomeric protein exhibiting a molecular mass of 60kDa in SDS-PAGE and gel filtration. The optimum pH and temperature of the enzyme with pNPGal as substrate were 5.0 and 53.5°C, respectively. It displayed great pH stability within the pH range 3.0–10.0. Besides, the enzyme harbored remarkable resistance to acid protease and varying degrees of tolerance to other proteases: trypsin>collagenase Type-I>α-chymotrypsin neutral protease>proteinaseK. It was strongly inhibited by K+, Cd2+, Cu2+, Hg2+, Al3+, Fe3+ and Ag + ions. The chemical modification reagents diethypyrocarbonate (DEPC), 2,3-butanedione (DIC) and trinitrophenol (TNBS) increased the activity of PDGI 1.5-fold whereas N-bromosuccinimide (NBS) and parachloro-mercuri-benzoate (PCMB) drastically suppressed its activity. PDGI displayed activity toward stachyose and raffinose. The Km values for hydrolysis of pNPGal, stachyose and raffinose were 0.76, 7.63 and 6.29mM, respectively. Furthermore, PDGI degraded raffinose and sthachyose. These results suggest that PDGI has great potential for elimination of the non-digestible and flatulence-causing oligosaccharides stachyose and raffinose from legumes.

A novel Ca2+-activated protease from germinating Vigna radiata seeds and its role in storage protein mobilization

Calcium (Ca(2+))-dependent/activated proteases make decisive cleavages in proteins, affecting their further degradation/activation. Few such Ca(2+)-dependent proteases have been reported from plants, and none during germination-related events. Seeds are woken up from their quiescent state upon imbibition of water. The subsequent process of germination is strongly influenced by hormones (mainly gibberellins) and light, with both resulting in change in intracellular Ca(2+). We have investigated the effect of Ca(2+) on protease activity in extracts prepared from dry Vigna radiata (L.) Wilczec seeds and cotyledons 4, 24, 48 and 72h post-imbibition. Ca(2+)-activated protease activity is present at a very low level in dry seeds, rises with imbibition and peaks 24h post-imbibition. Subsequently, the activity rapidly declines, even as total protease activity continues to rise. Calcium activation of proteolysis was reversed by ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 1,10, phenanthroline, chlorpromazine and by beta-mercaptoethanol in a concentration-dependent manner. Protease activity was also inhibited by para chloro mercuribenzoate (pCMB) and l-trans-epoxysuccinyl-leucylamido(4-guanidino) butane (E 64), while phenyl methyl sulfonyl fluoride (PMSF) and pepstatin did not effect Ca(2+) activation. The protease could be separated from the calmodulin fraction by size-exclusion chromatography, while retaining its ability for Ca(2+) activation, excluding the possibility of activation through calmodulin-based pathways. The presence of a Ca(2+)-activated protease in the cotyledons suggests its role in a predetermined program of germination involving elevation of cytosolic Ca(2+) levels during germination. This protease could be an important enzyme interfacing cytoplasmic signaling events and initiation of storage protein mobilization during seed germination.

Tissue distribution of a novel non‐AT1, non‐AT2 binding site for angiotensins in the mouse

We recently reported the existence of a novel, non‐AT1, non‐AT2 binding site for angiotensins II (Ang II) and III (Ang III) in the rat, mouse and human brain. While the absence of this binding site in the liver, adrenal, and kidney suggested that this binding site was brain‐specific, we now report its presence in other mouse tissues. 125I‐Sar1,Ile8 Ang II binding was determined as described previously (Karamyan et al., Eur J. Pharmacol., 2008, 590: 87) in tissue homogenates with or without 0.3 mM parachloromercuribenzoate (PCMB), in the presence of 10 μM losartan and 10 μM PD123319 to saturate AT1 and AT2 receptors, respectively. In the absence of 0.3 mM PCMB specific (3 μM Ang II displaceable) binding of 125I‐ Sar1,Ile8 Ang II could not be measured reliably. In the presence of 0.3 mM PCMB, saturable, high affinity binding was observed in the testis (4.1±2.2 pmol/g, KD = 5.0±2.7 nM) with comparable density to the brain (4.2±2.0 pmol/g), but slightly lower affinity (KD = 2.7±1.2 nM, p<0.05). Measurable binding was present in 5 of 6 pancreas and 4 of 7 spleen samples with KD values similar to the brain, but at lower (23 and 30%, respectively) binding density. Binding was generally undetectable in the lung and heart. The presence of this binding site in testis, pancreas and spleen may indicate additional functions for angiotensins in these tissues. Supported by the Peptide Radioiodination Service Center of the University of Mississippi.

Human brain contains a novel non-AT1, non-AT2 binding site for active angiotensin peptides

Aims To determine whether the novel non-AT1, non-AT2 binding site for angiotensins recently discovered in rodent brains occurs in the human brain. Main methods Radioligand binding assays of 125I-sarcosine 1, isoleucine 8 angiotensin II binding were carried out in homogenates of the rostral pole of the temporal cortex of human brains containing 0.3 mM parachloromercuribenzoate (PCMB), 10 µM losartan to saturate AT1 receptors, 10 µM PD123319 to saturate AT2 receptors, with or without 10 µM angiotensin II to define specific binding. Competition binding assays employed a variety of angiotensin peptides, specific angiotensin receptor antagonists, several neuropeptides and an endopeptidase inhibitor to determine pharmacological specificity for this binding site. Key findings The novel non-AT1, non-AT2 binding site was present in similar amounts in female and male brains: Bmax 1.77 ± 0.16 and 1.52 ± 0.17 fmol/mg initial wet weight in female and male brains, respectively. The K D values, 1.79 ± 0.09 nM for females, and 1.53 ± 0.06 nM for males were also similar. The binding site shows pharmacological specificity similar to that in rodent brains: sarcosine 1, isoleucine 8 angiotensin II > angiotensin III > angiotensin II > angiotensin I ’ angiotensin IV > angiotensin 1–7. Shorter angiotensin fragments and non-angiotensin peptides showed low affinity for this binding site. Significance The presence in human brain of this novel non-AT1, non-AT2 binding site supports the concept that this binding site is an important component of the brain angiotensin system. The functional significance of this binding site, either as a novel angiotensin receptor or a highly specific angiotensinase remains to be determined.

Effect of cadmium, mercury and copper on partially purified hepatic flavokinase of rat.

The effect of cadmium (Cd2+), mercury (Hg2+) and copper (Cu2+) was studied with partially purified flavokinase (ATP:riboflavin 5'-phosphotransferase EC 2.7.1.26) from rat liver. All the divalent heavy metal cations inhibited flavokinase activity in a concentration-dependent manner. The inhibitory effect of cadmium on the enzyme was completely reversed by increasing concentration, of Zinc (Zn2+) indicating a competition between Zn2+ and Cd2+ for binding with the enzyme. A competition between riboflavin and Cd2+ is also evident from the present investigation. These observations hint at the possibility that Zn2+ and Cd2+ probably compete for the same site on the enzyme where riboflavin binds. However, inhibition of flavokinase by Hg2+ could not be reversed by Zn2+. Our studies further reveal that hepatic flavokinase appears to contain an essential, accessible and functional thiol group(s) which is evident from a concentration dependent inhibition of activity by sulfhydryl reagents like parachloromercuribenzoate (PCMB), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM). Inhibition of flavokinase by sulfhydryl reagents were protected, except in case of NEM inhibition, when the enzyme was incubated with thiol protectors like glutathione (GSH) and dithiothreitol (DTT). Furthermore, the enzyme could also be protected from the inhibitory effect of Cd2+ and Hg2+ by GSH and DTT suggesting that Cd2+ probably interacts with a reactive thiol group at or near the active site of enzyme in bringing about its inhibitory effect.

The Turnover of Cytoplasmic Triacylglycerols in Human Fibroblasts Involves Two Separate Acyl Chain Length-dependent Degradation Pathways

Cultured fibroblasts from patients affected with the genetic metabolic disorder named neutral lipid storage disease (NLSD) exhibit a dramatic accumulation of cytoplasmic triacylglycerols (Radom, J., Salvayre, R., Nègre, A., Maret, A., and Douste-Blazy, L. (1987) Eur. J. Biochem. 164, 703-708). We compared here the metabolism of radiolabeled short-, medium- and long-chain fatty acids in these cells. Short/medium-chain fatty acids (C4-C10) were incorporated into polar lipids (60-80%) and triacylglycerols (20-40%) at a lower rate (5-10 times lower) than long-chain fatty acids. Pulse-chase experiments allowed to evaluate the degradation rate of cytoplasmic triacylglycerols in normal and NLSD fibroblasts and to discriminate between two catabolic pathways of cytoplasmic triacylglycerols. Short/medium-chain (C4-C10) triacylglycerols were degraded at a normal rate in NLSD fibroblasts, whereas long-chain (C12 and longer) triacylglycerols remained undegraded. These data are confirmed by mass analysis. The use of diethylparanitrophenyl phosphate (E600) and parachloromercuribenzoate (PCMB) inhibitors allows to discriminate between the two triacylglycerol degradation pathways. E600 inhibited selectively the in situ degradation of short/medium-chain triacylglycerols without inhibition of the degradation of long-chain triacylglycerols, whereas PCMB inhibited selectively the in situ hydrolysis of long-chain triacylglycerols without affecting the degradation of long-chain triacylglycerols. This was correlated with the in vitro properties of cellular triacylglycerol-hydrolyzing enzymes characterized by their substrate specificity and their susceptibility to inhibitors; the neutral lipase specific to long-chain triacylglycerols is inhibited by PCMB, but not by E600, in contrast to short/medium-chain lipase, which is inhibited by E600 but not by PCMB. The data of in vitro and in situ experiments suggest the existence in fibroblasts of two separate acyl chain length-dependent pathways involved in the degradation of cytoplasmic triacylglycerols, one mediated by a neutral long-chain lipase and another one mediated by a short/medium-chain lipase.

A membrane-associated auxin-binding site from chickpea ( Cicer arietinum L.) epicotyls

A membrane-associated indoleacetic acid (IAA)-binding site with high affinity, high specificity and finite capacity has been found in etiolated chickpea epicotyls. Dissociation constant ( K d ) and binding site concentration are 3 × 10 −7 M and 0.25 pmol/mg protein, respectively. IAA binding is optimum at pH 5.5 and 0°C. The sensitivity of specific binding to pretreatment with proteases including trypsin, sodium dodecylsulfate (SDS) and thiol-reactive agents such as parachloromercuribenzoate (PCMB) and mercuric chloride (HgCl 2) indicates that the binding moiety is a protein containing a sulfhydryl group.

Reversible dissociation of cortisol-transcortin complex by sodium para-chloromercuribenzoate

Mercurials are considered as sulphydryl group specific reagents and one of them, sodium para-chloromercuribenzoate (PCMB), is currently used for SH titration. It has been shown that cellular steroid receptors are reversibly inactivated by mercurials even when the binding site is occupied by the steroid (Coty, W.A. (1980) J. Biol. Chem. 255, 8035–8037). This is a striking difference with alkylating SH reagents such as iodoacetic acid or N-ethylmaleimide, since these reagents inactivate only steroid-free receptors. In order to explain this discrepancy, we tested, in the present study, the specificity of PCMB on a blood plasma steroid binding protein: human transcortin. This protein presents the advantages, over cellular receptors, of being well characterized and to be available in a pure state. The transcortin-cortisol complex was also reversibly inactivated by PCMB when the reaction was carried out at a high excess of reagent over protein; such conditions are those previously used with steroid receptors. The reversibility was obtained not only with a reducing agent (dithiothreitol) but also with EDTA, which suggests a poor stability of the protein mercurial bond and therefore a nonspecific action. The decrease of activity was the result of a loss of binding sites and Scatchard plot analysis did not reveal any detectable decrease of the affinity constant for cortisol. Transcortin possesses two SH groups per molecule, one of these being buried in native conformation. After blockage of the accessible SH group by aminoethylation, transcortin kept the same activity, but when this aminoethylated transcortin was incubated with PCMB a loss of activity was obtained, although the residual buried SH group was again titrable with Ellman's reagent. Therefore, we can conclude that the action of PCMB on proteins must be interpreted with precaution, since it can induce an inactivation that is SH-independent.

Evidence for active and passive urate transport in the rat proximal tubule

Studies utilizing the technique of simultaneous microperfusion of peritubular capillaries and tubular lumen of the proximal tubule of the rat were performed to determine if the absorption of urate was an active transport process and to determine the passive permeability coefficient for urate. When radioactive urate of equal specific activity and concentration was present in both perfusion solutions, the ratio of collected to initial concentrations of urate in the luminal perfusate (CO/CI) was 0.71 +/- 0.02. This gradient was higher than that predicted at equilibrium from the electrical potential difference determined in the in vitro perfused rabbit proximal tubule. The addition of para-chloromercuribenzoate (PCMB) to both solutions resulted in a significantly higher CO/CI of 0.90 +/- 0.02. This latter value is closer to the value predicted at electrochemical equilibrium. In separate studies, the unidirectional fluxes of urate were determined in the presence of PCMB. The calculated passive permeability coefficient averaged approximately 0.94 pmol . min-1 . mm-1 . mM-1 and was equal in both directions. These results indicate that in the rat proximal tubule urate absorption is an active transport process. In addition, there exists a passive permeation pathway for urate movement out of and into the proximal tubule.

Partial characterization of a phosphoryl group transferring enzyme in the membrane of catecholamine storage vesicles

Phosphoryl group transfer from ATP to ADP occurred in the isolated membrane of catecholamine storage vesicles. The reaction was accelerated by extraction of the membranes with 50% (v/v) acetone and by treatment with 1% (v/v) Triton X-100. The phosphoryl group transfer reaction was activated by Mg2+ and by Mn2+. The activation profile differed from that obtained for the ATPase activity. The Michaelis-Menten kinetics of the phosphoryl transfer reaction were not entirely linear. From the linear parts of the double reciprocal plots KmATP approximately equal to 1 mM and KmADP approximately equal to 0.4 mM was obtained. All lines of the double reciprocal plots intersected indicating a sequential reaction mechanism. The reaction exhibited a narrow specificity for nucleoside diphospate and a broader one for nucleoside triphosphate indicating that ADP was the true substrate. The transfer reaction was slightly inhibited by AMP, orthophosphate and P1, P5-di(adenosine-5')pentaphosphate. The thiol reagents, N-ethylmaleimide and para-chloromercuribenzoate (PCMB), affected the ATPase activity and the phosphoryl transfer activity differently: with the blockade of 2.4 essential thiol equivalents by N-ethylmaleimide the ATPase was inhibited 50% and net uptake of catecholamine ceased, while the phosphoryl transfer remained unimpaired. PCMB affected both, the ATPase activity and phosphoryl transfer reaction. Treatment of the membranes with dithioerythritol prevented the PCMB-induced inhibition of the phosphoryl transfer, but was ineffective in protecting the ATPase activity, indicating that different thiol groups must be involved in the both enzymatic activities.

EcoRI ★ activity: Enzyme modification or activation of accompanying endonuclease?

A study has been made of the factors and mechanism leading to appearance of the so-called EcoRI ∗ activity described by Polisky et al. (1975) in the restrictase EcoRI preparations. The preparations of purified restrictase EcoRI, precipitated at 0.9 ammonium sulphate saturation, as well as that obtained using standard techniques have been found to contain an admixture of an endonuclease which at neutral pH and high ionic strength multiply cleaves those DNAs which normally have only one recognition site for EcoRI. Under the standard conditions for EcoRI digestion this activity is found only when large amounts of freshly isolated enzyme are added to the incubation mixture and it is sharply enhanced by replacement of Mg 2+ with Mn 2+. The number and size of DNA fragments produced under such conditions practically do not differ from those found under the so-called EcoRI ∗ conditions, that is for alkaline pH values and low ionic strength. The optimum incubation mixture for the EcoRI ∗ activity has been found to be 10 mM Tris·HCl buffer (pH 8.8) + 2 mM Mn 2+. Similar activity is induced also by addition to EcoRI solution of 40–50% glycerol or a number of organic solvents (dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulphoxide (DMSO), sulphalane (SP) in concentrations from 1 to 6%. The EcoRI ∗ activity induced by 50% glycerol or at alkaline pH values and low ionic strength is suppressed or sharply inhibited by 2–3 mM parachloromercuribenzoate (PCMB), while EcoRI is not sensitive to this agent. The DNA fragments cleaved by EcoRI ∗ have cohesive termini and can be easily ligated. It is suggested that the EcoRI ∗ activity can be due not only (or largely not) to modification of the

Cholesterol ester metabolizing enzymes in human brain: properties, subcellular distribution and relative levels in various diseased conditions.

AbstractWe have in the present study examined the properties and subcellular distribution of cholesterol ester metabolizing enzymes in human brain, and compared the levels of these enzymes in brains from patients with phenylketonuria (PKU), metachromatic leucodystrophy (MLD), and Down's Syndrome (DS).Cholesterol esterification was optimal at pH 5.6, did not require ATP or CoA as cofactors and was inhibited by detergents (TWEEN‐20 and Triton X‐100) and bile acids (sodium taurocholate and sodium deoxycholate). The specific activity of the cholesterol esterifying enzyme was highest in the mitochondrial fraction. Cholesterol esterifying activity in brains from PKU, MLD, and DS patients was not significantly different.Cholesterol ester hydrolase activity in human brain peaked at two different pHs (4.5 and 6.5). The activity was optimal when the substrate was dispersed in Triton X‐100 and sonicated. The specific activity of the pH 4.5 hydrolase was highest in the mitochondrial fraction, while that of the pH 6.5 hydrolase was highest in myelin. The sulfhydryl group reagent parachloromercuribenzoate (PCMB) inhibited the activity of the hydrolase(s) but diisopropylfluorophosphate (DFP), a typical serine reagent, had no effect on hydrolase(s) activity. Addition of either phosphatidyl serine or phosphatidyl inositol significantly enhanced the hydrolase activity at both pHs.The level of cholesterol ester hydrolase(s) in PKU brains was lower than in the brains from DS patients, and the level of these enzymes in the brains from two patients with metachromatic leucodystrophy was lower than in the brains from PKU patients. It is concluded that the properties and subcellular distribution of cholesterol esterifying enzyme in human brain is similar to that in rat brain (Ero & Suzuki, 1971) but that the hydrolases in human brain differ from that in rat brain in several respects, and that the low levels of hydrolase(s) activity in MLD and PKU brain may be related to reduced myelin content of those brains.

The Role of Hydroxyl Radicals in Radiation-Induced Single-Strand Breaks of Bacterial DNA Sensitized by Parachloromercuribenzoate

Escherichia coli 15T/sup -/ cells have been irradiated by gamma rays at 21/sup 0/C at a dose rate of 1.3 krad/min in the presence and absence of parachloromercuribenzoate (PCMB) and the effects of hydroxyl radical scavengers on the yields of single-strand DNA breaks under atmospheric and anoxic conditions (the latter achieved with the aid of sodium dithionite) studied. In the absence of PCMB, the amount of breaks, 1.9 x 10/sup 14/ breaks/g of DNA/krad, is little affected by the removal of OH radicals or oxygen, and represents the result of broken DNA that has largely been rejoined under the conditions employed. PCMB increases single-strand breaks under atmospheric and anoxic conditions by 18- and 7-fold, respectively. A significant amount of such breaks can be reduced by OH radical scavengers, and a study of the kinetics of scavenging under atmospheric conditions by four such scavengers shows that OH radicals are responsible for 79 percent of the breaks in PCMB-treated cells. In the absence of PCMB, the cells are capable of repairing all the breaks produced by OH radicals as well as some by the direct effect. The sensitizing effect of PCMB is attributable to its inactivation of intracellular repair enzymes. Other plausiblemore » modes of action of PCMB are also discussed.« less

SH groups in the alpha-naphthyl acetate esterase in the thyroid of the guinea-pig. A histochemical study.

The alpha-naphthyl acetate esterase in both group I and group II thyroid cells is shown to contain SH groups since there is a decline in activity in both cell groups when certain sulfhydryl reagents [DTNB; 5,5'-Dithiobis-(2-nitrobenzoic acid)-AgNO3-Mersalyl-PCMB (parachloro mercuribenzoate) + urea] are added to the incubation media. Thus the inhibition is by far the greatest in group I cells, which also show the greatest activity after incubation in conventional media, when long fixation and storage times are used. In all cases the inhibiting effect was complete or almost completely reversed if cysteine was added to the incubation media in equivalent concentrations to the SH blocker. There were great differences among the sulfhydryl reagents used in their ability to bring about enzyme inhibition. The alkylating agents NEM (N-ethylmaleimide) and iodoacetamide had no or little effect while PCMB could only inhibit the activity of the alpha-naphthylacetate esterase if the enzyme was denaturated with 5 M urea. The maximal inhibitory effect of PCMB was only obtained when NaCl was added to the incubation media. The most effective inhibitor was AgNO3.

The activity of non-specific esterase in the thyroid epithelial cells of the guinea pig as influenced by various inhibitors and activators. A histochemical study.

The action of various inhibitors and activators upon esterase activity in the thyroid epithelial cells is demonstrated. The agents used were triorthocresylphosphate (TOCP), parachloromercuribenzoate (PCMB), Arsanillic acid, p-nitrophenyl dimethyl carbamate and bis p-nitrophenyl phosphate. TOCP was found to inhibit selectively the activity in the follicle cells proper when alpha naphthyl acetate was used as a substrate. Arsanillic acid (0,001 M) activated the follicle cells proper selectively, but if the concentration was raised to 0,01 M the effect was that of inhibition while the activity in the para-, inter- and intrafollicular cells was unchanged. The results obtained are related to previous biochemical and histochemical observations and the nature of esterases in the thyroid is discussed.

Studies of Parachloromercuribenzoate-Induced Radiosensitization in Escherichia coli and Bacteriophages

Treatment of Escherichia coli B/r and ${\rm B}_{{\rm s}-1}$ with parachloromercuribenzoate (PCMB) causes marked increases (a maximum of about 16- to 20-fold) in the gamma radiation-induced single-strand breaks in DNA with the attainment of the same high limiting yield (of the order of $3\times 10^{15}$ breaks/gDNA/krad) as that reported previously for E. coli $15{\rm T}^{-}$ . The amount of breaks in cells pretreated with $5\times 10^{-4} M$ PCMB and subsequently washed five times prior to irradiation is comparable to that obtained with cells irradiated in $5\times 10^{-5} M$ thus suggesting sensitization to involve largely cell-bound PCMB. However, no complex formation between PCMB and calf thymus DNA is detectable spectrophotometrically. Sensitization of breaks is observable at concentrations above $6.3\times 10^{-6} M$ , a concentration which is only minimally toxic to the cell...

Mercury(II) binding to s4U in E. coli tRNA(Val).

The accessibility of the s(4)U base in native tRNA(Val) from E.coli was monitored by studying the binding of various mercurials. The relative binding order HgBr(2)[unk]HgCl(2)>>CH(3)HgOAc[unk]CH(3)HgCl[unk]PCMB parallels approximately the steric requirements of linear HgX(2) or RHgX compounds for S(N)2 displacement by sulfur, although other factors are operative. Para-chloromercuri-benzoate (PCMB) does not bind the thiolated nucleotide unless the tertiary structure of the tRNA is opened up by removal of Mg(2+) ions and heating to 40 degrees . Under these conditions, equilibrium dialysis measurements using (14)C-labeled PCMB showed one binding site (n = 0.93) with an association constant, K(1), of 9 x 10(4)M(-1).

Effect of sulfur-containing compounds on experimental diabetes. XI. Effect of thiol and disulfide compounds on glucose transfer into red blood cells.

Studies were made on the influence of thiol and disulfide compounds on glucose uptake into red blood cells (RBC) of normal and alloxan diabetic rats. 1) Incorporation of glutathione (GSH), 2-mercaptopropionylglycine (MPG), oxidized GSH and oxidized MPG into RBC was examined. MPG was found to be incorporated into RBC. When MPG was allowed to react with RBC at 37° for 30 minutes, approximately 30% of MPG in the reaction solution was incorporated into RBC (alloxan diabetic (AD)-RBC). Four to ten per cent of the incorporated MPG existed in the oxidized form. 2) Incorporation of glucose into normal RBC and AD-RBC was examined. Incorporation of glucose into AD-RBC was inhibited. 3) In normal RBC, such compounds as MPG and cystein which were incorporated into RBC increased glucose uptake, while GSH showed a tendency to inhibit incorporation of glucose. Incorporation of glucose was inhibited by oxd GSH, oxd MPG and cystine. 4) Influence of various inhibitors was studied. Incorporation of glucose was inhibited by SH reagents (HgCl2, parachloromercuribenzoate (PCMB), parachloromercuribenzene sulfonate (PCMBS), NEM), but not by insulin. Potassium borohydride increased glucose uptake. 5) Disappearance of glucose from normal RBC and AD-RBC was studied. Fifty per cent of glucose disappeared from normal RBC 9 minutes later and from AD-RBC 19 minutes later. The time required for the disappearance of 50% of glucose from normal RBC and from AD-RBC was shortened to 7 minutes and to 16 minutes respectively by the addition of MPG. As mentioned above, there is a difference in incorporation of glucose into RBC between normal RBC and AD-RBC. Furthermore, promotion of glucose uptake by MPG and cysteine and inhibition of glucose uptake by oxd MPG and cystine were discussed.

Specific labelling of thiol groups in mammalian keratin suitable for electron microscope studies.

SUMMARYQuantitative Polarographic studies of the reaction between methylmercuric iodide and variously reduced wool keratin indicate that reaction occurs specifically with the thiol groups from cystine. Since loss of protein from fibres is negligible, this organomercurial is preferred as a selective amino acid label in electron microscope studies. Similar reactions of wool with parachloromercuribenzoate (PCMB) result in extensive structural modifications.The mercury mercaptides were shown to be thermally unstable in the electron beam and cooling of the specimen between — 140°C and — 88°C coupled with low beam intensities was found necessary to study the original distribution of the mercurials in the samples. Since a direct relationship between the electron scattering sites of the mercurial and cystine within the keratin was established, it has been possible to demonstrate unambiguously for the first time that:(a) There exists a radial distribution of cystine within cuticular cells. Indeed a new sulphur‐rich component at the outer edge of the ‘a’ layer in peripheral cuticular cells has been identified.(b) Para‐cortical cells of intact keratin contain a higher proportion of cystine than ortho‐type cells.(c) A dense concentration of cystine labelled by the electron scattering centres (mercurials) corresponds closely to an intermicrofibrillar‐matrix system in the cortex.Furthermore, a method has been devised for determining the cystine content of the exocuticle using data obtained from electron micrographs.

The Modification of the Yields of Single Strand Breaks in DNA of Gamma-Irradiated Escherichia coli

The yield of single strand breaks in DNA in the γ-irradiation of Escherichia coli $15 {\rm T}^{-}$, as measured by the alkaline sucrose gradient technique, is enhanced by two enzyme inactivating agents, namely, sodium cyanide and parachloromercuribenzoate (PCMB), but to different extents. The G-value of 0.26 obtained in air for the yield of single strand breaks in the absence of additives is increased to 1.4 by sodium cyanide and to 5.4 by PCMB. In nitrogen, the yield for sodium cyanide-treated cells is 0.55 and that for PCMB-treated cells 5.3. Irradiation of PCMB-treated cells in the presence of histidine gives a lower yield of 1.0. β-Mercaptoethylamine (MEA) completely neutralizes the enhancing effect of PCMB, the residual yield being 0.27. For cyanide-treated cells, both histidine and MEA give the same limiting yield of 0.45. The cell is apparently capable of rejoining by itself breaks attributable to free radical attack as well as some produced by the direct action of radiation. These results indicat...