Effect Of PCMB Research Articles

Toxicological and behavioral study of two potential antibacterial agents:4-chloromercuribenzoic acid and quercetin on Swiss-albino mice

Global dissemination of carbapenem resistant-Gram negative bacteria (CR-GNB) is supposed to be clinically alarming because it extremely delimits the treatment options against serious infections. 4-Chloromercuribenzoic acid (pCMB) is an efficient metallo-enzyme inhibitor, and quercetin is known for antioxidant, antiviral, anticancer, antimicrobial, and anti-inflammatory activities. These two compounds could be considered as potential candidates for the treatment of CR-GNB mediated infections. Hence, in this study, antibacterial activity of pCMB and quercetin was evaluated against CR-GNB through minimum inhibitory concentration (MIC) determination. Toxicity of pCMB and quercetin was evaluated by LC50 calculation through brine shrimp test (BST) and by investigating hematological, serum biochemical, and histopathological parameters in Swiss-albino mice. Moreover, aggressive–depressive–cognitive behavioral effects of pCMB and quercetin on murine model were evaluated. All the carbapenem resistant isolates (CR-GNB) exhibited MIC values in the range of 4–256 μg/ml, 16–256 μg/ml, and 64–1024 μg/ml for pCMB, quercetin, and meropenem, respectively. BST determined LC50 of pCMB and quercetin at 91.57 ± 0.35 mg/L and 448.45 ± 0.46 mg/L, respectively. Oral administration of low dose of pCMB and quercetin did not induce any significant changes in morphological, behavioral, hematological, serum biochemical, and histopathological parameters among Swiss-albino mice. But, a high dose of pCMB and quercetin exhibited slight toxicity. However, no death was reported for any dosage of pCMB and quercetin. Therefore, pCMB and quercetin might be considered for further investigations on alternative therapeutics to combat against CR-GNB.

PCMB Treatment Reveals the Essential Role of Cysteinyl Residues in Conferring Functional Competence to the Regulatory Subunit of Protein Kinase CK2

To assess the functional role of the four conserved cysteinyl residues in the regulatory β-subunit of protein kinase CK2, the effect of pCMB and other reagents of sulfhydryl groups has been investigated. The pCMB-treated β-subunit has lost its ability to form either homodimers or regular α2β2 heterotetramers with the catalytic subunit. It also fails to increase catalytic activity toward peptide substrates and to mediate the stimulatory effect of polylysine. The pCMB-treated β-subunit, however, is still able to prevent calmodulin phosphorylation and to physically interact with the α-subunit to form inactive complexes whose sedimentation coefficient is lower than that of CK2 holoenzyme. These inactive complexes upon treatment with reducing agents like DTT are converted into a fully active heterotetrameric holoenzyme.

Effects of sulfhydryl inhibitors on depolarizations-contraction coupling in frog skeletal muscle fibers.

We have studied the effects of the sulfhydryl reagents on contractile responses, using either electrically stimulated single muscle fibers or short muscle fibers that were voltage-clamped with a two-microelectrode voltage-clamp technique that allows the fiber tension in response to membrane depolarization to be recorded. The sulfhydryl inhibitors para-chloromercuribenzoic acid (PCMB) and parahydroximercuriphenyl sulfonic acid (PHMPS), at concentrations from 0.5 to 2 mM, cause loss of the contractile ability; however, before this effect is completed, they change the fiber contractile behavior in a complex way. After relatively short exposure to the compounds, < 20 min, before the fibers lose their contractile capacity, secondary tension responses may appear after electrically elicited twitches or tetani. After losing their ability to contract in response to electrical stimulation, the fibers maintain their capacity to develop caffeine contractures, even after prolonged periods (120 min) of exposure to PHMPS. In fibers under voltage-clamp conditions, contractility is also lost; however, before this happens, long-lasting (i.e., minutes) episodes of spontaneous contractile activity may occur with the membrane polarized at -100 mV. After more prolonged exposure (> 30 min), the responses to membrane depolarization are reduced and eventually disappear. The agent DTT at a concentration of 2 mM appears to protect the fibers from the effects of PCMB and PHMPS. Furthermore, after loss of the contractile responses by the action of PCMB or PHMPS, addition of 2 mM DTT causes recovery of tension development capacity.

Characterization of the inhibitor sensitivity of the coenzyme A transport system in isolated rat heart mitochondria.

The effect of protein labeling agents on coenzyme A (CoA) transport into isolated rat heart mitochondria was studied. CoA transport was substantially inhibited by sulfhydryl reagents (mersalyl, pCMB) as well as by the tyrosine-selective reagent N-acetylimidazole. The effect of pCMB was reversed by DTT. Moreover, CoA uptake was completely abolished by agents selective for lysine and amino terminal residues (pyridoxal 5-phosphate, dansyl chloride). In contrast arginine-selective reagents (2, 3-butanedione, phenylglyoxal) caused considerably less inhibition of CoA uptake. Moreover, partial inhibition of transport was observed with the stilbene disulfonic acid derivatives DIDS and SITS. Finally, measurement of the effects of the labeling agents on the mitochondrial membrane potential indicated that the inhibition of CoA transport into mitochondria is not a secondary effect that arises from an alteration in the electric potential gradient across the inner mitochondrial membrane. These results provide the first information on the types of amino acid residues that may be essential to the CoA transport mechanism and provide additional support for the existence of a CoA transport protein within the mitochondrial inner membrane. Furthermore, the identification of effective inhibitors of the CoA transport system will greatly facilitate the functional reconstitution of this transporter in a proteoliposomal system following its solubilization and purification.

Limited proteolysis of glutamate dehydrogenase in the liver of eel ( Anguilla japonica)

1. 1. Limited proteolyzed glutamate dehydrogenase (GDH) was purified from acetone powder prepared from fresh eel livers. 2. 2. The limited proteolyzed GDH has a subunit of 50,000 molecular weight as well as a subunit of 54,000 molecular weight, and the specific activity of the GDH was higher than that of the native GDH. 3. 3. The amino acid composition and sequence of four residues from the N- terminal of the limited proteolyzed GDH were the same as those of the trypsin-treated GDH (Tang et al., J. Biochem. 111, 655–661, 1992). 4. 4. Effects of PCMB, ADP and GTP on the limited proteolyzed GDH were comparable with those on the trypsin-treated GDH. 5. 5. Proteases extracted from the acetone powder of the fresh livers were applied to a typsin inhibitor-Sepharose column, and proteases retained on this column did not bring about limited proteolysis of the native GDH.

Effect of sulfhydryl compounds on ATP-stimulated H+ transport and Cl− uptake in rabbit renal cortical endosomes

The vacuolar H+ ATPase is inhibited by N-ethylmaleimide (NEM), a sulfhydryl compound, suggesting the involvement of a sulfhydryl group in this transport process. We have examined the effects of several sulfhydryl-containing compounds on the vacuolar H+. ATPase of rabbit renal cortical endosomes. A number of such compounds were effective inhibitors of endosomal H+ transport at 10(-5)-10(-6) M, including NEM, mersalyl, aldrithiol, 5,5' dithiobis (2-nitrobenzoic acid), p-chloromercuribenzoic acid (PCMB) and p-chloromercuriphenyl sulfonic acid (PCMBS). NEM, mersalyl, aldrithiol and PCMBS had no effect on pH-gradient dissipation, whereas PCMB decreased the pH gradient faster than control. In the absence of ATP, PCMB (10(-4) M) stimulated endosomal 36Cl- uptake, particularly in the presence of an inside-alkaline pH gradient (pHin = 7.6/pHout = 5.5). This result was not an effect of PCMB on the Cl(-)-conductive pathway. The less permeable PCMBS did not stimulate 36Cl- uptake. The effects of PCMB were concentration dependent and were prevented by dithioerithritol. ATP-dependent 36Cl- uptake was decreased by addition of PCMB. Finally, PCMB had no effect on 45Ca2+ uptake. These results support the presence of two functionally important sulfhydryl groups in this endosomal preparation. One such group is involved with ATP-driven H+ transport and must be located on the cytoplasmic surface of the endosomal membrane. The second sulfhydryl group must reside on the internal surface of the endosomal membrane and relates to a PCMB-activated Cl-/OH- exchanger that is functional both in the presence and absence of ATP. This endosomal transporter is similar to the PCMB-activated Cl-/OH- exchanger recently described in rabbit renal brush-border membranes.

Activation of Cl-/OH- exchange by parachloromercuribenzoic acid in rabbit renal brush-border membranes.

The effect of the sulfhydryl reagent parachloromercuribenzoic acid (PCMB) on chloride transport was examined in rabbit renal brush-border membrane vesicles (BBMV). PCMB had no effect on the chloride conductive pathway. In the presence of an inside-alkaline pH gradient and a K-/valinomycin voltage clamp, the addition of PCMB stimulated 36Cl uptake and induced a threefold overshoot above the equilibrium value, indicating Cl/OH exchange. The effect of PCMB was reversed by dithiothreitol. Cl/OH exchange was not observed in the absence of PCMB. PCMB-activated Cl/OH exchange persisted even when the membrane potential was made inside-negative relative to the controls, thus, demonstrating that PCMB's effect on 36Cl uptake under pH-gradient conditions is not mediated by parallel Cl- and H+ conductive pathways. PCMB-activated Cl/OH exchange was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) with IC50 values of 290 and 80 microM, respectively. These results demonstrate that modification of sulfhydryl groups by PCMB activates Cl/OH exchange in BBMV.

Effect of thiol reactive reagents and ionizing radiation on the permeability of erythrocyte membrane for non-electrolyte spin labels.

The paper presents some results on the effect of PCMB and NEM on the transport of non-electrolyte spin labels: TEMPO and TEMPOL across non-irradiated and irradiated porcine erythrocyte. Irradiated erythrocytes exhibited increased inhibitory effect of thiol reactive compounds in the TEMPO and TEMPOL transport compared to non-irradiated erythrocytes.

Effect of thiol reagents and ionizing radiation on the permeability of erythrocyte membrane for spin-labeled non-electrolytes.

Four different thiol reagents: p-chloromercuribenzoic acid (pCMB), mercuric chloride (HgCl2), N-ethylmaleimide (NEM), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) were employed as agents modifying the transport of a hydrophilic and hydrophobic non-electrolyte spin labels: 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) into bovine erythrocytes. Gamma-irradiation of erythrocytes amplified the effects of pCMB, HgCl2 and NEM of inhibition of TEMPOL transport and attenuated them in the case of TEMPO transport. These results suggest that the transport of TEMPOL across the erythrocyte membrane is controlled by both superficially and more deeply located membrane -SH groups while only superficial -SH groups control the transport of TEMPO. The lower extent of inhibition of TEMPO transport indicates a higher contribution of diffusion through the lipid phase to the transport of TEMPO across the erythrocyte membrane as compared with TEMPOL.

Relationship between alkaline proteases from the midgut lumen and epithelia of the silkworm: Partial purification and comparison of properties of both proteases-6B3

1. 1. Midgut epithelial protease of the silkworm, Bombyx mori was solubilized, partially purified and compared with the properties of corresponding lumenal protease-6B3. 2. 2. The optimum pH, pI and K m value for BAPNA of the epithelial protease were 11.4, 10.70 and 0.697 mM respectively. 3. 3. Both epithelial and lumenal enzymes were inactivated by PMSF, leupeptin, STI and LTI, indicating that they are serine-type and trypsin-like proteases. 4. 4. Thiol blocking reagents, PCMB, Cu 2+ and IAA inhibited both proteases. 5. 5. These proteases were different in the effect of PCMB, IAA, STI and some ions. 6. 6. The relationship between epithelial and lumenal proteases is discussed.

The effect of some oligo-amines and -guanidines on membrane permeability in higher plants

The effect of a series of oligo-amines and -guanidines on the membranes of higher plants has been tested by measuring the efflux of betacyanin from beet root discs, and the loss of total ions from beet root and swede discs, beet and spinach leaf discs and apple cells in suspension culture. All of the naturally occurring di- and polyamines tested showed relatively little toxicity. Betacyanin efflux from beet root discs was reduced by diamines [NH 2(CH 2) x NH 2] up to x = 10 or less. Ion efflux was minimal at x = 7. Within the triamine series [NH 2(CH 2) x NH(CH 2) 3NH 2] for x = 8 or less, betacyanin efflux was reduced or unaffected, although total ion loss was increased by the triamines when x = 4 or more and especially by the longer chain amines (to x = 10). Similar behaviour was found in the tetra-amine series [NH 2(CH 2) 3NH(CH 2) x NH(CH 2) 3NH 2] with betacyanin efflux reduced for x = 2–4 (spermine). Although spermine potentiated the toxicity effects of Guazatine {[NH 2C(NH)NH(CH 2) 8] 2NH} and Dodine [NH 2C(NH)NH(CH 2) 11,Me] in beet root discs, spermine and calcium ions reduced the ion efflux caused by these toxic guanidines and also by Synthalin B [NH 2C(NH)NH(CH 2) 12NHC(NH)NH 2] in swede discs, spinach leaves and apple cells. No significant reversal of ion loss was detected with putrescine, cadaverine or spermidine in swede discs. In the homologous series of monoguanidines [NH 2C(NH)NH(CH 2) x−1Me] for x up to 18, greatest toxicity was shown for x = 10 and 11 in both betacyanin loss and total ion efflux in beet root discs. Greatest ion efflux from the apple cell suspension was found with x = 11 and 12. In the homologous series of diguanidines [NH 2C(NH)NH(CH 2),NHC(NH)NH 2] for x = 2–12 greatest toxicity was shown for x = 12 (the longest chain tested) in beet root and in the efflux of ions from apple cell suspension. Technical Guazatine was considerably more phytotoxic than pure Guazatine in all systems tested. p-Chloromercuribenzoate ( p-CMB) potentiates the loss of betacyanin and total ions caused by Guazatine, Synthalin B, and Dodine in beet root discs. This effect of p-CMB is reversed by subsequent incubation in cysteine or mercaptoethanol, prior to treatment with the guanidines.

Heavy Meromysoin from Skipjack Tuna, Euthynus Pelamis Preparation and Enzymic Properties 1

A method was developed to obtain heavy meromyosin (HMM) from the tryptic digest of skipjack tuna dorsal myosin. The tuna HMM thus obtained was shown to be homogeneous on gel filtration-gel electrophoresis, and on ultracentrifugation. The sedimentation constant (S20,w) was estimated to be 6.1S for tuna HMM. The ATPase activity of tuna dorsal HMM was found to be very similar to that of rabbit skeletal HMM in many respects: KCl concentration dependence, pH dependence, effect of pCMB, kinetic parameters (Vmax and Ka) in actin activation, and Arrhenius activation energy. The only difference found between tuna HMM and rabbit HMM was in heat denaturation behavior: the ATPase activities of tuna HMM were approximately four times as sensitive to heat inactivation as those of rabbit HMM. Thus, tuna HMM should represent a good experimental material for investigations of the molecular basis of susceptibility to denaturation, and of the characteristics of fish myosins in general. A new type of heat denaturation of myosin was observed. It occurred in a very early stage of heat treatment of either tuna dorsal myosin or rabbit skeletal myosin; however, it did not occur upon heat treatment of HMM of either tuna or rabbit, and it was detectable in terms of the Mg-ATPase activity only when the activity was measured in the presence of untreated actin.

Physiology of the maxillary sugar sensitive cell in the red turnip beetle,Entomoscelis americana

The galeae of red turnip beetle larvae each have a single sensillum which is chemosensitive. One of the four chemosensitive cells in this sensillum responds to sugars, sucrose and mannose being the most effective of the sugars tested (Fig. 4). The electrophysiological threshold for the response to sucrose can be as low as 0.01 mM and the Kb for the response is approximately 1.0 mM (Figs. 1 and 2). The same cell also responds to several amino acids, alanine being the most effective (Table 2). When the characteristics of this sugar sensitive cell are compared with similar cells in adult flies, significant differences are found. The most obvious differences are related to amino acid sensitivity and the effect of PCMB on the response to sucrose (Table 1).

Glucose transport in Brucella abortus.

Brucella abortus British strain 19 transported glucose with an apparent K(m) of 0.16 mM and an apparent V(max) of 250 nmol per min per mg of N. The only common glucose analogue transported was 2-deoxyglucose (2-DOG), with an apparent K(i) of 0.73 mM. Alpha- or beta-methyl glucosides and 3-O-methylglucose were not transported. Transport was linear for 70 to 90 s, depending on the concentration of substrate used. 2-Deoxyglucose was transported as the free sugar and was not further metabolized once inside the cell. There was no glucose phosphoenolpyruvate phosphotransferase system (PEP-PTS) present, and there were no inhibitors present in Brucella cell-free extract that inhibited the Escherichia coli glucose PEP-PTS. N-Ethylmaleimide (NEM) and p-chloromercuribenzoate (pCMB) completely inhibited transport of glucose and 2-DOG. Glutathione, dithiothreitol, and beta-mercaptoethanol reversed the effects of pCMB but not of NEM. A pH optimum of 7.2 and a temperature optimum of 37 to 45 C were observed for both K(m) and V(max). The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, erythritol, or glucose. The electron transfer inhibitors carbonyl cyanide, m-chlorophenylhydrazone, NaN(3), 2,4-dinitrophenol, and KCN inhibited 2-DOG transport to a greater extent than did the metabolic energy inhibitors NaAsO(4), iodoacetate, KF, and 2-heptyl-4-hydroxyquinoline-N-oxide. Dicyclohexylcarbodiimide, an inhibitor of membrane-bound adenosine triphosphatases, inhibited transport by 100%.

Na+-K+-active inheritable adenosinetriphosphatase system: the effect of Triton-X-100 on the SH groups

A significant portion of the SH-groups of the rat-brain Na+-K+-ATPase preparation is masked; it can be unmasked by the treatment of Triton-X-100. The inhibitory effects of PCMB and NEM on the ATPase activity are different in the detergent-treated and untreated preparations.

The Effect of Sulfhydrylinhibitors and Glycine Derivatives on Fibrin Polymerization and the Physical Strength of Fibrin in Plasma

SummaryThe influence of p-CMB, iodoacetamide, iodoacetic acid, N-ethylmaleimide, glycerinederivatives and of EDTA on thrombingeneration on fibrinpolymeriza- tion and on the physical strength of clots as expressed by spectrophotometrical and thrombelastographic measurements respectively has been examined.P-CMB and N-ethylmaleimide significantly inhibit the thrombin generation.EDTA retards the initial steps of the fibrinpolymerization and it increases the final turbidity. Its effect is neutralized by calcium and magnesium. The sulfhydrylinhibitors do not inhibit the polymerization neither do the glycinederivatives.To judge from these results does FSF not influence the polymerization but calcium and magnesium do. This statement is verified using FSF deficient plasma.The four SH-inhibitors tested inhibit the development of a normal thromb- elastogram, specially in reducing the mA values. Cysteine, having no effect by itself, reverses the effect of p-CMB and iodoacetamide.p-CMB has a similar effect when added to FSF deficient plasma and the main effect of this SH inhibition is suggested to be on platelet enzymes. p-CMB and iodoacetic acid also reduce the m A values of platelet-free normal plasma possibly by inhibiting FSF.

The kinetics of dissociation of the first oxygen molecule from fully saturated oxyhaemoglobin in sheep blood solutions.

A detailed description is given of a stopped-flow apparatus, which is specially suitable for measuring the rates of the rapid reactions of haemoglobin with oxygen and carbon monoxide. The apparatus has been chiefly applied to determinations of the rate of displacement of oxygen,r, from combination with haemoglobin by carbon monoxide. It is shown theoretically, and confirmed experimentally, that the reciprocal ofrwhen plotted against the oxygen pressure initially in equilibrium with the haemoglobin gives a straight line from whichk4, the velocity constant of the reactionHb4O8→Hb4O6+O2, can be readily calculated. The value ofk4is scarcely affected by pH over the range 9.1 to 7.1, but has a temperature coefficient at both pH values of about 3.2 per 10° C. Measurements have also been made of the overall velocity constants of combination of oxygen (k') and of carbon monoxide (l') with sheep haemoglobin, and of the overall rate of dissociation of sheep oxyhaemoglobin in presence of Na2S2O4(k). The numerical values tally well with those obtained by previous rougher methods. The effect ofp-chloromercuribenzoate (PCMB) onk4,k',l' andkhas been determined. This reagent has only a slight effect on the combination velocity constantsk' andl', but increases markedly the value ofk4. Its effect onkis very complicated, suggesting that the velocity constants of the reactionsHb4O6→Hb4O4+O2andHb4O4→Hb4O2+O2may be increased, whereas that of the reactionHb4O2→Hb4+O2may be greatly reduced. These curious effects are qualitatively in line with the effects of PCMB on the equilibrium between oxygen and haemoglobin.