SH-group Reagents Research Articles

A hemorrhagic metalloprotease of Egyptian Cerastes vipera venom: Biochemical and immunological properties.

A hemorrhagic metalloprotease (CVHT1) was isolated from Cerastes vipera (CV) viper venom and characterized in a set of biochemical and immunological assays. A simple two-step purification procedure included gel filtration and ion-exchange increase the purity of enzyme 39-fold with specific activity of 20,200 Umg-1 compared to 520 Umg-1 for crude venom. CVHT1 is a dimer enzyme with two subunits of ~60 kDa. The LC-MS/MS analysis of CVHT1 revealed that the identified peptides show high homology to other P-III snake venom zinc-metalloproteases. The activity of CVHT1 showed stability at pH (6.5-8.5) and temperature (30-60 °C) with optima at pH 8.5 and 60 °C. Activators for CVHT1 included Mg+2, Zn+2, Ca+2, K+, Ba+2 and Na+, while the full inhibition was given by other tested ions, SH-group reagents and metalloproteinase inhibitors. The CVHT1 potentially digested gelatin, fibrinogen, fibronectin and inhibited the platelet aggregation. The hemorrhagic and proteolytic activities of medically important Egyptian viper venoms were highly cross-neutralized by anti-CVHT1. The anti-CVHT1 increased the survival time of mice injected with lethal dose of CV venom to 23 ± 2.5 h compared to the mice injected with venom alone 0.52 ± 0. 05 h. This study could be useful for production of safer and more efficient therapeutic anti-venom.

Microtubules as a Critical Target for Arsenic Toxicity in Lung Cells in Vitro and in Vivo

To understand mechanisms for arsenic toxicity in the lung, we examined effects of sodium m-arsenite (As3+) on microtubule (MT) assembly in vitro (0–40 µM), in cultured rat lung fibroblasts (RFL6, 0–20 µM for 24 h) and in the rat animal model (intratracheal instillation of 2.02 mg As/kg body weight, once a week for 5 weeks). As3+ induced a dose-dependent disassembly of cellular MTs and enhancement of the free tubulin pool, initiating an autoregulation of tubulin synthesis manifest as inhibition of steady-state mRNA levels of βI-tubulin in dosed lung cells and tissues. Spindle MT injuries by As3+ were concomitant with chromosomal disorientations. As3+ reduced the binding to tubulin of [3H]N-ethylmaleimide (NEM), an -SH group reagent, resulting in inhibition of MT polymerization in vitro with bovine brain tubulins which was abolished by addition of dithiothreitol (DTT) suggesting As3+ action upon tubulin through -SH groups. In response to As3+, cells elevated cellular thiols such as metallothionein. Taxol, a tubulin polymerization agent, antagonized both As3+ and NEM induced MT depolymerization. MT–associated proteins (MAPs) essential for the MT stability were markedly suppressed in As3+-treated cells. Thus, tubulin sulfhydryls and MAPs are major molecular targets for As3+ damage to the lung triggering MT disassembly cascades.

Purification and properties of a new S-adenosyl-L-methionine:flavonoid 4'-O-methyltransferase from carnation (Dianthus caryophyllus L.).

A new enzyme, S-adenosyl-l-methionine:flavonoid 4'-O-methyltransferase (EC 2.1.1.-) (F 4'-OMT), has been purified 1 399-fold from the tissues of carnation (Dianthus caryophyllus L). The enzyme, with a molecular mass of 43-45 kDa and a pI of 4.15, specifically methylates the hydroxy substituent in 4'-position of the flavones, flavanones and isoflavones in the presence of S-adenosyl-l-methionine. A high affinity for the flavone kaempferol was observed (Km = 1.7 micro m; Vmax = 95.2 micro mol.min-1.mg-1), while other 4'-hydroxylated flavonoids proved likewise to be suitable substrates. Enzyme activity had no apparent Mg++ requirement but was inhibited by SH-group reagents. The optimum pH value for F 4'-OMT activity was found to be around neutrality. Kinetic analysis of the enzyme bi-substrate reaction indicates a Ping-Pong mechanism and excludes the formation of a ternary complex. The F 4'-OMT activity was increased, in both in vitro and in vivo carnation tissues, by the inoculation with Fusarium oxysporum f. sp. dianthi. The enzyme did not display activity towards hydroxycinnamic acid derivatives, some of which are involved, as methylated monolignols, in lignin biosynthesis; the role of this enzyme could be therefore mainly defensive, rather than structural, although its precise function still needs to be ascertained.

Transport of L-carnitine in isolated cerebral cortex neurons.

The accumulation of carnitine was measured in cerebral cortex neurons isolated from adult rat brain. This process was found to be lowered by 40% after preincubation with ouabain and with SH-group reagents (N-ethylmaleimide and mersalyl). The initial velocity of carnitine transport was found to be inhibited by 4-aminobutyrate (GABA) in a competitive way (Ki = 20.9 +/- 2.4 mM). However, of various inhibitors of GABA transporters, only nipecotic acid and very high concentrations of 1-[2-([(diphenylmethylene)amino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO-711) acid decreased carnitine accumulation while betaine, taurine and beta-alanine had no effect. The GABA transporters expressed in Xenopus laevis oocytes did not transport carnitine. Moreover, carnitine was not observed to diminish the accumulation of GABA in cerebral cortex neurons, which further excluded a possible involvement of the GABA transporter GAT1 in the process of carnitine accumulation, despite the expression of this protein in the cells under study. The absence of carnitine transporter OCTN2 in rat cerebral cortex neurons (K. A. Nałecz, D. Dymna, J. E. Mroczkowska, A. Broër, S. Broër, M. J. Nałecz and R. Cecchelli, unpublished results), together with the insensitivity of carnitine accumulation towards betaines, implies that a novel transporting protein is present in these cells.

Significance of sulfhydryl groups in the activity of urease from pigeonpea (Cajanus cajan L.) seeds.

Titration of urease from pigeonpea (Cajanus cajan L.), a hexameric protein (mol. wt. 480000; subunit mol. wt. 80000), with 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) reveals the presence of 5.82+/-0.13 'accessible' sulfhydryl groups per molecule of the enzyme protein (i.e. about one 'accessible' SH group per subunit). Denatured enzyme was found to titrate for 12.1+/-0.1 SH groups per molecule (i.e. about two SH groups per subunit). Half of the 'accessible' groups react faster than the remaining at pH 8.5 as well as pH 7.5. However, the reaction was slower at pH 7.5 than 8.5. Time-dependent loss of enzyme activity with DTNB was also found to be biphasic. The enzyme was inactivated at low concentration of p-chloromercuribenzoate (p-CMB), N-ethyl maleimide (NEM) and iodoacetamide. The inactivation reactions were biphasic, with half of the activity lost more rapidly than the remaining half. The loss of activity with p-CMB was linearly related to the blocking of accessible SH groups. Inactivation by p-CMB is largely reversible by addition of excess of cysteine. Fluoride ion strongly protects the enzyme against NEM inactivation, however, substrate urea provides much weaker protection against SH group reagents. The significance of these results is discussed.

Blood–Brain Barrier Controls Carnitine Level in the Brain: A Study on a Model System with RBE4 Cells

Transport of carnitine was studied with immortalized rat brain endothelial cells (RBE4), an in vitro model of the blood–brain barrier. The experiments on uptake and efflux through the luminal membrane excluded any involvement of choline and amino acids transporters, as well as that of glycoprotein P. Acetyl-, octanoylcarnitine, and betaine were without any effect; the only compound decreasing both processes was butyrobetaine. An exposure of the abluminal membrane resulted in a 40% inhibition of carnitine uptake by the substrates of neutral amino acid transporter L, while its efflux through the basolateral membrane, occurring in a form of free carnitine, was sensitive to SH group reagent, mersalyl, and was diminished by butyrobetaine. These features of carnitine transport did not fully correspond to the known characteristics of the proteins transporting carnitine in other tissues (OCTN2 and CT1); however, they did not exclude an involvement of a transporter belonging to the same superfamily. Moreover, such a protein in brain endothelium would fulfill a regulatory role in the transport of carnitine through the blood–brain barrier.

Transient Stimulation of Oxygen Uptake Induced by Sulfhydryl Reagents in Egeria densa and Potamogeton crispus Leaves

Abstract:A vigorous and transient increase of O2 uptake associated with a simultaneous release of CO2 was elicited in Egeria densa and in Potamogeton crispus leaves by treatment with N‐ethylmaleimide (NEM) and by other ‐SH group reagents (iodoacetate, p‐(chloromercuri)benzenesulfonate (p‐CMBS), Ag+, Hg2+, Cu2+). The NEM‐induced respiratory burst was apparent even in the absence of photosynthesis (darkness, or presence of DCMU) as well as in the presence of the respiration inhibitors cyanide and propyl gallate or SHAM, separate or in combination. In contrast, a complete suppression of the respiratory burst was induced by diphenylene iodonium and by quinacrine, inhibitors of the plasma membrane NADPH oxidase activated in the pathogen‐elicited oxidative burst in granulocytes and in plants. The respiratory burst induced by NEM and by the uncoupler CCCP were additive. The intensity of the respiratory burst was markedly decreased by increasing the pH of the medium from 5 to 8, and partially decreased by the presence of K+ in the medium. Azide inhibited the burst (as well as basal respiration) at pH 6.5 but not at pH 5. The stimulation of QO2 by SH reagents was associated with an early, pronounced membrane depolarization together with a rapid increase of the release into the medium of K+ and other electrolytes, and with a rapid decrease of the intracellular ATP, ADP and G6 P contents. The possible relationships between this SH reagent‐induced respiratory burst and the associated effects on Em and electrolyte leakage are discussed.

Mitochondrial protein import: modification of sulfhydryl groups of the inner mitochondrial membrane import machinery in Solanum tuberosum inhibits protein import.

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F1 beta subunit of the ATP synthase (pF1 beta) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu2+ were inhibitory. Inhibition by Cu2+ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondrial than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu2+ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF1 beta with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu2+ were inhibitory. NEM, MPB, DTNB and Cu2+ inhibited import of the NEM-modified pF1 beta into intact mitochondria. Import of pF1 beta through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu2+ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.

Ferric chelate reduction by sunflower (Helianthus annuusL.) leaves: influence of light, oxygen, iron-deficiency and leaf age

The presence of ferric chelate reducing activity in sunflower [Helianthus annuus L.) leaves has been studied by submerging leaf discs in a solution with Fe(III)-ethylenediaminetetra-acetate (FeEDTA), batho-phenanthroline disulphonate (BPDS) and vacuum infiltration. The effect of different factors on the Fe(III) reduction rate was studied. Ferric reduction rate was about 10-fold higher in the light than in darkness. The light effect was greatly inhibited by 3-(3,4-dichloro-phenyl)-1,1-dimethylurea (DCMU), a photosystem II inhibitor. Several inhibitors of redox systems [cis-platinum (II) diamine dichloride (cis-platin), p-nitro-phenylacetate (p-NPA) and p-hydroxymercuribenzoic acid (pHMB)] decreased the FeEDTA reduction rate. The greatest inhibition was produced by the - SH group reagent pHMB (17% of control, in light). The FeEDTA reduction rate was much higher in the absence of O2 than with air or 100% O2. Superoxide dismutase (SOD) decreased FeEDTA reduction with air in the light. Young leaves reduced Fe(III)-chelate at a higher rate than did older leaves. In iron-deficient plants, leaves did not exhibit enhanced ferric chelate-reducing activity as was observed in roots. It is suggested that at least two different redox systems or two states of the same redox system work in the light and in darkness.

Acute stimulation of glucose transport by histamine in cardiac microvascular endothelial cells

The purpose of the present work was to study the acute regulation of glucose uptake in cultured cardiac endothelial cells (CEC). Two types of potential stimuli were considered: (1) agents that are known to acutely stimulate glucose transport (i.e., within minutes) in fat and muscle tissues and (2) agents that influence endothelial cell function. Among the former agents, neither insulin, nor catecholamines (adrenaline, dopamine, phenylephrine), nor serotonin affected the rate of glucose transport in CEC, while SH-group reagents (phenylarsine oxide, diamide or menadione) were inhibitory. Among the factors of the second group that were tested (heparin, ADP, histamine, bradykinin), histamine was found to stimulate glucose transport in CEC by 10–50%. This effect was concentration-dependent (with an EC 50 value ⋟ 12 μ M ) and reached a maximum within 5 min upon histamine addition. This stimulation of glucose transport was suppressed by pyrilamine (100 nM), a specific H 1-receptor antagonist, but not by cimetidine (100 μM), a H 2-selective antagonist. Northern blot and Western blot analysis of CEC extracts revealed the presence of the ubiquitous glucose transporter isoform GLUT1 mRNA and protein, but not of the ‘insulin-regulatable’ isoform GLUT4. In conclusion, this is the first report on an acute stimulation of glucose transport in cardiac endothelial cells, in particular, and in an insulin-unresponsive cell type, in general. The effect of histamine is most likely mediated by H 1-receptors and cannot be accounted for by a recruitment of GLUT4.

Starvation-induced increase of hepatic alanine uptake is related to changes in sensitivity to SH-group reagents.

Changes in L-alanine transport in plasma membrane vesicles from livers of control and 24- and 48-h starved adult rats and the sensitivity of alanine uptake to sulfhydryl group reagents [N-ethylmaleimide (NEM) and p-chloromercuribenzenesulfonate (p-CMBS)] were studied. The portal concentration of certain amino acids was measured, and the relationship between L-alanine transport kinetic parameters and amino acid levels was analyzed. Starvation only induced a decrease in portal concentration of these amino acids that are mainly carried by Na(+)-dependent systems (85 and 61% for 24- and 48-h starved rats, respectively). Portal alanine concentration was lower in 24-h starved animals than in control rats (370 vs. 587 microM) and further decreased after 48 h of fasting (228 microM). Starvation induced an increase in maximum velocity (Vmax) values of Na(+)-dependent L-alanine transport (7.19, 8.97, and 12.38 pmol.U 5'-nucleotidase-1.10 s-1 for control and 24- and 48-h starved rats, respectively) with slight, but not significant, changes in the apparent Michaelis constant (Km) values (3.35, 2.63, and 2.20 mM for control and 24- and 48-h starved rats, respectively). Portal alanine showed a directly close correlation with Km values and inverse with Vmax values. The mean affinity constant values for the effects of NEM and p-CMBS on Na(+)-dependent L-alanine transport were lower in 48- (2.57 and 0.13 mM, respectively) and 24-h starved rats (3.59 and 0.32 mM, respectively) than in control rats (8.56 and 0.59 mM, respectively) and showed a directly strong correlation with kinetic characteristics of L-alanine transport and portal alanine concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of respiration inhibitors on the oxidative burst induced inElodea densaleaves by -SH group reagents

Effects of respiration inhibitors on the oxidative burst induced in<i>Elodea densa</i>leaves by -SH group reagents

Stimulation of oxygen consumption in Elodea densa leaves by -SH group reagents

Stimulation of oxygen consumption in <i>Elodea densa</i> leaves by -SH group reagents

Effects of SH-Group Reagents on Ca 2+ and K + Channel Currents of Pancreatic B-Cells

The whole-cell patch-clamp technique has been applied to elucidate the effects of H 2O 2 and the SH-group reagent 2,2′-dithio-bis(5-nitropyridine) (DTBNP) on Ca 2+ currents of mouse pancreatic B-cells. We also tested the effect of DTBNP on the whole-cell K + ATP current. DTBNP (20 and 100 μmol/l) blocked both the current through Ca 2+ channels (with Ba 2+ as the charge carrier) and the K + ATP current in a concentration-dependent manner. The results suggest that the channels themselves or regulating proteins possess free SH-groups which may be of functional relevance. However, H 2O 2 did not influence the Ca 2+ channel currents under standard whole-cell conditions as well as when recorded with the perforated patch technique. These results indicate that the two agents act differently on ion channel currents in B-cells although both are known to oxidize SH-groups.

Control of sorbitol metabolism in renal inner medulla of diabetic rats: regulation by substrate, cosubstrate and products of the aldose reductase reaction

Streptozotocin diabetes induces a 4-fold increase in the maximal velocity of inner medullary aldose reductase as determined in vitro but increases sorbital synthesis in intact inner medullary collecting duct (IMCD) cells only 1.3-fold [1]. In order to resolve this discrepancy we investigated the importance of intracellular factors in controlling the role of cellular sorbitol synthesis. These factors include glucose concentration, sorbitol concentration, the activity of the NADPH-regenerating pentose phosphate pathway, intracellular NADP and NADPH content, and intracellular reduced (GSH) and oxidized glutathione (GSSG). It was found that the apparent K m of cellular sorbitol production for glucose was identical in control and diabetic rats ( 56 ± 18 vs. 59 ± 14 mmol/l d-glucose ), whereas V max increased by 31% in diabetes. In inner medullary collecting duct cells of diabetic rats containing 146 ± 5 μmol sorbitol/g protein, sorbitol synthesis slightly lower (−15%), compared to cells which had been sorbitol-depleted prior to the experiment (87 ± 4 μmol sorbitol/g protein). However, no inhibitory effect of sorbitol (up to 200 mmol/l) was observed on aldose reductase activity in vitro. In diabetic rats the content of NADPH was about 32% lower than in the control rats (3.8 ± 0.3 vs. 5.6 ± 0.4 μmol/g protein) and the ratio of NADPH/NADP was decreased from 25.6 ± 5.1 to 8.6 ± 1.7. In homogenates of the inner medulla the activity of 6-phospho-gluconate dehydrogenase (EC 1.1.1.43) was identical in both experimental groups, so the pentose phosphate shunt seems to be unaltered. GSH content in diabetic rats was also diminished (4.2 ± 0.67 μmol/g protein vs. 7.41 ± 0.5 μmol/g protein) and the GSH/GSSG ratio fell from 92.6 to 57.4. In enzyme tests in vitro an apparent K m of 7.3 ± 1.9 μmol/l of the aldose reductase for NADPH was found; NADP acted as competitive inhibitor with a apparent K i of 183 ± 31 μmol/l. Aldose reductase activity was also found to be strongly inhibited by the SH-group reagent p-chloromercurybenzoesulfonate (apparent K i = 0.85 · 10 −6 mol/l). Combining the results obtained on the properties of the aldose reductase in vitro and the observation made in the intact cells, the investigators suggest that the decrease in NADPH/ NADP ratio, as well as changes in the redox state in the cells of diabetic animals, can play a significant role in the control of sorbitol synthesis.

Sequence analysis of the catalytic subunit of H +-ATPase from porcine renal brush-border membranes

The catalytic subunit of the H +-ATPase from brush-border membranes of porcine renal proximal tubules was labeled with the hydrophobic SH-group reagent 10- N-(bromoacetyl)amino-1-decyl- β-glycopyronoside) (BADG) which irreversibly inhibits proton pump activity in the absence but not in the presence of ATP. The labeled protein was purified and digested with proteinases. After isolation and sequencing of proteolytic peptides two BADG-labeled cysteines were identified. The amino acid sequences of the obtained proteolytic peptides were homologous to the catalytic subunit of V-ATPases. From mRNA of porcine kidney cortex a catalytic H +-ATPase subunit was cloned. 181 of 183 amino acids which overlap in the sequence derived from the cDNA and the proteolytic peptides were identical, and the two deviations are due to single base exchanges. A comparison of the amino acid sequence derived from the cloned cDNA with sequences of catalytic H +-ATPase subunits communicated by other laboratories revealed 98%, 96% and 94% identity with sequences from bovine adrenal medulla, from bovine kidney medulla and from clathrin-coated vesicles of bovine brain. Between 64% and 69% identity was obtained with sequences from fungi and plants. The data show that the catalytic subunit of V-ATPases is highly conserved during evolution. They indicate organ and species specificity in mammalians.

Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations.

We have studied the induction of the mitochondrial cyclosporin A-sensitive permeability transition pore (PTP) by the bifunctional SH group reagent phenylarsine oxide (PhAsO). Addition of nanomolar concentrations of the electroneutral H(+)-K+ ionophore nigericin to nonrespiring mitochondria in sucrose medium determines a dramatic increase of the time required for PTP induction by PhAsO, while no effect of nigericin is apparent in KCl medium. Using mitochondria loaded with the internal pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, we show that the effect of nigericin is mediated by the ionophore-induced acidification of matrix pH. Indeed, experimental manipulation of pHi by a number of treatments indicates that PTP induction is directly related to matrix pH, in that the PTP induction process becomes slower as pHi decreases at constant pHo. PTP induction by PhAsO in respiration-inhibited mitochondria is stimulated by Ca2+ and inhibited by a series of divalent cations. Since PhAsO induces the PTP even in the presence of excess EGTA and in the absence of respiration (Lenartowicz, E., Bernardi, P., and Azzone, G.F. (1991) J. Bioenerg. Biomembr. 23, 679-688), we have been able to study the Ca2+ dependence of the induction process. We show that the apparent Km for Ca2+ activation is about 10(-5) M and that Ca2+, cyclosporin A, and inhibitory Me2+ ions behave as if they were competing for the same binding site(s) on the pore. Since similar results are obtained from patch-clamp experiments on the mitochondrial megachannel (Szabó, I., Bernardi, P., and Zoratti, M. (1992) J. Biol. Chem. 267, 2940-2946), we suggest that (i) the PTP and the mitochondrial megachannel are the same molecular structures and (ii) the same factors affect both the process of pore induction and its open-closed orientation.

Phenylarsine oxide induces the cyclosporin A-sensitive membrane permeability transition in rat liver mitochondria

This paper reports an investigation on the effects of the hydrophobic, bifunctional SH group reagent phenylarsine oxide (PhAsO) on mitochondrial membrane permeability. We show that PhAsO is a potent inducer of the mitochondrial permeability transition in a process which is sensitive to both the oxygen radical scavanger BHT and to cyclosporin A. The PhAsO-induced permeability transition is stimulated by Ca2+ but takes place also in the presence of EGTA in a process that maintains its sensitivity to BHT and cyclosporin A. Our findings suggest that, at variance from other known inducers of the permeability transition, PhAsO reacts directly with functional SH groups that are inaccessible to hydrophilic reagents in the absence of Ca2+.

Partial Purification and Some Properties of Flavonol 7-Sulfotransferase from Flaveria bidentis

A novel flavonol-specific sulfotransferase was partially purified from the shoot tips of Flaveria bidentis var. Angustifolia O.K. (Asteraceae) by chromatography on 3'-phosphoadenosine 5'-phosphate-agarose affinity column and chromatofocusing on Mono P. The latter step resulted in the separation of two isoforms, both of which exhibited expressed specificity for position 7 of quercetin 3,3'- and quercetin 3,4'-disulfate. The 7-sulfotransferase isoforms I and II had a pH optimum of 7.5 in phosphate buffer, apparent pl values of 6.5 and 6.3, and an M(r) of 35,000. They had no requirement for divalent cations and were not inhibited by EDTA or SH group reagents. Their K(m) values for both the sulfate donor and flavonol acceptor were of the same order of magnitude (0.20-0.46 micromolar). This enzyme, together with the recently reported flavonol 3-, 3'-, and 4'-sulfotransferases from F. chloraefolia (L Varin, RK Ibrahim [1989] Plant Physiol 90: 977-981) form the complement involved in the biosynthesis of polysulfated flavonols in this genus. A proposed sequential order for the enzymatic sulfation in both species is described.

Significance and redox state of SH groups in pyruvate carrier isolated from bovine heart mitochondria

The role and properties of -SH groups of purified pyruvate (monocarboxylate) carrier were investigated. After isolation, this protein has all -SH groups in the oxidized state. Upon reduction, the carrier can be labelled with eosin-5-maleimide. The shift in apparent M r after the labelling points to the presence of at least two cysteine residues. Pyruvate uptake in the reconstituted system is inhibited by both permeable (eosin-5-maleimide at 1 mM concentration) and impermeable (mersalyl, p-chloromercuribenzoate) -SH group reagents. Phenylarsine oxide inhibits pyruvate transport only slightly (20%), but the inhibition is enhanced after preincubation with the substrate.