Modification of the Activity of the Plasma Membrane Redox System of Zea mays L. Roots by Vitamin K3 and Dicumarol

The correlation of the effects of vitamin K₃ and dicumarol (ananti-vitamin K in pharmaceutical applications) on the transplasmamembrane electrical potential difference of maize roots withthe reduction of the artificial electron acceptors hexacyanoferrate(III) or hexabromoiridate (IV) and the concomitant enhancementof acidification of the incubation medium was investigated. Vitamin K₃ depolarized the plasma membrane of Zea mays L. roots,while dicumarol had no significant effect on the membrane potential.Plants treated with vitamin K₃ for 30 min followed by intenserinsing showed higher reduction of hexabromoiridate (IV) thanhexacyanoferrate (III), as well as a stimulated acidificationof the incubation medium. Depolarization of the plasma membraneby hexacyanoferrate (III) or hexabromoiridate (IV) decreasedafter an incubation with vitamin K₃. Pretreatment with dicumarolcaused an inhibition of hexacyanoferrate (III) reduction andmedium acidification as well as depolarization by K₃. The reductionof hexabromoiridate (IV) was not affected by dicumarol pretreatment.The proton secretion associated with the reduction was slightlylowered. According to our results, it seems possible that vitaminK₃ acts as an electron acceptor for the plasmalemma electrontransport system of maize roots whereas dicumarol appears toinhibit electron and proton transport. Key words: Vitamin K3, dicumarol, plasmalemma redox system, Zea mays L., membrane potential     

Effects of the administration of anticoagulants on the activity of the enzyme-reduced NAD(P)H dehydrogenase in rat livers, hepatomas and precarcinomatous rat liver lesions

The anticoagulants dicumarol, warfarin and diphenadione, are in vitro inhibitors of the enzyme reduced NAD(P)H dehydrogenase of rat liver. These chemicals were administered by intragastric gavage to determine whether the same inhibitory effects could be observed in Sprague-Dawley male rats. Doses of 2 and 10 mg/100 g body weight were used. Our results indicate that only dicumarol inhibited the enzyme, whereas warfarin did not produce a significant effect, and diphenadione at large doses produced an increase in the activity of the enzyme. Dicumarol was further tested to see if it would alter the activity of the enzyme in hyperplastic nodules and liver hepatomas. A similar inhibitory effect was found. The three strains of rats tested in this work have different levels of reduced NAD(P)H dehydrogenase activity. Thus, our results indicate that dicumarol is the only anticoagulant that inhibits in vivo the reduced NAD(P)H dehydrogenase of rat liver and liver neoplasms.     

A microelectrochemical technique to measure trans‐plasma membrane electron transport in plant tissue and cells in vivo

A novel electrochemical technique was developed to enable high‐resolution measurements of trans‐plasma membrane reductase activity in vivo in growing plant tissue and single cells. Carbon fibre microelectrodes (CFMEs) with a tip diameter of 5 µm were used for electrochemical mapping of the reduction of the external impermeant electron acceptor ferricyanide along the root tip surface of 4‐d‐old maize seedlings. Ferricyanide reduction was detected in all locations along the first 12 mm of the growing root apex. However, a distinct peak in activity was detected at the proximal end of the elongation zone (1·5–4·5 mm from the apex), where reductase activity was three times greater than in more apical or distal regions. The inhibition of the ferricyanide reduction at all locations along the growing apex, by the vitamin K antagonists warfarin and dicumarol, supports previous data showing that electron transfer by the constitutive trans‐plasma membrane reductase is achieved via a quinone shuttle. We demonstrate that in addition to their utility in whole‐tissue/‐organ studies, CFMEs are sensitive enough to monitor trans‐plasma membrane electron transport in single cells.     

The Effects of Vitamin K3 and Dicumarol on the Plasma Membrane Redox System and H+ Pumping Activity of Zea mays L Roots Measured over a Long Time Scale

The effects of vitamin K₃ or dicumarol on plasma membrane boundhexacyanoferrate (III) and hexabromoiridate (IV) reductase activityand on the H⁺ pumping rate were investigated. Incubation withvitamin K₃ followed by intense rinsing stimulated the subsequentreduction of hexabromoiridate (IV) and hexacyanoferrate (III)as well as proton secretion induced by external electron-acceptors,while pretreatment with dicumarol inhibited proton secretioninduced by redox activity and hexacyanoferrate (III) reductionrate, but not the effects of hexabromoiridate (IV). A 30 minincubation in 0·2 mM K₃ or dicumarol, followed by rinsing,inhibited H⁺ secretion for about 2 d. Incubation for more than12 h in 0·1 mM dicumarol or 0·2 mM K₃ caused lethalinjury to the root cells. Key words: Vitamin K.₃, dicumarol, plasmalemma redox system, Zea mays L., proton pump     

Interaction between electron transport at the plasma membrane and nitrate uptake by maize (Zea mays L.) roots

Summary In the present study nitrate uptake by maize (Zea mays L.) roots was investigated in the presence or absence of ferricyanide (hexacyanoferrate III) or dicumarol. Nitrate uptake caused an alkalization of the medium. Nitrate uptake of intact maize seedlings was inhibited by ferricyanide while the effect of dicumarol was not very pronounced. Nitrite was not detected in the incubation medium, neither with dicumarol-treated nor with control plants after application of 100 M nitrate to the incubation solution. In a second set of experiments interactions between nitrate and ferricyanide were investigated in vivo and in vitro. Nitrate (1 or 3 mM) did neither influence ferricyanide reductase activity of intact maize roots nor NADH-ferricyanide oxidoreductase activity of isolated plasma membranes. Nitrate reductase activity of plasma-membrane-enriched fractions was slightly stimulated by 25 M dicumarol but was not altered by 100 M dicumarol, while NADH-ferricyanide oxidoreductase activity was inhibited in the presence of dicumarol. These data suggest that plasma-membrane-bound standard-ferricyanide reductase and nitrate reductase activities of maize roots may be different. A possible regulation of nitrate uptake by plasmalemma redox activity, as proposed by other groups, is discussed.Abbreviations ADH alcohol dehydrogenase - HCF III hexacyanoferrate III (ferricyanide) - ME NADP-dependent malic enzyme - NR nitrate reductase - PM plasma membrane - PM NR nitrate reductase copurifying with plasma membranes     

Inhibition of trans-membrane hexacyanoferrate III reductase activity and proton secretion of maize (Zea mays L.) roots by thenoyltrifluoroacetone

Summary Intact plants can reduce external oxidants by an appearingly trans-membrane electron transport. In vivo an increase in net medium acidification accompanies the reduction of the apoplastic substrate. Up to now, several NAD(P)H dehydrogenases,b-type cytochromes, and a phylloquinone have been identified and partially purified from plant plasma membranes. The occurrence of a quinone in the plasma membrane of maize roots supports the hypothetical model of a proton-transferring redox system, i.e., an electron transport chain with a quinone as mobile electron and proton carrier. In the present study the trans-membrane electron transport system of intact maize (Zea mays L.) roots was investigated. Flow-through and ionostat systems have been used to estimate the electron and proton transport activity of this material. Application of 4,4,4-trifluoro-1-(2-thienyl)-butane-1,3-dione (thenoyltrifluoroacetone) inhibited the reduction of ferricyanide in the incubation solution of intact maize roots up to 70%. This inhibition could not be washed off by rinsing the roots with fresh incubation medium. The acidification of the medium induced after ferricyanide application was inhibited to about 62%. The effects of thenoyltrifluoroacetone on proton fluxes in the absence of ferricyanide have been characterized in a pH-stat system. The net medium acidification by maize roots was inhibited up to 75% by thenoyltrifluoroacetone in the absence of ferricyanide, while dicumarol inhibited net acidification completely. The inhibition of H⁺-ATPase activity was estimated with plasma membrane vesicles isolated by phase partitioning and treated with 0.05% (w/v) Brij 58. ATP-dependent proton gradients and P_i release were measured after preincubation with the effectors. The proton pumping activity by those plasma membrane vesicles was inhibited by dicumarol (53.6%) and thenoyltrifluoroacetone (77.8%), while the release of P_i was unaffected by both inhibitors.Abbreviations Brij 58 polyoxyethylene 20-cetyl ether - duroquinone tetramethyl-p-benzoquinone - HCF III hexacyanoferrate III - TTFA thenoyltrifluoroacetone - vitamin K₁ 2-methyl-3-phytyl-1,4-naphthoquinone - vitamin K₃ 2-methyl-1,4-naphthoquinone     

Plasma membrane electron transport coupled to Na(+) extrusion in the halotolerant alga Dunaliella

The halotolerant alga Dunaliella adapts to exceptionally high salinity and maintains low [Na(+)](in) at hypersaline solutions, suggesting that it possesses efficient mechanisms for regulating intracellular Na(+). In this work we examined the possibility that Na(+) export in Dunaliella is linked to a plasma membrane electron transport (redox) system. Na(+) extrusion was induced in Dunaliella cells by elevation of intracellular Na(+) with Na(+)-specific ionophores. Elevation of intracellular Na(+) was found to enhance the reduction of an extracellular electron acceptor ferricyanide (FeCN). The quinone analogs NQNO and dicumarol inhibited FeCN reduction and led to accumulation of Na(+) by inhibition of Na(+) extrusion. These inhibitors also diminished the plasma membrane potential in Dunaliella. Anaerobic conditions elevated, whereas FeCN partially decreased intracellular Na(+) content. Cellular NAD(P)H level decreased upon enhancement of plasma membrane electron transport. These results are consistent with the operation of an electrogenic NAD(P)H-driven redox system coupled to Na(+) extrusion in Dunaliella plasma membrane. We propose that redox-driven Na(+) extrusion and recycling in Dunaliella evolved as means of adaptation to hypersaline environments.     

The development of cytochrome b-245 in maturing human macrophages.

Cytochrome b-245, the putative terminal component of the specialized cidal oxidase system of phagocytes, was measured in human monocytes in culture. There was a dramatic synthesis of the cytochrome, which increased by 27.3 +/- 2.0 pmol/day per 10(7) cells. This represents an increase of about 40%/day in the early stages and an overall 7-fold increase after 16 days. The protein content increased 3-fold over the same period, resulting in a doubling of the specific content of the cytochrome b. The newly synthesized cytochrome b was identified as that specifically located in the microbicidal oxidase electron-transport chain, as titration demonstrated that, at day 16 of maturation, 70% of the total membrane cytochrome b had a very low midpoint potential (-260 to -220 mV), characteristic of that found in this oxidase system. This cytochrome distributed with the plasma membrane on analytical subcellular fractionation, and a close relationship was observed between the maturation-induced increase in the concentration of this molecule and the capacity of the cells to produce superoxide.     

Redox reactions of tonoplast and plasma membranes isolated from soybean hypocotyls by free-flow electrophoresis

Redox reactions were studied in more than 90% pure tonoplast and plasma membranes isolated by free-flow electrophoresis from soybean (Glycine max) hypocotyls. Both types of membrane contained a b-type cytochrome (alpha max = 561 nm) and a noncovalently bound flavin, two possible components of a transmembrane electron-transport chain. Isolated tonoplast and plasma membranes reduced ferricyanide, indophenol and various iron complexes with NADH or NADPH as electron donors. The redox activity was inhibited in tonoplast membranes by about 60% by 10 microM p-chloromercuribenzene sulfonate, 8% by 500 microM lanthanum nitrate and 10% by 100 microM nitrophenyl acetate. In contrast, the redox activity of isolated plasma membranes was inhibited by about 60% by 500 microM lanthanum nitrate or 100 microM nitrophenyl acetate, but only 25% by 10 microM p-chloromercuribenzene sulfonate. The results show that both tonoplast and plasma membranes of soybean contain active electron-transport systems, but that the two systems respond differently to inhibitors.     

A novel plasma membrane quinone reductase and NAD(P)H:quinone oxidoreductase 1 are upregulated by serum withdrawal in human promyelocytic HL-60 cells

We have studied changes in plasma membrane NAD(P)H:quinone oxidoreductases of HL-60 cells under serum withdrawal conditions, as a model to analyze cell responses to oxidative stress. Highly enriched plasma membrane fractions were obtained from cell homogenates. A major part of NADH-quinone oxidoreductase in the plasma membrane was insensitive to micromolar concentrations of dicumarol, a specific inhibitor of the NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase), and only a minor portion was characterized as DT-diaphorase. An enzyme with properties of a cytochrome b ₅ reductase accounted for most dicumarol-resistant quinone reductase activity in HL-60 plasma membranes. The enzyme used mainly NADH as donor, it reduced coenzyme Q₀ through a one-electron mechanism with generation of superoxide, and its inhibition profile by p-hydroxymercuribenzoate was similar to that of authentic cytochrome b ₅ reductase. Both NQO1 and a novel dicumarol-insensitive quinone reductase that was not accounted by a cytochrome b ₅ reductase were significantly increased in plasma membranes after serum deprivation, showing a peak at 32 h of treatment. The reductase was specific for NADH, did not generate superoxide during quinone reduction, and was significantly resistant to p-hydroxymercuribenzoate. The function of this novel quinone reductase remains to be elucidated whereas dicumarol inhibition of NQO1 strongly potentiated growth arrest and decreased viability of HL-60 cells in the absence of serum. Our results demonstrate that upregulation of two-electron quinone reductases at the plasma membrane is a mechanism evoked by cells for defense against oxidative stress caused by serum withdrawal.     

Redox reactions of tonoplast and plasma membranes isolated from soybean hypocotyls by free-flow electrophoresis

Redox reactions were studied in more than 90% pure tonoplast and plasma membranes isolated by free-flow electrophoresis from soybean (Glycine max) hypocotyls. Both types of membrane contained a b-type cytochrome (_max = 561 nm) and a noncovalently bound flavin, two possible components of a transmembrane electron-transport chain. Isolated tonoplast and plasma membranes reduced ferricyanide, indophenol and various iron complexes with NADH or NADPH as electron donors. The redox activity was inhibited in tonoplast membranes by about 60% by 10 μM p-chloromercuribenzene sulfonate, 8% by 500 μM lanthanum nitrate and 10% by 100 μM nitrophenyl acetate. In contrast, the redox activity of isolated plasma membranes was inhibited by about 60% by 500 μM lanthanum nitrate or 100 μM nitrophenyl acetate, but only 25% by 10 μM p-chloromercuribenzene sulfonate. The results show that both tonoplast and plasma membranes of soybean contain active electron-transport systems, but that the two systems respond differently to inhibitors.     

The influence of spinach thylakoid lumen volume and membrane proximity on the rotational motion of the spin label Tempamine

Phosphorylating electron-transport reactions, using either Photosystem I or Photosystem II, were found to be competent in driving the light-induced increase in rotational hindrance of the spin label Tempamine. The uncoupler gramicidin prevented the change induced by either photosystem while the electron-transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) only prevented the light-induced change associated with Photosystem II. The internal lumen microviscosity is termed ‘η’ and was found to be inversely proportional to the lumen volume. Substances which should compete for Tempamine-binding sites on the thylakoid membrane were found to be largely ineffectual in changing η. These results further support the hypothesis that the magnitude of η is directly related to the proximity of Tempamine to a membrane surface.     

Spin-labeled stearates as probes for microenvironment of murine thymocyte adenylate cyclase-cyclic adenosine 3':5'-monophosphate system.

The interaction of various spin-labeled compounds with the murine thymocyte adenylate cyclase-cyclic AMP system was investigated. Electron paramagnetic resonance spectra from spin-labeled compounds were used to calculate the order parameter, S, and indicated that the thymocyte plasma membrane is a relatively rigid structure. Increasing concentrations of spin-labeled stearates, but not their corresponding methyl esters, resulted in increased membrane fluidity, partial lysis, and concomitant complete inhibition of cholera toxin-mediated increases in cyclic AMP content. Upon subsequent isolation of plasma membranes from these cells, cholera toxin-stimulated adenylate cyclase activity was also completely inhibited. Direct addition of spin-labeled stearates, but not spin-labeled methyl stearates, to thymocyte homogenates caused a dramatic reduction of basal, cholera toxin-, isoproterenol-, NaF-, and prostaglandin E1-stimulated adenylate cyclase activity. Inhibition was complete within the first minute of addition to homogenates and required approximately 0.2 mM spin-labeled stearate I(12,3) for half-maximal inhibition. This inhibition occurred in the presence or absence of an ATP-regenerating system and was not readily reversible. Furthermore, since the membrane cyclic phosphodiesterase activity was not altered by spin-labeled stearates, their inhibition was attributed to a direct action of stearate spin labels on adenylate cyclase. Neither stearate, methyl stearate, spin-labeled methyl stearates nor 2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempo) altered cell viability or enzyme activities at the concentrations studied. Spin-labeled stearates seemed to intercalate into different areas of the plasma membrane than their corresponding methyl esters. Furthermore, the action of spin-labeled stearates appeared to be on the exterior of the plasma membrane rather than the interior. These results illustrate the presence of multilipid domains and the importance of selected lipids and lipid-protein interactions in the adenylate cyclase-cyclic AMP system. Thymocyte adenylate cyclase is described in terms of a current model for membrane proteins.     

Electron transport components of the MnO2 reductase system and the location of the terminal reductase in a marine Bacillus.

The response of MnO2 reduction by uninduced and induced whole cells and cell extracts of Bacillus 29 to several electron transport inhibitors was compared. MnO2 reduction with glucose by uninduced whole cells and cell extracts was strongly inhibited at 0.1 mM dicumarol, 100 mM azide, and 8 mM cyanide but not by atebrine or carbon monoxide, suggesting the involvement of a vitamin K--type quinone and a metalloenzyme in the electron transport chain. MnO2 reduction with ferrocyanide by uninduced cell extracts was inhibited by 5 mM cyanide and 100 mM azide but not by atebrine, dicumarol, or carbon monoxide, suggesting that the metalloenzyme was associated with the terminal oxidase activity. MnO2 reduction with glucose by induced whole cells and cell extracts, was inhibited by 1 mM atebrine, 0.1 mM dicumarol, and 10 mM cyanide but not by antimycin A, 2n-nonyl-4-hydroxyguinoline-N-oxide) (NOQNO), 4,4,4-trifluoro-1-(2-thienyl),1,3-butanedione, or carbon monoxide. Induced cell extract was also inhibited by 100 mM azide, but stimulated by 1 mM and 10 mM azide. Induced whole cells were stimulated by 10 mM and 100 mM azide. These results suggested that electron transport from glucose to MnO2 in induced cells involved such components as flavoprotein, a vitamin K-type quinone, and metalloenzyme. The stimulatory effect of azide on induced cells was explained on the basis of a branching in the terminal part of the electron transport chain, one branch involving a metalloenzyme for the reduction of MnO2 and the other involving a metalloenzyme for the reduction of oxygen. The latter was assumed to be the more azide sensitive. Spectral studies showed the presence of a-, b-, and c-type cytochromes in membrane but not in soluble fractions. Of these cytochromes, only the c type may be involved in electron transport of MnO2, owing to the lack of inhibition by antimycin A or 2n-nonyl-4-hydroxyquinoline-N-oxide. The terminal MnO2 reductase appears to be loosely attached to the cell membrane of Bacillus 29 because of cell fractionation it is found associated with both particulate and soluble fractions. Electron photomicrographs of bacilli attached to synthetic Fe-Mn oxide revealed an intimate contact of the cell walls with the oxide particles.     

Electron transport components of the MnO2 reductase system and the location of the terminal reductase in a marine Bacillus.

The response of MnO2 reduction by uninduced and induced whole cells and cell extracts of Bacillus 29 to several electron transport inhibitors was compared. MnO2 reduction with glucose by uninduced whole cells and cell extracts was strongly inhibited at 0.1 mM dicumarol, 100 mM azide, and 8 mM cyanide but not by atebrine or carbon monoxide, suggesting the involvement of a vitamin K--type quinone and a metalloenzyme in the electron transport chain. MnO2 reduction with ferrocyanide by uninduced cell extracts was inhibited by 5 mM cyanide and 100 mM azide but not by atebrine, dicumarol, or carbon monoxide, suggesting that the metalloenzyme was associated with the terminal oxidase activity. MnO2 reduction with glucose by induced whole cells and cell extracts, was inhibited by 1 mM atebrine, 0.1 mM dicumarol, and 10 mM cyanide but not by antimycin A, 2n-nonyl-4-hydroxyguinoline-N-oxide) (NOQNO), 4,4,4-trifluoro-1-(2-thienyl),1,3-butanedione, or carbon monoxide. Induced cell extract was also inhibited by 100 mM azide, but stimulated by 1 mM and 10 mM azide. Induced whole cells were stimulated by 10 mM and 100 mM azide. These results suggested that electron transport from glucose to MnO2 in induced cells involved such components as flavoprotein, a vitamin K-type quinone, and metalloenzyme. The stimulatory effect of azide on induced cells was explained on the basis of a branching in the terminal part of the electron transport chain, one branch involving a metalloenzyme for the reduction of MnO2 and the other involving a metalloenzyme for the reduction of oxygen. The latter was assumed to be the more azide sensitive. Spectral studies showed the presence of a-, b-, and c-type cytochromes in membrane but not in soluble fractions. Of these cytochromes, only the c type may be involved in electron transport of MnO2, owing to the lack of inhibition by antimycin A or 2n-nonyl-4-hydroxyquinoline-N-oxide. The terminal MnO2 reductase appears to be loosely attached to the cell membrane of Bacillus 29 because of cell fractionation it is found associated with both particulate and soluble fractions. Electron photomicrographs of bacilli attached to synthetic Fe-Mn oxide revealed an intimate contact of the cell walls with the oxide particles.     

Generation of action potentials in a mathematical model of corticotrophs.

Corticotropin-releasing hormone (CRH) is an important regulator of adrenocorticotropin (ACTH) secretion from pituitary corticotroph cells. The intracellular signaling system that underlies this process involves modulation of voltage-sensitive Ca2+ channel activity, which leads to the generation of Ca2+ action potentials and influx of Ca2+. However, the mechanisms by which Ca2+ channel activity is modulated in corticotrophs are not currently known. We investigated this process in a Hodgkin-Huxley-type mathematical model of corticotroph plasma membrane electrical responses. We found that an increase in the L-type Ca2+ current was sufficient to generate action potentials from a previously resting state of the model. The increase in the L-type current could be elicited by either a shift in the voltage dependence of the current toward more negative potentials, or by an increase in the conductance of the current. Although either of these mechanisms is potentially responsible for the generation of action potentials, previous experimental evidence favors the former mechanism, with the magnitude of the shift required being consistent with the experimental findings. The model also shows that the T-type Ca2+ current plays a role in setting the excitability of the plasma membrane, but does not appear to contribute in a dynamic manner to action potential generation. Inhibition of a K+ conductance that is active at rest also affects the excitability of the plasma membrane.     

The stimulatory effects of carbon tetrachloride on peroxidative reactions in rat liver fractions in vitro. Interaction sites in the endoplasmic reticulum

1. The actions of various inhibitors of the microsomal NADPH-cytochrome P-450 electron-transport chain have been studied on the stimulatory effect of carbon tetrachloride on malonaldehyde production. 2. Carbon monoxide, p-chloromercuribenzoate, beta-diethylaminoethyl-3,3'-diphenylpropyl acetate (SKF 525A) and nicotinamide did not decrease the stimulatory action of carbon tetrachloride on malonaldehyde production when present in concentrations shown to be capable of strongly inhibiting the demethylation of aminopyrine. 3. In contrast with the effects of the substances mentioned above, low concentrations of cytochrome c strongly depressed the stimulatory action of carbon tetrachloride on malonaldehyde production while increasing the endogenous rate of peroxidation. 4. Aging the microsomal suspensions at 0 degrees C caused a rapid decrease in aminopyrine demethylation activity and in lipid peroxidation catalysed by ADP and Fe(2+). The stimulation of malonaldehyde production by carbon tetrachloride was relatively unaffected, however, by aging the microsomes at 0 degrees C for 3 days; during this period cytochrome P-450 decreased by more than 30%. 5. The conclusion is reached that the interaction between carbon tetrachloride and the NADPH-cytochrome P-450 electron-transport chain necessary for the stimulation of malonaldehyde production involves a locus near to if not identical with the NADPH-cytochrome c reductase flavoprotein.     

Ion fluxes changes during early stages of Schistosoma mansoni. Evaluation of complement effect

The relationship between the average membrane potential (delta psi av) and sensitivity to complement action of the Schistosoma mansoni parasite was explored. The average membrane potential was estimated by measuring the uptake of [3H]tetraphenyl phosphonium ([3H]Ph4P+). The parasites take up Ph4P+ indicating the existence of a negative internal plasma potential which is in part dependent on the transmembrane K+ gradient, maintained by an active Na+/K+-ATPase. Values for Ph4P+ uptake could be corrected for mitochondrial accumulation by employing the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which collapses the mitochondrial potential. The plasma membrane potential derived by this technique was in the range of -60 mV. Transformation of this parasite, from its early cercaria stage to the adult worm, was associated with changes in the average membrane potential. The apparent hyperpolarization, which accompanies transformation, may be related to changes in ionic permeability and morphology which occur concomitantly. Complement acting through both the classical and alternative pathways was found to affect the potential of the parasite in its early development stages. The correlation between effects on delta psi av and sensitivity to complement action, indicates that the complement-induced changes in delta psi av are indeed tightly associated with its mode of action. Treatment of the parasite with complement resulted in net hyperpolarization of the membrane indicating that hyperpolarization rather than depolarization of the membrane is linked to the primary non-lethal action of complement.     

Direct quantitative determinations by multiple metastable peak monitoring. 1--Warfarin in plasma

A technique for the direct quantitative determination of plasma warfarin concentrations has been developed using multiple metastable peak monitoring on a double focusing mass spectrometer of normal geometry. The method uses a direct insertion probe and requires no sample purification or derivatization. Deuterated warfarin was added as an internal standard, and the first field free region metastable fragmentations for the loss of an acetyl radical from the molecular ion of both warfarin and internal standard were monitored. These independent daughter ions were focused by a simple combination of linking the magnetic and electric sectors, accelerating voltage switching and selected ion monitoring using standard instrumentation. Results were acquired and processed by normal selected ion monitoring software. Repeated determinations of plasma warfarin concentrations were made with plasma extracts representing approximately 4 microliter of plasma, each sample taking two minutes to process by this technique.     

Stimulus-response coupling in a cell-free platelet membrane system. GTP-dependent release of Ca2+ by thrombin, and inhibition by pertussis toxin and a monoclonal antibody that blocks calcium release by IP3

The Ca2+-mobilizing action of thrombin was demonstrated in a cell-free platelet membrane system consisting of open sheets of plasma membrane plus sealed membrane vesicles that accumulate Ca2+ and release Ca2+ in response to IP3. Thrombin plus GTP, acting on plasma membrane (not vesicles), produced a soluble factor (destroyed by alkaline phosphatase) that released Ca2+ from the vesicles. This effect of thrombin/GTP was blocked by a monoclonal antibody that binds to vesicles and prevents Ca2+ release by IP3. Pertussis toxin plus NAD ADP-ribosylated plasma membrane polypeptides of 39 and 41 kDa and blocked Ca2+ release by thrombin/GTP, but not by IP3.