Myrosinase in Brassicaceae: I. LOCALIZATION OF MYROSINASE IN CELL FRACTIONS OF ROOTS OF Sinapis alba L.

Myrosinases (thioglucoside glucohydrolases E.C. 3.2.3.1 [EC] .), whichcatalyse the hydrolysis of glucosinolates present in Brassicaceae,were isolated from Sinapis alba L. seeds. The crude enzyme extractwas purified using gel and ion-exchange chromatography, isoelectricfocusing, and polyacrylamide gel electrophoresis. The separationof two myrosinase isoenzymes was obtained after gel chromatographyon Sephadex G-100. Further purification of the main myrosinasecomponents was achieved when the combined isoenzymes were separatedon the anion-exchanger DEAE-Sephadex A-50 followed by polyacrylamidegel electrophoresis. A similar purification was obtained when the crude extract wasgroup-fractionated on Sephadex G-50 followed by DEAE-cellulosechromatography on Whatman DE-52 and gel chromatography on SephadexG-200. The enzyme from the last step was further separated byisolectric focusing into two isoenzymes with isoelectric points4.9 and 6.2. In order to clarify where the myrosinase was localized in theroot tip cells, cell fractionation studies were performed usingaldehydes as pre-fixatives to stabilize the enzymes and thecell organelles. Biochemical tests of crude and purified samplesof the isolated myrosinases showed that when glutaraldehydeor formaldehyde were used as pre-fixatives at a final concentrationof 1% (w/v), they did not inhibit the enzyme activity. Relativelyhomogeneous cell organelle fractions were obtained using ultracentrifugationand stepwise sucrose gradients. The myrosinase activity expressedon the basis of the protein content was found to be highestin the dictyosome and smooth endoplasmic reticulum fractions     

Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties

Myrosinases (EC 3.2.1.147) are β-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1–TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS–PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation–inhibition responses towards ascorbic acid with maximal activity around 0.7–1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-β-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.     

Purification and Characterisation of the Light and Dark Forms of Phosphoenolpyruvate Carboxylase from the Dicot Plant Amaranthus viridis L. An Examination of Its Kinetic and Regulatory Properties in the Presence of Water-Alcohol Binary Solvents

The light and dark forms of phosphoenolpyruvate (PEP) carboxylase(PEPC) from the dicot plant Amaranthus viridis L. were purifiedand their kinetic properties were studied in water-based orbinary alcohol-water solvents. At pH 7.3, the specific activityof the purified light form was about 2.7-fold higher than thatpresented by the dark form of PEPC under optimal conditions,while K_m remained virtually unchanged in both forms. The enzyme'slight form was better activated by glucose 6-phosphate and lessinhibited by L-malate than the dark PEPC. From the organic solventsstudied, methanol showed the most important effect, enhancingPEPC activity by two-fold at 20% (v/v). Ethanol, ethylene glycol,tert-butanol and 2-propanol were also activators to a lesserdegree, but at high concentrations (typically greater than 20%,v/v) the effect was reduced or turned to inhibition. K_m (PEP)was reduced by an order of magnitude in the presence of 20%(v/v) methanol (i.e. from 0.32 to 0.022 mM for the light formof the enzyme). The inhibitory effect of malate at low PEP waslessened by methanol for both forms (i.e. I₅₀ 0.25 mM in aqueousmedium to 0.48 mM in binary mixture for the dark form), whileglucose-6-P activation of PEPC was not affected by methanol.The results suggest that the kinetics of PEPC in a medium thatmimics more closely in vivo conditions are different from thoseobserved by standard procedures consisting of aqueous media,and provide a new insight on the properties of PEPC as relatedto its regulation in vivo. (Received June 26, 1995; Accepted August 24, 1995)     

Light-induced Changes in Expression of Pathogenesis-related Anionic Peroxidase in Cucumber Seedlings

The effect of irradiance on the expression of the Cucumis sativus pathogenesis-related (srPRX) anionic peroxidase was studied in germinating seeds at the period when seedlings start to be photosynthetically active. The diversity in the expression patterns of srPRX was observed in both dark- and light-grown seedlings using activity staining and immunoblotting: beside the three srPRX isoenzymes also other three, serologically unrelated, peroxidase isoforms were accumulated in dark-grown seedlings and one in light-grown seedlings. Furthermore, in light-grown seedlings, it was observed a marked difference in the expression of particular srPRX isoenzymes at higher irradiance (up to 260 W m-2, 400 - 700 nm) in comparison to low irradiance. By tissue printing on nitrocellulose paper it was demonstrated that irradiance during germination induced changes in the temporal and spatial distribution of the srPRX.     

Characterization of a Brassica napus myrosinase expressed and secreted by Pichia pastoris

In Brassica napus three different gene families with different temporal and tissue-specific expression and distribution patterns encode myrosinases (thioglucoside glucohydrolases, EC 3.2.3.1). Myrosinases encoded by the MA gene family are found as free and soluble dimers, while myrosinases encoded by the MB and MC gene families are mainly found in large insoluble complexes associated with myrosinase-binding proteins and myrosinase-associated proteins. These large complexes impede purification and characterization of MB and MC myrosinases from the plant. We used Pichia pastoris to express and secrete functional recombinant MYR1 myrosinase from B. napus to allow further characterization of myrosinase belonging to the MB gene family. The purified recombinant myrosinase hydrolyzes sinigrin with a K(m) of 1.0 mM; the specific activity and calculated k(cat)/K(m) were 175 U/mg and 1.9 x 10(5) s(-1) M(-1), respectively. A novel in-gel staining method for myrosinase activity is presented.     

Growth responses of sunflower hypocotyl disks inoculated with Escherichia coli and Agrobacterium tumefaciens I. Morphological observations

Disks of sunflower hypocotyls 1 mm thick grown in light anddarkness on agar containing mineral salts and sucrose to whichIAA was added in varying concentrations, were inoculated withE. coli, A. tumefaciens or sterile synthetic medium. Light-growndisks inoculated with E. coli proliferated from the lower surfaceand formed numerous long roots but dark-grown disks were usuallyinhibited in comparison with uninfected disks. Inoculation withA. tumefaciens induced proliferation mainly from the upper surfaceand a few short roots were formed. Uninfected disks grown with0.01 ppm IAA proliferated in a manner similar to that of E.coli-infected light-grown disks on simple medium but in thedark similar treatments produced quite different morphologicaleffects. The form of proliferation induced by E. coli underall conditions of growth could not be equated with that inducedby A. tumefaciens or by different concentrations of IAA. (Received December 5, 1967; )     

Myrosinase in Brassicaceae (Cruciferae): II. MYROSINASE ACTIVITY IN DIFFERENT ORGANS OF SINAPIS ALBA L.

Seeds of Sinapis alba L. were germinated in darkness for 3 dand a part of the etiolated seedlings were transferred to long-dayconditions for 6 weeks. Myrosinase solutions were prepared forcotyledons, hypocotyls, primary roots, leaves, stems, inflorescences,and seeds, and used to hydrolyse sinigrin. Glucose, one of the cleavage products, was determined by fourdifferent spectrophotometric methods, and their usefulness forcalculations of myrosinase activity in crude plant extractsis considered. Specific activity was calculated in relations to protein, andit was found to be about 30% higher in seedlings and also higherin seeds than in adult plants. Of the organs, those with thehighest activity were the hypocotyls and the stems. The differentparts of the plant contained different numbers of isoenzymes,as shown by polyacrylamide gel electrophoresis. Seedlings yieldedtwo, and adult plants four or five isoenzymes.     

In vitro inhibition of alkaline phosphatase activities from intestine, bone, liver, and kidney by phenobarbital.

A kinetic study of the inhibition of several alkaline phosphatase (AP isoenzyme activities by phenobarbital was carried out using p-nitrophenylphosphate (10 mM) as a substrate at pH 9.8 in a 300-mM Hepes buffer. AP from bovine kidney, calf intestine, bovine liver, and rat bone was used. Over a phenobarbital concentration range of 20-400 mM, all these isoenzymes were inhibited in an uncompetitive manner with a Ki of 200 mM for intestinal AP, and in a linear mixed-type manner for all the other isoenzymes tested. The Ki values were 10, 40 and 55 mM for kidney, bone and liver AP, respectively. The use of 15 mM carbonate-bicarbonate or 400 mM diethanolamine buffer did not modify the degree of inhibition of intestinal AP activity. Dixon plots of the reciprocal of reaction velocity versus inhibitor concentration either at different substrate concentration or at different DEA concentration indicate uncompetitive inhibition for the intestinal enzyme. This in vitro inhibitory effect of phenobarbital is in contrast to its in vivo stimulating action on AP. However, in the whole animal, the effects of phenobarbital administration probably represent the sum of multiple effects.     

Regulated Expression of a Gene-Fusion Product Derived from the Gene for Phytochrome I from Pisum sativum and the uidA Gene from E. coli in Transgenic Petunia hybrida

The 5'-upstream region (2.4 kb) of the gene for phytochromeI from Pisum sativum (phyl) was fused to the uidA gene fromEscherichia coli that encodes ß-glucuronidase (GUS).The resulting PHY-GUS fusion was introduced into Petunia hybridaand was used as a reporter of the expression of the phyI genewhich was recognized by GUS activity. The PHY-GUS fusion wasexpressed at a relatively high level when transgenic plantswere grown in the dark, while leaves and stems of light-grownplants showed background activity. Flowers of light-grown plantswere shown to have significant levels of GUS activity but rootsdid not have such activity. When light-grown transgenic plantswere transferred to the dark, they expressed the activity atlevels that corresponded to those of dark-grown plants. Lighttreatment prior to growth in darkness revealed red/far-red reversibilityof recovery of the activity. Thus, the 2.4-kb fragment fromthe 5' region of the phyI gene carries the information necessaryfor the light-repressible autoregulation. (Received March 30, 1991; Accepted May 20, 1991)     

Effects of fatty acids on BK channels in GH(3) cells

Ca²⁺-activated K⁺ (BK) channels inGH₃ cells are activated by arachidonic acid (AA). Becausecytosolic phospholipase A₂ can produce other unsaturatedfree fatty acids (FFA), we examined the effects of FFA on BK channelsin excised patches. Control recordings were made at several holdingpotentials. The desired FFA was added to the bath solution, and thevoltage paradigm was repeated. AA increased the activity of BK channelsby 3.6 ± 1.6-fold. The cis FFA, palmitoleic, oleic,linoleic, linolenic, eicosapentaenoic, and the triple bond analog ofAA, eicosatetraynoic acid, all increased BK channel activity, whereasstearic (saturated) or the trans isomers elaidic,linolelaidic, and linolenelaidic had no effect. The cisunsaturated FFA shifted the open probability vs. voltage relationshipsto the left without a change in slope, suggesting no change in thesensitivity of the voltage sensor. Measurements of membrane fluidityshowed no correlation between the change of membrane fluidity and thechange in BK channel activation. In addition, AA effects on BK channelswere unaffected in the presence of N-acetylcysteine.Arachidonyl-CoA, a membrane impermeable analog of AA, activateschannels when applied to the cytosolic surface of excised patches,suggesting an effect of FFAs from the cytosolic surface of BK channels.Our data imply a direct interaction between cis FFA and theBK channel protein.

     

Genotoxicity of acrylamide and glycidamide in human lymphoblastoid TK6 cells

The recent finding that acrylamide (AA), a potent carcinogen, is formed in foods during cooking raises human health concerns. In the present study, we investigated the genotoxicity of AA and its metabolite glycidamide (GA) in human lymphoblastoid TK6 cells examining three endpoints: DNA damage (comet assay), clastogenesis (micronucleus test) and gene mutation (thymidine kinase (TK) assay). In a 4 h treatment without metabolic activation, AA was mildly genotoxic in the micronucleus and TK assays at high concentrations (> 10 mM), whereas GA was significantly and concentration-dependently genotoxic at all endpoints at > or = 0.5 mM. Molecular analysis of the TK mutants revealed that AA predominantly induced loss of heterozygosity (LOH) mutation like spontaneous one while GA-induced primarily point mutations. These results indicate that the genotoxic characteristics of AA and GA were distinctly different: AA was clastogenic and GA was mutagenic. The cytotoxicity and genotoxicity of AA were not enhanced by metabolic activation (rat liver S9), implying that the rat liver S9 did not activate AA. We discuss the in vitro and in vivo genotoxicity of AA and GA.     

Phytochrome Control of Turion Formation in Spirodela polyrhiza L. Schleiden

Turion yield in Spirodela polyrhiza, strain SJ, is increasedby increasing the daily light period. This effect is more pronouncedin autotrophic than in mixotrophic conditions. Night-break irradiation(15 mins) increased turion yield by 150 % under the conditionsof an 8-h daily light period. Besides the effect of night-breakirradiation, end-of-day far-red irradiation decreased turionyield with increasing photoperiod, whereas end-of-day red irradiationwas without any effect. This demonstrates the promoting effectof the P_fr form of phytochrome on formation of light-grown turions. Formation of dark-grown turions was increased by about 240%by a single red light pulse and was reversed by an immediatelyapplied far-red light pulse. Consequently, under heterotrophicconditions phytochrome modulates the turion formation process. Spirodela polyrhiza L. Schleiden, duckweed, Lemnaceae, photomorphogenesis, phytochrome, turion     

Purification, biochemical properties and active sites of N-acetyl-beta-D-hexosaminidases from human seminal plasma.

Two isoenzymes of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) (Hex A and Hex B) from human seminal plasma were purified to homogeneity with specific activities of 26 and 60 units/mg of protein respectively. N-Acetyl-beta-D-glucosaminidase activity was inseparable from N-acetyl-beta-D-galactosaminidase activity in both Hex A and Hex B by various conventional chromatographic procedures. Although Km values of N-acetyl-beta-glucosaminidase activity of Hex A and Hex B were similar (1.33 mM), those of N-acetyl-beta-galactosaminidase activity were 0.14 mM for Hex A and 0.40 mM for Hex B. However, pH optima and temperature optima were identical for N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities of both isoenzymes; Hex A was far more heat-sensitive than Hex B. Thiol-reactive compounds such as silver salts, mercuric salts, p-chloromercuribenzoate and thimerosal strongly inhibited the N-acetyl-beta-glucosaminidase activities of both isoenzymes. GSH protected the enzyme activities from inactivation caused by these reagents, confirming the presence of thiol groups at the active centres. Inhibitions of N-acetyl-beta-glucosaminidase activities of both isoenzymes by metal salts and organic anions were comparable; acetate and arsenite were effective inhibitors for both isoenzymes. In contrast, inhibitions of N-acetyl-beta-glucosaminidase activities of the two isoenzymes by iodoacetic acid, iodoacetamide and ethylmaleimide were not comparable; Hex B was more susceptible to inhibition by these agents at 20 mM concentration. The N-acetyl-beta-glucosaminidase activities of both isoenzymes are strongly inhibited, in decreasing order, by N-acetyl-galactosamine, mannosamine, disaccharic acid lactone, N-acetylglucosamine and gluconolactone. The Ki values of the N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities for N-acetylhexosamines and results from mixed-substrate kinetics indicated that the activities for the two substrates are located at different sites in Hex A and at the same site in Hex B. The Mr values of Hex A and Hex B were determined to be 195,000 and 210,000 respectively by gel filtration through Sephadex G-200. SDS/polyacrylamide-gel electrophoresis revealed that Hex A and Hex B are each composed of four subunits corresponding to Mr about 50,000 each. No further polypeptide chain was obtained after reduction and alkylation of Hex A and Hex B with 10 mM-dithiothreitol and 10 mM-iodoacetamide.     

Salt Tolerance in Suaeda maritima (L.) Dum: THE EFFECT OF SODIUM CHLORIDE ON GROWTH, RESPIRATION, AND SOLUBLE ENZYMES IN A COMPARATIVE STUDY WITH PISUM SATIVUM L

The effect of sodium, chloride on the growth of a halophyte,Suaeda maritima (L.) Dum., was compared with its effect on Pisumsativum L. cv. Alaska under controlled environmental conditions.The salt stimulated the growth of Suaeda maximally at concentrationsof 170 to 340 mM while the growth of Pisum was inhibited evenby 100 mM. Both species accumulated ions in the tops and themaximum concentrations of Na⁺ and Cl^– rose in Suaeda to860 mM (based on the water content) and 730 mM and in Pisumto 170 mM and 300 mM respectively. Respiration in both specieswas inhibited as the NaCl level in the culture solution wasraised. Four supernatant enzymes (malic dehydrogenase, glucose-6-phosphatedehydrogenase, peroxidase, and acid phosphatase) prepared fromPisum and from Suaeda (grown either in the absence of addedNaCl or in the presence of 340 mM NaCl) were assayed in variouslevels of sodium chloride. The dehydrogenases were markedlyinhibited by increasing salt concentrations while there wasa smaller effect on the peroxidase and acid phosphatase. Therewas no difference in the effect of salt on the enzymes preparedfrom the two species although one is halophilic and the otherhalophobic.     

Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C.

The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.     

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel

We usedsingle-channel recording techniques to identify and characterize alarge-conductance,Ca²⁺-independentK⁺ channel in the colonicsecretory cell line T84. In symmetric potassium gluconate, this channelhad a linear current-voltage relationship with a single-channelconductance of 161 pS. Channel open probability(P_o) wasincreased at depolarizing potentials. Partial substitution of bathK⁺ withNa⁺ indicated a permeability ratioof K⁺ toNa⁺ of 25:1. ChannelP_o was reduced byextracellular Ba²⁺. Event-durationanalysis suggested a linear kinetic model for channel gating having asingle open state and three closed states: C₃C₂C₁O.Arachidonic acid (AA) increased theP_o of thechannel, with an apparent stimulatory constant(K_s)of 1.39 µM. Neither channel open time (O) nor the fast closed time(C₁) was affected by AA. Incontrast, AA dramatically reduced mean closed time by decreasing bothC₃ andC₂. Thecis-unsaturated fatty acid linoleate increased P_oalso, whereas the saturated fatty acid myristate and thetrans-unsaturated fatty acid elaidatedid not affectP_o. This channelis activated also by negative pressure applied to the pipette duringinside-out recording. Thus we determined the effect of thestretch-activated channel blockers amiloride and Gd³⁺ on theK⁺ channel after activation by AA.Amiloride (2 mM) on the extracellular side reduced single-channelamplitude in a voltage-dependent manner, whereasGd³⁺ (100 µM) had no effect onchannel activity. Activation of this K⁺ channel may be important duringstimulation of Cl secretionby agonists that use AA as a second messenger (e.g., vasoactiveintestinal polypeptide, adenosine) or during the volume regulatoryresponse to cell swelling.

     

A novel approach for reliable activity determination of ascorbic acid depending myrosinases

Up to now, a wide array of methods for the determination of myrosinase activity has been described. These vary from the simple photometric estimation to highly sophisticated assays using radioactively labelled substrates. However, ascorbic acid--an effective activator of myrosinases--interferes with most of these enzyme tests. Unfortunately, in the past, such interferences were disregarded in many scientific examinations of myrosinases. Whereas such failings have less effects when the activation of myrosinases is not very distinctive, they are quite relevant in all cases where myrosinases are completely inactive in the absence of ascorbic acid. In this paper, the current methods for myrosinase determination are reviewed critically with special emphasis on putative interferences with ascorbic acid. Thereafter, an alternative and interference-free HPLC-based quantification method of the enzymatically produced glucose is presented. Due to the benzoylation of glucose, it becomes possible to quantify even those exiguous glucose concentrations, which are indispensable for correct determination of kinetic enzyme data in the presence of ascorbic acid. Using this new method, the activity of Tropaeolum majus myrosinase towards glucotropaeolin was analyzed. This enzyme shows a distinctive activation by ascorbic acid with maximal activation at a concentration of about 2 mM.     

Alleviation of Seed Dormancy in the Desert ForbZygophyllum simplexL. from Pakistan

Zygophyllum simplexL. is a succulent annual that grows on thecoastal and inland saline flats around Karachi, Pakistan. Theseeds are moderately salt tolerant during germination. GerminationofZygophyllum simplexseeds under various salinity, proline,betaine, GA and kinetin treatments was determined. Proline (0.1and 1 mM) and betaine (0.1 and 1 mM) alleviated the innate dormancyof seeds, and germination reached 60–70% compared to 12%in the control set. At low salinity compatible osmotica alleviatedsome effects of salinity, but at higher NaCl concentrationsboth proline and betaine were ineffective. Gibberellic acid(0.3 and 3 mM) and kinetin (0.05 and 0.5 mM) substantially alleviatedboth innate as well as salinity-induced seed dormancy. At highersalinity (125 mM), low concentrations of kinetin (0.05 mM) andhigh concentrations of GA (3 mM) were more effective. GA completelyalleviated the effect of salinity at all concentrations used. Betaine; desert; dormancy; forb; GA; germination; halophyte; kinetin; proline; seeds; Zygophyllum simplex     

Arachidonic acid both inhibits and enhances whole cell calcium currents in rat sympathetic neurons

We recently reported thatarachidonic acid (AA) inhibits L- and N-type Ca²⁺ currentsat positive test potentials in the presence of the dihydropyridine L-type Ca²⁺ channel agonist (+)-202-791 in dissociatedneonatal rat superior cervical ganglion neurons [Liu L and RittenhouseAR. J Physiol (Lond) 525: 291-404, 2000]. In thisfirst of two companion papers, we characterized the mechanism ofinhibition by AA at the whole cell level. In the presence of either-conotoxin GVIA or nimodipine, AA decreased current amplitude,confirming that L- and N-type currents, respectively, were inhibited.AA-induced inhibition was concentration dependent and reversible withan albumin-containing wash solution, but appears independent of AAmetabolism and G protein activity. In characterizing inhibition, anAA-induced enhancement of current amplitude was revealed that occurredprimarily at negative test potentials. Cell dialysis with albuminminimized inhibition but had little effect on enhancement, suggestingthat AA has distinct sites of action. We examined AA's actions oncurrent kinetics and found that AA increased holdingpotential-dependent inactivation. AA also enhanced the rate of N-typecurrent activation. These findings indicate that AA causes multiplechanges in sympathetic Ca²⁺ currents.