Role of Carbonic Anhydrase in Photosynthesis of Blue-Green Alga (Cyanobacterium) Anabaena variabilis ATCC 29413

The affinity for NaHCO₃ (CO₂) in photosynthesis of Anabaenavariabilis ATCC 29413 was much higher in the cells grown underordinary air (low-CO₂ cells) than in those grown in air enrichedwith 2–4% CO₂ (high-CO₂ cells) (pH 8.0, 25?C). Ethoxyzolamide(50 µM) increased the K_m(NaHCO₃ in low-CO₂ cells aboutnine times (from 14.3 to 125), while the maximum rate of photosynthesisdecreased about 20%. When high-CO₂ cells were transferred tolow-CO₂ conditions, carbonic anhydrase (CA) activity increased,while K_m(NaHCO₃) in photosynthesis decreased from 140 to 30µM within about 5 h. The addition of CA to the suspensionof both high- and low-CO₂ cells enhanced the rates of photosyntheticO₂ evolution under CO₂-limiting conditions. The rate of ¹⁴CO₂fixation was much faster than that of H¹⁴CO₃^– fixation.The former reaction was greatly suppressed, while the latterwas enhanced by the addition of CA. These results indicate thatthe active species of inorganic carbon utilized for photosynthesiswas free CO₂ irrespective of the CO₂ concentration given duringgrowth. It is suggested that CA plays an active role in increasingthe affinity for CO₂ in photosynthesis of low-CO₂ cells of thisblue-green alga. (Received January 24, 1984; Accepted October 22, 1984)     

Inorganic Carbon Transport and Fixation in Cells of Anabaena variabilis Adapted to Mixotrophic Conditions

The Cyanobacterium Anabaena variabilis ATCC 29413 grown at lowCO₂ concentration under mixotrophic conditions with fructoseshowed a repression in the ability to fix inoganic carbon. Thisrepression was not due to a diminution in the ability to transportexternal inorganic carbon but could be explained by a decreaseof two enzymatic activities involved in the assimilation ofinorganic carbon: carbonic anhydrase and Rubisco. Carbonic anhydraseactivity was close to 50% lower in mixotrophic than in autotrophiccells. Moreover growth under mixotrophic conditions reducedRubisco activity at all dissolved inorganic carbon concentrationsassayed (5–60 mM). Maximum Rubisco activity (V_max decreasedfrom µmol CO₂ mg protein^-1h^-1 in autotrophic cells to2.3 µmol CO₂ mg protein^-1h^-1 in mixotrophic cells. Nosignificant differences in K_m(C₁) between autotrophic and mixotrophiccells were however observed. The possible mechanisms involvedin the inhibition of Rubisco are discussed. (Received November 8, 1994; Accepted October 12, 1995)     

Uptake and Utilization of Fructose by Anabaena variabilis ATCC 29413. Effect on Respiration and Photosynthesis

Anabaena variabilis ATCC 29413 showed a constitutive mechanismfor fructose uptake which was further enhanced by growing thecells with fructose. The uptake process was energydependentas indicated the inhibitory effect of the uncoupler carbonylcyanidem-chlorophenylhydrazone (CCCP) and the reduction induced byincubating the cells in the dark or in the light with DCMU.Cells adapted to growth on fructose showed increased rates ofrespiration both in the dark and in the light. The rate of ¹⁴CO₂evolved from radiolabelled fructose was lower in the light thanin the dark or in the presence of DCMU. This fact can be partiallyexplained by the photosynthetic reutilization of respiratory¹⁴CO₂. Modifications in photosynthesis were observed in fructose-growncells. PS I and PS II activity measured in spheroplasts obtainedby lysozyme treatment were enhanced by fructose probably asa way to compensate the lower concentration of chorophyll showedby fructose-grown cells. The photosynthetic affinity for externalCO₂ and the rate of photosynthesis dependent on external inorganiccarbon were reduced by fructose. (Received September 24, 1991; Accepted February 14, 1992)     

Adenosine 5'-Phosphosulf ate Sulfotransferase from Euglena: Enzyme-Bound Intermediates

Adenosine 5'-phosphosulfate sulfotransferase (APSST) purifiedfrom Euglena gracilis Klebs var. bacillaris mutant W₁₀BSmL byammonium sulfate precipitation, Sephadex G-100 gel filtration,reactive blue agarose, reactive dye agarose and DEAE-cellulosecan be labeled by incubation with AP³⁵S and separated from smallradioactive compounds on Sephadex G-50. Most of the label isnot exchangeable with nonradioactive APS and therefore is notassociated with bound substrate. On non-inactivating SDS-PAGE,a radioactive band at the position of native APSST tetramershows APSST activity (measured as acid-volatile radioactivity).Labeled protein hydrolyzed with Pronase yields radioactive S-sulfocysteine,indicating that at least one cysteine residue of APSST acceptsa sulfo group from APS to form E-S-SO₃^–. A labeled lowmolecular weight compound can be separated from the proteinby paper electrophoresis or by treatment with acidic proteindenaturing reagents such as trifluoroacetic acid (TFA) or trichloroaceticacid (TCA). This labeled compound (perhaps the sulfo-carrier)behaves as a strong acid on paper electrophoresis and is stabilizedby iodoacetamide or acidic conditions but degrades to thiosulfate,sulfate and other compounds as the pH is raised. The radioactivityin APSST is exchangeable with sulfite or thiosulfate. AMP inhibitsAPSST in the formation of acid-volatile radioactivity by competingwith APS, but APA inhibits APSST activity uncompetitively. AK_m of 0.1 µM for APS and K_i of 0.1 mM for AMP and 0.6mM for APA are obtained when a saturating amount of dithiothreitol(DTT) is used as the thiol. A mechanism is proposed for theinitial reaction(s) catalyzed by APSST. ¹Present address: Boyce Thompson Institute for Plant Research,Tower Road, Ithaca, NY 14853, U.S.A.     

DCEBIO stimulates Cl- secretion in the mouse jejunum

We investigated the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one(DCEBIO) on the Cl^– secretory response of the mouse jejunum using the Ussing short-circuit current (I_sc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in I_sc (EC₅₀ 41 ± 1 µM). Pretreating tissues with 0.25 µM forskolin reduced the concentration-dependent increase in I_sc by DCEBIO and increased the EC₅₀ (53 ± 5 µM). Bumetanide blocked (82 ± 5%) the DCEBIO-stimulated I_sc consistent with Cl^– secretion. DCEBIO was a more potent stimulator of Cl^– secretion than its parent molecule, 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated I_sc by >80% indicating the participation of CFTR in the DCEBIO-stimulated I_sc response. Clotrimazole reduced DCEBIO-stimulated I_sc by 67 ± 15%, suggesting the participation of the intermediate conductance Ca²⁺-activated K⁺ channel (IK_Ca) in the DCEBIO-activated I_sc response. In the presence of maximum forskolin (10 µM), the DCEBIO response was reduced and biphasic, reaching a peak response of the change in I_sc of 43 ± 5 µA/cm² and then falling to a steady-state response of 17 ± 10 µA/cm² compared with DCEBIO control tissues (61 ± 6 µA/cm²). The forskolin-stimulated I_sc in the presence of DCEBIO was reduced compared with forskolin control tissues. Similar results were observed with DCEBIO and 8-BrcAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated I_sc, providing evidence that DCEBIO increased Cl^– secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl^– secretion of the mouse jejunum and that DCEBIO targets components of the Cl^– secretory mechanism. 1-ethyl-2-benzimidazolinone; forskolin; glibenclamide; clotrimazole; H89     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

Chloride secretion by semicircular canal duct epithelium is stimulated via beta 2-adrenergic receptors

The ductalepithelium of the semicircular canal forms much of the boundary betweenthe K⁺-rich luminal fluid and the Na⁺-richabluminal fluid. We sought to determine whether the net ion fluxproducing the apical-to-basal short-circuit current(I_sc) in primary cultures was due to anionsecretion and/or cation absorption and under control of receptoragonists. Net fluxes of ²²Na, ⁸⁶Rb, and³⁶Cl demonstrated a basal-to-apical Clsecretion that was stimulated by isoproterenol. Isoproterenol andnorepinephrine increased I_sc with anEC₅₀ of 3 and 15 nM, respectively, and isoproterenolincreased tissue cAMP of native canals with an EC₅₀ of 5 nM. Agonists for adenosine, histamine, and vasopressin receptors had noeffect on I_sc. Isoproterenol stimulation ofI_sc and cAMP was inhibited by ICI-118551(IC₅₀ = 6 µM for I_sc) but notby CGP-20712A (1 µM) in primary cultures, and similar results werefound in native epithelium. I_sc was partially inhibited by basolateral Ba²⁺ (IC₅₀ = 0.27 mM) and ouabain, whereas responses to genistein, glibenclamide, andDIDS did not fully fit the profile for CFTR. Our findings show that thecanal epithelium contributes to endolymph homeostasis by secretion ofCl under ₂-adrenergic control with cAMP assecond messenger, a process that parallels the adrenergic control ofK⁺ secretion by vestibular dark cells. The current workpoints to one possible etiology of endolymphatic hydrops in Meniere'sdisease and may provide a basis for intervention.

     

Quantitative Analysis of ATP-Dependent H+ Efflux and Pump Current Driven by an Electrogenic Pump in Nitellopsis obtusa

Internodal cells of Nitellopsis were made tonoplast-free byperfusion with a medium containing EGTA. Cytoplasmic concentrationsof solutes were controlled by a second perfusion with mediaof known composition. The electrogenic pump current (I_p), whichwas calculated from electrical data obtained from cells withand without ATP, was compared with the current carried by H⁺(I_H⁺) across the plasma membrane. A close correlation betweenI_p and I_H⁺ was found under various internal and external conditions.(1) I_p and I_H⁺ depended on the internal ATP and showed Michaelis-Mententype saturation curves. For I_p, K_m was 120 µM and themaximum current V_max was 15.1 mA m^–2, while for I_H⁺, K_mwas 160 µM and V_max was 16.6 mA m^–2. (2) I_p andI_H⁺ showed almost the same I_H²⁺ dependence. The Mg²⁺-dependentI_p was 19.5 mA m^–2, while the Mg²⁺-dependent I_H²⁺ was17.7 mA m^–2. (3) I_H²⁺ was maximal at an external pH of8 and decreased both in acidic and alkaline pH ranges. I_p wasnearly equal to I_H⁺ in the pH range between 8 and 5. (4) I_H⁺became maximal at an internal pH of 7.3, which is nearly thesame as the pH for maximal electrogenecity found by Mimura andTazawa (1984). All these facts support the idea proposed in our previous paper(Takeshige et al. 1985) that the electrogenic ion pump locatedin the plasma membrane of Nitellopsis is the H⁺ pump. ¹ Dedicated to Professor Dr. Erwin Bünning on the occasionof his 80th birthday. (Received June 21, 1985; Accepted December 20, 1985)     

Alicyclic acid metabolism in plants 10. Partial purification and some properties of 3-dehydroquinate synthase from Phaseolus mungo seedlings

Dehydroquinate synthase from Phaseolus mungo seedlings was purified120-fold by DE-23, hydroxylapatite and Sephadex G-100 columnchromatography. The final preparation was free of dehydroquinatehydro-lyase and NAD(P)H₂ oxidase. The dehydroquinate synthaserequired Co²⁺ and NAD as cofactors. Co²⁺ could be replaced byCu²⁺ at 0.1 mM, but Cu²⁺ at higher levels was inhibitory. Noneof the other metal ions tested activated the enzyme. Some activitywas observed in the absence of added Co²⁺ and this activitywas inhibited by EDTA but not by diethyldithiocarbamate, NaN₃or NaCN. Heavy metal ions, such as Ag⁺ and Hg²⁺, and p-chloromercuribenzoatestrongly inhibited the enzyme activity. Of the pyridine nucleotidestested only NAD was required for the maximum activity of theenzyme. In the absence of NAD, the enzyme retained 30 to 40%of the activity obtained with added NAD. The apparent Km valuefor DAHP at pH 7.4 was about 23 µM. The enzyme activityappeared to be maximum at about pH 8.5. However, the characteristicsof the enzyme were studied at pH 7.4, because of the labilityof the enzyme under alkaline conditions. An Arrhenius plot ofthe enzyme reaction showed a break at about 21?C, and belowthis critical temperature the activation energy increased. (Received March 4, 1977; )     

Beta-adrenergic stimulation does not activate Na+/Ca2+ exchange current in guinea pig, mouse, and rat ventricular myocytes

The effect of -adrenergic stimulation on cardiac Na⁺/Ca²⁺ exchange has been controversial. To clarify the effect, we measured Na⁺/Ca²⁺ exchange current (I_NCX) in voltage-clamped guinea pig, mouse, and rat ventricular cells. When I_NCX was defined as a 5 mM Ni²⁺-sensitive current in guinea pig ventricular myocytes, 1 µM isoproterenol apparently augmented I_NCX by 32%. However, this increase was probably due to contamination of the cAMP-dependent Cl^– current (CFTR-Cl^– current, I_CFTR-Cl), because Ni²⁺ inhibited the activation of I_CFTR-Cl by 1 µM isoproterenol with a half-maximum concentration of 0.5 mM under conditions where I_NCX was suppressed. Five or ten millimolar Ni²⁺ did not inhibit I_CFTR-Cl activated by 10 µM forskolin, an activator of adenylate cyclase, suggesting that Ni²⁺ acted upstream of adenylate cyclase in the -adrenergic signaling pathway. Furthermore, in a low-extracellular Cl^– bath solution, 1 µM isoproterenol did not significantly alter the amplitude of Ni²⁺-sensitive I_NCX at +50 mV, which is close to the reversal potential of I_CFTR-Cl. No change in I_NCX amplitude was induced by 10 µM forskolin. When I_NCX was activated by extracellular Ca²⁺, it was not significantly affected by 1 µM isoproterenol in guinea pig, mouse, or rat ventricular cells. We concluded that -adrenergic stimulation does not have significant effects on I_NCX in guinea pig, mouse, or rat ventricular myocytes. cystic fibrosis transmembrane conductance regulator; nickel ion     

Detection and Characterization of UDP-Glucose:Flavonoid O-Glucosyltransferases in the Leaves of Prunus ? yedoensis Matsum

A novel O-glucosyltransferase (I4'GT) which catalyzes the transferof D-glucose from UDP-D-glucose to position 4' of prunetin (4',5-dihydroxyl-7-methoxyisoflavone)was isolated from the leaves of Prunus ? yedoensis Matsum. andpurified 66-fold by precipitation with ammonium sulfate andchromatography on DEAE-cellulose. UDP-glucose:flavonol 3-O-glucosyltransferase(F3GT) was also isolated and purified 50-fold in the same manner.The molecular weights of both I4'GT and F3GT were estimatedby elution from a column of Sephadex G-100 to be about 51,000Da. The pH optima for I4'GT and F3GT activities were 8.0 and7.5, respectively. The specificities of I4'GT and F3GT for thesugar donor were quite strict, and only UDP-glucose could serveas glucosyl donor, both ADP-D-glucose and GDP-D-glucose beingineffective. The apparent K_m values for UDP-glucose and prunetinwere 10.0µM and 1.20µM, respectively, for I4'GT.The K_m values for UDP-glucose and quercetin were 9.8 µMand 1.21 µM, respectively, for F3GT. The activities ofboth I4'GT and F3GT were stimulated by 1 mM Mg*⁺ and stronglyinhibited by 1 mM Cu²⁺, 1 mM Zn²⁺ and various reagents thatreact with sulfhydryl groups. (Received May 16, 1990; Accepted September 3, 1990)     

Ion Effects on the H+-Translocating Adenosine Triphosphatase and Pyrophosphatase Associated with the Tonoplast of Chora corallina

H⁺-translocating ATPase and pyrophosphatase (PPase) associatedwith the tonoplast of Chara corallina were isolated with theaid of a perfusion technique, and the effects of ions on theiractivities were studied. All the alkali metal cations testedstimulated the ATPase and ATPdependent H⁺ pumping activitiesonly by 10 to 40%. Anions, on the other hand, strongly affectedthe activities. Potassium salts of Cl^- and Br^- stimulated them,while F^- and NO₃^- inhibited them. By contrast, the H⁺-translocatingPPase was insensitive to anions but sensitive to cations. Theorder of cation stimulation was Rb⁺=K⁺>Cs⁺>Na⁺=Li⁺>choline⁺.NO₃^- (50 mil), thought to be a specific inhibitor of the tonoplast-typeH⁺-ATPase, inhibited the ATPdependent H⁺ pumping almost completelybut the ATPase activity by only about 50%. Na⁺ inhibited thePP₁-dependent H⁺ pumping (I_5O=5OmM) in the presence of 50 mMKCl but not the ATP-dependent one. The PPase was more sensitiveto F^- (I₅₀=400µM) than the ATPase. Both the H⁺-ATPaseand the H⁺-PPase required Mg²⁺ for their activities, althoughan excess was inhibitory to both. The different sensitivitiesof the PP₁-dependent and the ATP-dependent H⁺- pumping enzymesto ions correspond to the tonoplast enzymes of higher plantsand may be used as "markers" to distinguish between these enzymesin characean cells (Received October 2, 1987; Accepted May 18, 1988)     

Purification and Properties of Sweet Potato NADPH-Cytochrome c (P-450) Reductase

NADPH-cytochrome c reductase, strictly NADPH-cytochrome P-450reductase, was purified by chromatography through DEAE-cellulose,2',5'-ADP-Sepharose, and Sephadex G-100 columns after solubilizationfrom microsomes from Ceratocystis fimbriata-infected sweet potatoroot tissue with Emulgen 913. The enzyme existed in three formsafter solubilization which migrated to positions correspondingto molecular weights of 81,000, 75,000 and 72,000 on an SDS-polyacrylamidegel. Trypsin treatment of the enzyme species with the largestpolypeptide yielded the species with the smallest one. Aftersucrose density gradient centrifugation of the pellet fractionobtained by centrifugation at 100,000?g of the crude extract,the enzyme species with the largest polypeptide was presentin the particulate fractions, whereas that with the smallestone was only found at the top of the gradient. We conclude thatthe enzyme species with the largest polypeptide is in an intact,amphipathic form, whereas that with the smallest one, and probablyalso the other species, is its hydrophilic domain produced byan endogenous protease(s). The K_m values of the enzyme in theintact form for NADPH and cytochrome c were 7.7 and 2.3 µM,respectively. ¹ Present address: Laboratory of Food Hygienics, Faculty ofAgriculture, Kagawa University, Miki-cho, Kida-gun, Kagawa 761-07,Japan. (Received September 6, 1984; Accepted December 27, 1984)     

Partial Purification and Some Properties of Flavonol 3-O-Glycosyltransferases from Seedlings of Vigna mungo, with Special Reference to the Formation of Kaempferol 3-O-Galactoside and 3-O-Glucoside

A novel enzyme, UDP-D-galactose:flavonol 3-O-galactosyltransferase(F3GaT), catalyzing the transfer of D-galactose from UDP-D-galactoseto the 3 position of 5,7,4'-trihydroxyflavonol (kaempferol),was detected in and purified about 404-fold from seedlings ofVigna mungo by precipitation with ammonium sulfate, chromatographyon Sephadex G-100 and chromatofocusing. The enzyme was separatedby this procedure from a coexisting UDP-D-glucose:flavonol 3-O-glucosyltransferase(F3GT), which was simultaneously purified about 189-fold. F3GaTwas isolated as a soluble enzyme with pH optima of 8.0 in imidazole-HClbuffer and 7.5 in histidine-HCl buffer. F3GT had the same pHoptima. The M_r of both F3GaT and F3GT, which had isoelectricpoints of 5.1 and 6.1, respectively, was estimated by elutionfrom a column of Sephadex G-100 to be about 43,000. The activitiesof F3GaT and F3GT were stimulated by 14 mM 2-mercaptoethanoland strongly inhibited by 1 mM Cu²⁺, 1 mM Zn²⁺, and variousreagents that react with sulfhydryl groups. Among various possiblesubstrates for F3GaT that were tested, kaempferol, isorhamnetinand quercetin were the best. The K_m values for kaempferol andUDP-D-galactose were determined to be 0.40 µM and 125µM, respectively. Similarly, F3GT had low K_m values of0.69 µM for kaempferol and 1.67 mM for UDP-D-glucose.F3GaT and F3GT mediated the transfer of galactose and glucose,respectively, to the 3-hydroxyl groups exclusively of kaempferol,isorhamnetin and quercetin. Rhamnetin also functioned as a galactosylacceptor though less efficiently. (Received October 12, 1992; )     

Lipid Biosynthesis in the Blue-Green Alga (Cyanobacterium), Anabaena variabilis III. UDPglucose:Diacylglycerol Glucosyltransferase Activity in vitro

The synthesis of glyceroglycolipids was studied in membraneand soluble fractions of Anabaena variabilis. The membrane fractionexhibited a high activity of UDPglucose: diacylglycerol glucosyltransferase,but practically no activity of UDPgalactose: diacylglycerolgalactosyltransferase. The glucosyltransferase activity wasmaximal at about pH 7.0 and dependent on Mg²⁺ The Michaelisconstant (K_m) for UDPglucose was 45?10^–6 M. The solublefraction catalyzed the incorporation of galactose from UDP galactoseinto digalactosyl diacylglycerol. These in vitro results werecompatible with the biosynthetic pathway of glyceroglycolipidsin this alga that we previously elucidated on the basis of tracerexperiments in vivo. ¹ Present address: Department of Biology, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. (Received June 1, 1982; Accepted July 1, 1982)     

ATP-dependent regulation of inwardly rectifying K+ current in bovine retinal pigment epithelial cells

Inwardlyrectifying K⁺ current(I_Kir) infreshly isolated bovine retinal pigment epithelial (RPE) cells wasstudied in the whole cell recording configuration of the patch-clamptechnique. When cells were dialyzed with pipette solution containing noATP, I_Kir randown completely in <10 min [half time(t_1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustainedI_Kir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mMATP was used,I_Kir ran down by~71%. Mg²⁺ was a criticalcofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg²⁺(t_1/2 = 1.8 min).I_Kir also randown when the pipette solution contained 4 mMMg²⁺ + 4 mM5'-adenylylimidodiphosphate(t_1/2 = 2.7 min)or 4 mM adenosine 5'-O-(3-thiotriphosphate)(t_1/2 = 1.9 min),nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. Weconclude that the sustained activity ofI_Kirin bovine RPE requires intracellular MgATP and that the underlyingmechanism may involve ATP hydrolysis.

     

Regulation of transcervical permeability by two distinct P2 purinergic receptor mechanisms

Micromolar concentrations ofATP stimulate biphasic change in transepithelial conductance acrossCaSki cultures, an acute increase (phase I response) followed by aslower decrease (phase II response). Phase I andphase II responses involve two distinct calcium-dependentpathways, calcium mobilization and calcium influx. To test thehypothesis that phase I and phase II responsesare mediated by distinct P2 purinergic receptors, changes inpermeability were uncoupled by blocking calcium mobilization with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(BAPTA) or by lowering extracellular calcium, respectively. Under theseconditions ATP EC₅₀ was 25 µM for phase Iresponse and 2 µM for phase II response. The respectiveagonist profiles were ATP > UTP > adenosine5'-O-(3-thiotriphosphate) (ATP-S) = N⁶-([6-aminohexyl]carbamoylmethyl)adenosine5'-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-S. Suramin blocked phase Iresponse and ATP-induced calcium mobilization, whereas pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid (PPADS) blocked phase II response and ATP-augmented calcium influx. ATP time course andpharmacological profiles for phase II response and augmented calcium influx were similar, with a time constant of 2 min and asaturable concentration-dependent effect (EC₅₀ of 2-3µM). RT-PCR experiments revealed expression of mRNA for both theP2Y₂ and P2X₄ receptors. These results suggestthat the ATP-induced phase I and phase IIresponses are mediated by distinct P2 purinergic receptor mechanisms.

     

Purification and some properties of polyphenol oxidase from root-forming carrot callus tissues

1. Polyphenol oxidase (o-diphenol : O₂ oxidoreductase; E.C.1.10.3.1 [EC] ) was isolated from the other phenolases which werepresent in root-forming carrot callus, and its properties wereexamined. 2. The enzyme was purified about 45-fold over crudeextracts (precipitates between 40–70% saturation widiammonium sulfate) by a combination of Bio-gel filtration, protein-bagfiltration, and carboxymethyl cellulose chromatography. Thepurified oxidase was homogeneous according to polyacrylamidegel electrophoresis and Sephadex gel filtration. It was confirmedby CM-cellulose chromatography that the enzyme was absent incallus tissues without accompanying redifferentiation. 3. Themolecular weight of this oxidase was estimated to be 110,000-120,000 from molecular weight-mobility profiles on polyacrylamidegels containing sodium dodecyl sulfate and molecular size-elutionvolume correlations on Sephadex G-150 columns. 4. The enzymeoxidized o-diphenols but showed no detectable activity againstmonophenols. Pyrocatechol, dopamine, caffeic acid, and chlorogenicacid were effectual substrates of the enzyme with K_m valuesranging from 10^–3 M to 10^–5M. The enzyme effectivelycatalyzed the oxidation of o-diphenols over the range of pH6.0 to 7.0 and was readily inactivated by heating. The enzymeactivity was slightly influenced by increasing ionic strength.The initial rate of the enzymic reaction was enhanced by additionof Cu²⁺, Co²⁺ and Mn²⁺ ions, and was reduced in the presenceof DTT, PCMPS, glycylglycine, and DIECA. (Received June 17, 1978; )     

Studies on the Hill reaction of membrane fragments of blue-green algae II. The reaction steps inactivated at high water concentration

DPIP-photoreduction by membrane fragments of Anabaena cylindricaand A. variabilis was studied to determine which step(s) ofthe Hill reaction system is inactivated on incubation of themembrane fragments in a medium with a high water concentration(cf. 1). Supplementary experiments were done with Anacystisnidulans and Plectonema boryanum. After inactivation of the Hill system at a high water concentration,DPIP-photo-reducing activity was strongly enhanced in the A.variabilis system but less so in the A. cylindrica system byadding DPC, NH₂OH, Mn⁺⁺ or H₂0₂. The activity supported by theadded electron donor was inhibited by DCMU. The steady statelevel of chlorophyll fluorescence was lowered by the inactivationtreatment. In the A. variabilis system, the fluorescence yieldincreased to the original level on the addition of an electrondonor. In the A. cylindrica system, the yield was not so stronglyenhanced as in the A. variabilis system. We inferred that, in A. variabilis, inactivation occurs in thereaction system before the site which receives electrons fromartificial donors, probably including the water oxidation system.In A. cylindrica, besides this site, a site at or near the photochemicalsystem is also blocked. Similar types of inactivation were observed in DPIP-Hill reactionsusing Anacystis nidulans and Plectonema boryanum preparations.The characteristic stability of the Hill reaction system observedin two Anabaena preparations is probably common to the blue-greenalgae. (Received December 10, 1971; )     

Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport

We report, for the epithelialNa⁺ channel (ENaC) in A6 cells,the modulation by cell pH (pH_c)of the transepithelial Na⁺ current(I_Na), thecurrent through the individual Na⁺channel (i), the openNa⁺ channel density(N_o), and thekinetic parameters of the relationship betweenI_Na and theapical Na⁺ concentration. Thei andN_o were evaluatedfrom the Lorentzian I_Na noise inducedby the apical Na⁺ channel blocker6-chloro-3,5-diaminopyrazine-2-carboxamide.pH_c shifts were induced, understrict and volume-controlled experimental conditions, byapical/basolateral NH₄Cl pulses orbasolateral arrest of theNa⁺/H⁺exchanger (Na⁺ removal; block byethylisopropylamiloride) and were measured with the pH-sensitive probe2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Thechanges in pH_c were positivelycorrelated to changes inI_Na and theapically dominated transepithelial conductance. The sole pH_c-sensitive parameter underlyingI_Na wasN_o. Only thesaturation value of theI_Na kinetics wassubject to changes in pH_c.pH_c-dependent changes inN_o may be causedby influencingP_o, the ENaC openprobability, or/and the total channel number,N_T = N_o/P_o.