L-Leucine Transport in Isolated Protoplasts of Vinca Suspension Culture: Characterization of Uptake

The uptake of L-leucine into Vinca protoplasts was studied undervarious conditions. The uptake was highly pH-dependent, withthe optimal pH between 3.0 and 4.0. The uptake was also energydependent, since azide, 2,4-dinitrophenol (DNP), carbonyl cyanidem-chlorophenyl hydrazone (CCCP), and iodoacetate inhibited theuptake. Oligomycin, N,N'-dicycIohexyI carbodiimide (DCCD) andvanadate, but not ouabain, inhibited the uptake, suggestingthat ATPase for H⁺ electrogenic extrusion was necessary to theuptake of L-leucine. The uptake showed stereospecificity, butwas partially inhibited by other L-amino acids. A kinetic studyof the uptake showed that the uptake was multiphasic with threesaturable phases and one unsaturable phase which occurred atconcentrations of L-leucine over 1 mM. The K_m values of thethree affinity sites were 1.4 x 10^–3 M, 1.3 x 10^–4M, 4.3 x 10^–5 M; the maximum velocity values were 3.3x 10^–8, 4.5 x 10^–9, 1.8 x 10_–9 mol/10 min/4x 10⁶ cells. (Received April 18, 1981; Accepted August 25, 1981)     

Soluble Dissimilatory Nitrate Reductase Containing Cytochrome c from a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

Dissimilatory nitrate reductase [nitrite: (acceptor) oxidoreductase.EC 1.7.99.4 [EC] ] from a denitrifying photosynthetic bacterium, Rhodopseudomonassphaeroides forma sp. denitrificans proved to be a soluble enzymethat could be purified 47-fold. It was labile, and containedcytochrome c, based on the results of specific staining forheme on polyacrylamide gel electrophoresis and on its absorptionspectrum. Its physiological molecular weight was determinedto be 112k, although heterogeneous molecular weights of 112k,100k, 73k and 60k were found for different preparations. Theoptimum for enzyme activity was about pH 6, and the K_m for thenitrate was 1.6 mM. As an electron donor, benzyl viologen wasvery good; but NADH, NADPH, FAD, FMN, cytochromes b₂ and c₂,dichlorophenolindophenol and phenazine methosulfate were noteffective. Bathophenanthroline and thiocyanate inhibited enzymaticactivity. The addition of 1 mM tungstate to the growing culturein place of molybdate decreased the nitrate reductase in thecells, but a further addition of 1 mM molybdate stopped it.This nitrate reductase is believed to be a molybdo-iron proteinsimilar to the enzymes from other bacteria with a nitrate respiratingability. (Received February 29, 1980; Accepted January 29, 1981)     

Inhibition of nitrate reductase from spinach (Spinacea oleracea L.) leaf by adenosine nucleotides

NADH-dependent nitrate reductase of spinach showed ping-pongKinetics. Inhibition by ADP in the absence of thiols increasedduring a transient phase during which Kinetics changed fromweak (K₁ 20 mM), non-competitive and linear to comparativelystrong (K₁ 0.09 mM at 10 mM ADP), mixed with respect to NADHand non-linear (n_H = 1.7). Thiol compounds transformed the inhibitionto competitive (K₁ 0.18 mM at 10 mM ADP) and non-linear (n_H= 2.3). Effective concentrations increased in order: GSH>mercaptoethanol= thioglycollate>cysteine, and were increased by dissolvedoxygen or disulphide. Non-competitive inhibition by ribose-5-phosphate(K₁ 3 nua) was abolished by glutathione. Cyclic 3',5'-AMP inhibitednon-linearly (nH=2.4) and comparably to or more than ADP (K₁0.22 mM at 2.5 mM) but predominantly competitively except atlow NADH concentrations. Cyclic 2',3'-AMP showed similar butweaker inhibition (K₁ 1.4 mM) and more nearly linear (n_H =1.3).5'-AMP inhibited competitively (K₁ 1.2 mM at 10 mM) (n_H = 1.3).5'-ATP showed weak mixed inhibition (K₁ 5 nun at 10 mM) (n_H= l.3). Activity with BV° was scarcely inhibited by ADP,but phosphate activation with BV° or NADH were both inhibitedby ADP. Physiological significance of inhibition by ADP is discussedand might lie between 3% in light and 25% in dark in specialcircumstances of thiol supply. ¹ Present address: Department of Soil Microbiology, RothamstedExperimental Station, Harpenden, Herts. (Received May 22, 1975; )     

Distribution of a cryptating agent in excreta and its influence on urinary elimination of Na, K, Mg, Ca and Zn in the rat.

After a single s.c. dose of 13, 16, 21, 24-hexaoxa-1, 10-diazabicyclo- (8, 8, 8)-hexacosane, A-(222), rats eliminate 42% of this cryptating agent via urine during the first 24h and about 34% via feces during the second 24h. Excretion seems to be complete within 72h. A-(222) impairs significantly the urinary excretion of sodium-potassium- and calcium-ions during the first 24h. The excretion of magnesium- and zinc-ions is not affected. The observed impairments cannot be interpreted as a chelation effect of A-(222) with these cations.     

Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers

Blood stream forms (BSF) of Trypanosoma brucei brucei GUT at 3.1 were propagated in vitro in the absence of feeder layer cells at 37 C, using a modified Iscove's medium (HMI-18). The medium was supplemented with 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate. 0.16 mM thymidine, and 20% (v/v) Serum Plus (SP) (Hazleton Biologics, Lenexa, Kansas). The latter contained a low level of serum proteins (13 micrograms/ml). Each primary culture was initiated by placing 3.5-4 x 10(6) BSFs isolated from infected mice in a flask containing 5 ml of the medium (HMI-9) supplemented with 10% fetal bovine serum (FBS) and 10% SP. The cultures were maintained by replacing the medium every 24 hr for 5-7 days. During this period, many BSFs died. However, from day 4 onward, long slender BSFs increased in number. On days 5-7, trypanosome suspensions were pooled and cell debris was removed by means of diethylaminoethyl cellulose (DE52) column chromatography. Blood stream forms then were collected by centrifugation, resuspended in fresh medium at 7-9 x 10(5)/ml, and transferred to new flasks. Subcultures were maintained by readjusting the BSF density to 7-9 x 10(5)/ml every 24 hr. Concentrations of FBS were reduced gradually at 5-7-day intervals by alternating the amounts of FBS and SP in HMI-9 with 5% FBS and 15% SP, with 2% FBS and 18% SP, and finally with 20% SP (HMI-18). By this method, 2-3 x 10(6) VSFs/ml were obtained consistently every 24 hr. for more than 80 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pyruvate dehydrogenase complex and acetyl-CoA carboxylase in pea root plastids: their characterization and role in modulating glycolytic carbon flow to fatty acid biosynthesis

The pyruvate dehydrogenase complex (PDC) and acetyl-CoA carboxylase(ACC, EC 6.4.1.2 [EC] ) have been characterized in pea root plastids.PDC activity was optimum in the presence of 1.0 mM pyruvate,1.5 mM NAD⁺ 0.1 mM CoA, 0.1 mM TPP, 5 mM MgCl₂, 3.0 mM cysteine-HCl,and 0.1 M Tricine (pH 8.0) and represents approximately 47%of the total cellular activity. ACC activity was greatest inthe presence of 1.0 mM acetyl-CoA, 4 mM NaHCO₃ mM ATP, 10 mMMgCl₂, 2.5 mM dithiothreitol, and 100 mM Tricine (pH 8.0). Bothenzymes were stimulated by reduced sulphydryl reagents and inhibitedby sulphydryl inhibitors. ACC was also inhibited by malonyl-CoAwhile PDC was inhibited by both malonyl-CoA and NADH. Both enzymeswere stimulated by DHAP and UDP-galactose while ACC was alsostimulated by PEP and F₁,6P. Palmitic acid and oleic acid bothinhibited ACC, but had essentially no effect on PDC. Palmitoyl-CoAinhibited both enzymes while PA and Lyso-PA inhibited PDC, butstimulated ACC. The results presented support the hypothesisthat PDC and ACC function in a co-ordinated fashion to promoteglycolytic carbon flow to fatty acid biosynthesis in pea rootplastids. Key words: Pisum sativum L., pyruvate dehydrogenase complex, acetyl-CoA carboxylase, roots, non-photosynthetic plastids     

Effect of alcohol and kolanut interaction on brain sodium pump activity in Wistar Albino rats.

Effect of alcohol-kolanut interaction on Sodium Pump activity in wistar albino rats was studied. Thirty wistar albino rats were divided into six groups of five (5) rats per group and used for the study. The control group (1) received via oral route a placebo (4ml of distilled water). Groups 2 to 6 were treated for a period of 21 days, with (10% v/v) of alcohol (group 2), 50mg/kg body weight of kolanut (group 3), 50 mg/kg body weight of caffeine (group 4), 4ml of 10% v/v of alcohol and 50 mg/kg body weight kolanut (group 5), 4ml of 10% v/v of alcohol and 50 mg/kg body weight of caffeine in 4.0ml of the vehicle via gastric intubation respectively. A day after the final exposure, the brain of each rat was harvested and processed to examine several biochemical parameters, i.e., total ATpase, ouabain-insensitive ATpase, ouabain sensitive ATpase (Na(+)-K(+)ATPase), non-enzymatic breakdown of ATP and inorganic phosphate (Pi) released. The results showed that the essential enzyme of the brain responsible for neuronal function, Na(+)-K(+)ATPase, was inhibited by alcohol-kolanut co-administration relative to control, resulting in a decrease in Na(+)-K(+)ATPase activity, ATP production, ion transport and action potential, leading to loss of neuronal activities.     

Solubilization by lysolecithin and purification of the plasma membrane ATPase of the yeast Schizosaccharomyces pombe.

Purified plasma membranes of Schizosaccharomyces pombe were obtained by precipitation at pH 5.2 of a crude particulate fraction, followed by differential centrifugations and isopycnic centrifugation in a discontinuous sucrose gradient. The specific activity of the Mg2+-requiring plasma membrane ATPase activity (EC 3.6.1.3) was enriched from 0.3 mumol min-1 x mg-1 of protein in the homogenate to 26 in the purified membranes. The optimal conditions for solubilization of the ATPase activity by lysolecithin were found to be: 2 mg/ml of lysolecithin, a lysolecithin to protein ratio of 8 at pH 7.5, and 15 degrees C in the presence of 1 mM ATP and 1 mM ethylenediaminetetraacetic acid. A 6- to 7-fold purification of the solubilized ATPase activity was obtained by centrifugation of the lysolecithin extract in sucrose gradient. Part of the ATPase activity which was inactivated during the centrifugation in the sucrose gradient could be restored by addition of a micellar solution of 50 microgram of lysolecithin/ml during the assay. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the purified enzyme showed only one band of Mr = 105,000 stained with Coomassie blue. Another ATPase component of apparent molecular weight lower than 10,000 was stained by periodic Schiff reagent but not colored by Coomassie blue. The purified enzyme was 85% inhibited by 50 micrometer N,N'-dicyclohexylcarbodiimide and 94% inhibited by 53 microgram of Dio-9/ml.     

Partial characterization of the ouabain-insensitive, Na+-stimulated ATPase activity of kidney basal-lateral plasma membranes

The present paper characterizes the Na+-stimulated ATPase activity present in basal-lateral plasma membranes from guinea-pig kidney proximal tubular cells. These characteristics are compared with those of the (Na+ + K+)-stimulated ATPase activity, and they are: (A) Na+-ATPase activity: (1) requires Mg2+; (2) may be activated by mu molar quantities of Ca2+; (3) optimal ratio Mg:ATP = 5:1-2 and Ka for Mg:ATP = 3:0.60 mM; (4) Ka for Na+:8 mM; (5) does not require K+; (6) is only stimulated by Na+ and Li+ (in a lower extent); (7) is similarly stimulated by the Na+ salt of different anions; (8) hydrolyzes only ATP; (9) optimal temperature: 47 degrees C; (10) optimal pH: 6.9; (11) is ouabain insensitive; (12) is totally inhibited by 1.5 mM ethacrynic acid, 2 mM furosemide and 0.75 mM triflocin. (B) (Na+ + K+)-ATPase activity: (1) also requires Mg2+; (2) is inhibited by Ca2+; (3) optimal ratio Mg:ATP = 1.25:1 and Ka for Mg:ATP = 0.50: 0.40 mM; (4) Ka for Na+: 14 mM (data not shown); (5) needs K+ together with Na+; (6) K+ may be substituted by: Rb+ greater than NH+4 greater than Cs+; (7) is anion insensitive; (8) hydrolyzes mostly ATP and to a lesser extent GTP, ITP, UTP, ADP, CTP; (9) optimal temperature: 52 degrees C; (10) optimal pH: 7.2; (11) 100% inhibited by 1 mM ouabain; (12) 63% inhibited by 1.5 mM ethacrynic acid, 10% inhibited by 2 mM furosemide and insensitive to 0.75 mM triflocin.     

The Transport of Sugars in Developing Fruits of Satsuma Mandarin

Transport of sugars to the juice sacs of developing satsumamandarin (Citrus unshui Marc) has been studied in attached fruitsand in isolated fruit pieces. ¹⁴CO₂ fed to the leaves resultedin [¹⁴C]sugar accumulation in the juice sacs, mainly as [¹⁴C]sucrose.Uptake of sucrose and glucose by the excised fruit pieces proceededlinearly with time. Sucrose uptake was linearly related to sucroseconcentration over the range 25–300 mM, with no indicationof saturation. This uptake was insensitive to pH (5, 7 or 9),Ca²⁺(3 mM), PCMBS (2.5 mM), DNP (1 mM) or vanadate (0.1 mM)but was slightly reduced by erythrosin (21 % by 0–1 mM;27 % by 1 mM). No competitive effect of glucose (up to 100 mM)was detectable on sucrose uptake from 100 mM solution. Mostof the [¹⁴C]sucrose uptake observed was reversible, althoughconsiderable hydrolysis and metabolic conversion were evidenced.A vanadate-sensitive ATPase was demonstrated by EM localizationon the plasma membrane of the juice sac cells. These resultsare interpreted in relation to the accumulation of assimilatesby the developing fruit. Transport: sugar, satsuma mandarin, juice sacs     

{beta}-Naphthyl oligophosphates: Inhibitors of photophosphorylation and H+-ATPase of spinach chloroplasts

ß-Naphthyl di-, tri- or tetraphosphate inhibits photophosphorylationof spinach chloroplasts competitively with ADP, whereas ß-naphthylmonophosphate inhibits it competitively with Pi. The apparentKi of ß-naphthyl diphosphate for the ADP site was300 µM and that of ß-naphthyl monophosphatefor the Pi site was 1.45 mM. At 10 mM, both of these two organicphosphates inhibited photophosphorylation more than 90%. Noneof the above four ß-naphthyl phosphates were phosphorylatedby chloroplasts. ß-Naphthyl di-, tri- or tetraphosphateinhibits ATPase activity of isolated chloroplast coupling factor1 (CF₁) (EC 3.6.1.3 [EC] ) and light-triggered ATPase activity ofchloroplasts competitively with ATP, whereas ß-naphthylmonophosphate acts non-competitively. None of the four ß-naphthylphosphates were hydrolyzed by these two ATPase activities. Atconcentrations equal to ADP or ATP, ß-naphthyl di-,tri- or tetraphosphate inhibited these three reactions in theorder; ATPase of isolated CF₁> photophosphorylation>light-triggeredATPase of chloroplasts. The results suggest that the effect of the monophosphate isprincipally on the Pi site(s) and that of the di-, tri- or tetraphosphateis on the adenine nucleotide site(s) on the active center ofCF₁. ¹Part of this work was reported at the 1979 Annual Meeting ofthe Japanese Society of Plant Physiologists (Nagoya, April 7,1979) and the 52nd Annual Meeting of the Japanese BiochemicalSociety (Tokyo, October 7, 1979). This work was supported inpart by Grants-in-Aid for Scientific Research from the Ministryof Education, Science and Culture, Japan (311808 and 311909). (Received November 14, 1979; )     

Identification of residues within the drug-binding domain of the human multidrug resistance P-glycoprotein by cysteine-scanning mutagenesis and reaction with dibromobimane

P-glycoprotein (P-gp) can transport a wide variety of cytotoxic compounds that have diverse structures. Therefore, the drug-binding domain of the human multidrug resistance P-gp likely consists of residues from multiple transmembrane (TM) segments. In this study, we completed cysteine-scanning mutagenesis of all the predicted TM segments of P-gp (TMs 1-5 and 7-10) and tested for inhibition by a thiol-reactive substrate (dibromobimane) to identify residues within the drug-binding domain. The activities of 189 mutants were analyzed. Verapamil-stimulated ATPase activities of seven mutants (Y118C and V125C (TM2), S222C (TM4), I306C (TM5), S766C (TM9), and I868C and G872C (TM10)) were inhibited by more than 50% by dibromobimane. The activities of mutants S222C (TM4), I306C (TM5), I868C (TM10), and G872C (TM10), but not that of mutants Y118C (TM2), V125C (TM2), and S776C (TM9), were protected from inhibition by dibromobimane by pretreatment with verapamil, vinblastine, or colchicine. These results and those from previous studies (Loo, T. W. and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948; Loo, T. W. and Clarke, D. M. (1999) J. Biol. Chem. 274, 35388-35392) indicate that the drug-binding domain of P-gp consists of residues in TMs 4, 5, 6, 10, 11, and 12.     

In Vitro NADH Oxidation as an Early Indicator for Growth Reduction in Spinach Exposed to H2S in the Ambient Air

H₂S reduced growth of spinach at levels higher than 0.075 µl/literand inhibited NADH oxidation by shoot extracts. Inhibition ofNADH oxidation was maximum after a 48-h exposure. Oxidationof NADH was equally sensitive to sulfide and cyanide. NADH oxidationshowed promise as an early indicator for growth reduction byH₂S. ¹Present address: Centre for Agrobiological Research, P.O. Box14, 6700 AA Wageningen, The Netherlands. (Received May 18, 1987; Accepted February 9, 1988)     

The Influence of Apoplastic Ascorbate on the Activities of Cell Wall-Associated Peroxidase and NADH Oxidase in Needles of Norway Spruce (Picea abies L.)

Cell wall-associated peroxidases (EC 1.11.1.7 [EC] ) were extractedfrom the current year's needles of Norway spruce trees (Piceaabies L.) in two fractions, namely soluble apoplastic peroxidasesand covalently wall-bound peroxidases. Peroxidase activitieswere determined with two substrates: coniferyl alcohol, whichis important for lignification, and NADH, which is necessaryfor the production of H₂O₂. Coniferyl alcohol peroxidase activitywas detected in both the soluble apoplastic fraction and thewall-bound fraction, whereas NADH oxidase activity was foundonly in the soluble apoplastic fraction. Net oxidation of coniferylalcohol and NADH was inhibited by ascorbate, which reduced theoxidized intermediates of the peroxidase- and oxidase-catalyzedreactions. Since ascorbate itself was oxidized in these reactions,the inhibition was not persistent and it was released once theascorbate present in the assay mixture had been oxidized. Ascorbatedelayed the oxidation of NADH 10-fold more efficiently thanthe oxidation of coniferyl alcohol. Although the level and theredox state of apoplastic ascorbate were lower in lignifyingneedles than in mature needles, the concentration, which was1.17 mM in apoplastic washing fluids, was sufficiently highto inhibit peroxidase activity in vitro. These results suggestthat peroxidases can catalyze lignification only if local differencesexist in the concentration of reduced ascorbate between lignifyingand non-lignifying tissues. (Received April 21, 1994; Accepted September 26, 1994)     

Effects of some Cyclic 'Crown' Polyethers on Potassium Uptake, Efflux and Transport in Excised Root Segments and Whole Seedlings

The ionophores benzo-18-crown-6 (18-C-6), t-butylbenzo-18-crown-6(TBB) and di-t-butyldibenzo-30-crown-10 (30-C-10) were testedfor their effects on potassium ion absorption in onion rootsegments, and in wheat and mung bean seedlings. Potassium uptake,efflux and transport were progressively reduced in onion rootsegments and seedlings by 18-C-6 over the range 0.1–1.0mM. The effects of TBB (up to 0.3 mM) were more severe but otherwisegenerally similar to those of 18-C-6 in seedlings. Both ionophoresreduced growth, and at the highest concentrations, resultedin root potassium ion content falling below initial values after48 h treatment. The effects of 30-C-10 were evident at muchlower concentrations, inhibition of net potassium uptake occurringabove 10^–4 M. Between 10^–4 and 10^–4 M 30-C-10,however, a modest but significant stimulation of potassium uptakewas observed in onion roots and seedlings; growth of seedlingswas largely unaffected. The reductions in potassium absorptionwere attributed to the promotion, by the ionophores, of facilitateddiffusion down the electrochemical diffusion gradient, counteringthe efficiency of the potassium ion influx pump. Stimulationof uptake at certain concentrations of 30-C-10 was consideredmore likely to be due to an inhibition of passive potassiumefflux, rather than a stimulation of active influx. The importanceof stability constants, bonding and lipophilicity, in determiningthe relative effectiveness of the ionophores, is discussed. Allium cepa L, onion, Triticum aestivum L, wheat, Phaseolus aureus L, mung bean, cyclic ‘crown’ polyethers, potassium fluxes, ion transport     

Diphosphorylation of platelet myosin by myosin light chain kinase.

Recently, one of the authors (K.I.) and other investigators reported that myosin light chain (MLC) of smooth muscle (gizzard, arterial and tracheal) was diphosphorylated by myosin light chain kinase (MLCK) and that diphosphorylated myosin showed a marked increase in the actin-activated myosin ATPase activity in vitro and ex vivo. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated diphosphorylation of MLC of platelet by MLCK in vitro. Our results are as follows. (1) Platelet MLC was diphosphorylated by a relatively high concentration (greater than 20 micrograms/ml) of MLCK in vitro. As a result of diphosphorylation, the actin-activated myosin ATPase activity was increased 3 to 4-fold as compared to the monophosphorylation. (2) Both di- and monophosphorylation reactions showed similar Ca2+, KCl, MgCl2-dependence. Maximal reaction was seen at [Ca2+] greater than 10(-6) M, 60 mM KCl and 2 mM MgCl2. This condition was physiological in activated platelets. (3) Di- and monophosphorylated myosin showed similar Ca2+, KCl-dependence of ATPase activity but distinct MgCl2-dependence. Diphosphorylated myosin showed maximal ATPase activity at 2 mM MgCl2 and monophosphorylated myosin showed a maximum at 10 mM MgCl2. (4) The addition of tropomyosin stimulated actin-activated ATPase activity in both di- and monophosphorylated myosin to the same degree. (5) ML-9, a relatively specific inhibitor of MLCK, inhibited the aggregation of human platelets induced by thrombin ex vivo in a dose-dependent manner. Moreover, this drug also partially inhibited both di- and monophosphorylation reactions and actin-activated ATPase activity. On the other hand, H-7, a synthetic inhibitor of protein kinase C, had little effect on the aggregation of human platelets induced by thrombin ex vivo. From these results, we conclude that diphosphorylation of platelet myosin by MLCK may play an important role in activated platelets in vivo.     

Purification and Properties of L-Arginine Decarboxylase of Evernia prunastri

An L-arginine decarboxylase was isolated from Evernia prunastrithallus. The enzyme was purified about 117-fold and showed apH optimum of 7.1 and a temperature optimum at 26°C. Itsmolecular weight was estimated as 300,000. The Evernia argininedecarboxylase was significantly inhibited by L-ornithine, ureaand putrescine. The K_m for L-arginine was about 12.5 mM. (Received March 9, 1981; Accepted June 26, 1981)     

Reconstitution of Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

Reconstituted proteoliposomes of tonoplast ATPase are formedon solubilization of tonoplast membranes from mung bean (Vignaradiata L.) with deoxycholate (DOC) in the presence of a mixtureof soybean phospholipids (asolectin), after removal of DOC bypassage through a PD-10 column (Pharmacia). This method is idealbecause of its simplicity and rapidity. Selective insertionof sets of tonoplast H⁺-ATPase polypeptides (68 kDa, 60 kDa,16 kDa and several minor polypeptides) into liposomes usingthis method was confirmed by SDS-PAGE and immuno-blotting withantibodies raised against 68-kDa and 60-kDa polypeptides. Pumping of protons across the membranes of the proteoliposomeswas demonstrated by quinacrine-fluorescence quenching in thepresence of ATP-Mg²⁺. ATP-Mg²⁺ was shown to be the preferredsubstrate in both reconstituted and native tonoplast vesicles,and its optimum concentration was 0.75 to 3.0 mM. Quenchingwas completely abolished by a channel-forming ionophore, gramicidinD, and an inhibitor of tonoplast H⁺-ATPase, KNO₃. Antibodiesto 68-kDa and 60-kDa peptides partially inhibited the pumpingof protons. The rate of pumping of protons increased with thenumber of proteoliposomes, the maximal concentration of whichwas equivalent to 250 µg of protein per reaction mixture.The optimum pH for pumping was 6.5 when inside of proteoliposomeswere loaded pH at 7.2. The rate of pumping of protons was reducedwhen proteoliposomes were made using asolectin and cholesterolat 3 : 1 (w/w), as compared with those made with asolectin alone. The ATPase activity in reconstituted proteoliposomes was inhibitedby KNO₃, with half-maximal inhibition at approximately 7 mM.The enzyme actively hydrolyzed ATP in preference to GTP, CTP,UTP, and ADP, but it did not hydrolyze pNPP or AMP. Antibodiesagainst the 60-kDa polypeptide strongly inhibited ATPase activityas compared to antibodies against the 68-kDa polypeptide. Theresults obtained in this study demonstrate directly that functionaltonoplast H⁺-ATPase can be inserted selectively into liposomes. (Received August 31, 1990; Accepted April 18, 1991)     

Pyridoxal 5-phosphate, Phenyl Phosphate and Acetyl Phosphate, as Inhibitors of Photophosphorylation Competitive with Phosphate

Pyridoxal 5-phosphate, phenyl phosphate and acetyl phosphate,as well as rß-naphthyl monophosphate, inhibited photophosphorylationof spinach chloroplasts competitively with P_i and noncompetitivelywith ADP. The apparent dissociation constant of the inhibitor-enzymecomplex (K_i) values of pyridoxal 5-phosphate, phenyl phosphateand acetyl phosphate for the P_i site were 1.1, 3.8 and 2.4 _mM,respectively. These organic phosphates inhibited Ca²⁺-ATPaseof the isolated coupling factor 1 (CF₁) (EC 3.6.1.3 [EC] ) noncompetitivelywith ATP. AMP, creatine phosphate, fructose 1,6-bisphosphate,glucose 6-phosphate, 3-phosphoglyceric acid, ribose 5-phosphateand PP_i did not significantly inhibit photophosphorylation.Like rß-naphthyl monophosphate, pyridoxal 5-phosphateand phenyl phosphate inhibited photophosphorylation and thecoupled electron transport, but were almost without effect onthe basal electron transport. On the other hand, acetyl phosphateconsiderably inhibited photophosphorylation, but had almostno effect on the coupled electron transport rate and the basalrate. The results suggest that these organic phosphates inhibitphotophosphorylation by binding at the P_i site on the activecenter of CF₁ and that their binding inhibits the ATPase activityof isolated CF₁. These four organic phosphates which inhibited photophosphorylationcompetitively with Pi could not substitute for ADP or ATP ininhibiting ferricyanide photoreduction by decreasing H⁺-permeabilitythrough CF₁ and in protecting the ATPase of isolated CF₁ againstcold-anion inactivation. ¹ This work was supported in part by Grants-in-Aid for ScientificResearch from the Ministry of Education, Science and Culture,Japan to H.S. (Received May 25, 1981; Accepted September 28, 1981)