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; )     

Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

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)     

Modulation by Bicarbonate of Catalytic and Regulatory Properties of C4Phosphoenolpyruvate Carboxylase from Amaranthus hypochondriacus: Desensitization to Malate and Glucose 6-Phosphate and Sensitization to Mg2+

The catalytic and regulatory properties of phosphoenolpyruvate(PEP) carboxylase (PEPC) are modulated remarkably by the increasein the level of bicarbonate in the assay medium. The activityof PEPC increased by two-fold as the concentration of bicarbonatewas raised from 0.05 to 10 mM. During this state, there wasonly marginal effect on K_m for PEP, while the affinity of PEPCto Mg²⁺ increased by >2 fold. In contrast, the sensitivityof PEPC to malate decreased with increasing concentration ofHCO₃^–. Similarly, the stimulation by glucose 6-phosphate(G-6-P) at optimal concentration (10 mM) of HCO₃^– wasmuch less than that at suboptimal concentration (0.05 mM). K₁for malate increased by about 3 fold and K_a for G-6-P risedby fourfold as bicarbonate concentration was rised from 0.05to 10 mM. These results suggest that HCO₃^– desensitizesPEPC to both malate and G-6-P. Further, these changes were manifestedin both dark- as well as light-forms of the enzyme. Similarresults were obtained with PEPC in leaf extracts or in purifiedform. We therefore propose that bicarbonate-induced changesare independent of phospho-rylation and possibly through a significantchange in the conformation of the enzyme. This is the firstdetailed report indicating marked modulation of regulatory andcatalytic properties of PEPC by bicarbonate, one of its substrate. (Received April 14, 1998; Accepted September 22, 1998)     

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; )     

Equilibration of Adenylates by Maize Leaf Adenylate Kinase: Effects of Magnesium on Apparent and True Equilibria

In this study, the effect of magnesium on equilibration of adenylatesby purified maize leaf adenylate kinase (AK) was investigated.The equilibration was expressed in terms of either apparentequilibrium constant, defined as K_app=(ATP_total)(AMP_total)/(ADP_total)²,or true equilibrium constant, defined as K_true=(Mg-ATP)(AMP_free)/(Mg-ADP)(ADP_free).At a fixed concentration of free magnesium (1?8–1?9 mM),the K_app, and K_true were constant at 0?76?0?10 and 6?02?0?75,respectively. On the other hand, at the free magnesium rangeof 0?00l4 to 8?3 mM, the K_app varied from 0?30 to 1?27, whileremained constant at 5?93?0?31. The data indicate that, contraryto previous speculations, leaf AK does not maintain an equilibriumof total adenylates. Rather, the enzyme governs an equilibriumof Mg-ADP, free ADP, Mg-ATP, and free AMP, which are the truesubstrates/products of the AK reaction. Some implications ofthis finding for studies on energy metabolism in plant tissuesare discussed. Key words: Adenylate kinase, adenylate energy charge, adenylates, C₄-photosynthesis, magnesium     

The Effect of Isonicotinyl Hydrazide on the Photosynthetic Incorporation of Radioactive Carbon Dioxide into Ethanol-Soluble Compounds of Chlorella

The effect of 10^-2M. isonicotinyl hydrazide (isoniazid) on theincorporation of radioactive carbon dioxide by Chlorella duringphotosynthesis has been studied under steady-state conditionsat two carbon dioxide concentrations. Isoniazid treatment resultsin increased radioactivity in sucrose, glycollic acid, and glycineand decreased radioactivity in sugar monophosphates, serine,and alanine. An unidentified compound which is strongly radioactiveafter short-term exposures to ¹⁴CO₂ is present in isoniazid-treatedcells. It is suggested that isoniazid pre-dominantly inhibitsthe conversion of glycine to serine.     

The Localization in Cultured Carrot Cells of Factors that Induce their Growth

Various previously recognized parts of the complex of growthfactors present in the liquid endosperm of the coconut or inimmature fruits of Aesculus woerlitzensis were generally tritiated.The labeled growth factors were applied singly to culture mediawhich contained balanced requirements that had caused carrotexplants to proliferate and grow in accordance with combinationsof growth factors supplied. By the usc of electron microscopyand autoradiography, the radioactivity from each source wasdetected in the cells and its density and distribution, in theform of developed grains over different cellular compartmentsand organelles, was determined. The tabulated data relate tofour labeled sources as observed over seven cellular compartmentsunder six experimental treatments. Electron micrographs alsoshow how the radioactivity from the various sources relatedto organization of the cells. The distribution of radioactivity within the cells varied withthe source. Both ³H-myo-inositol and the tritiated growth factorsfrom Aesculus (³H-AF_1Aesc) with which it interacts (as in so-calledGrowth Promoting System I) contributed radioactivity, preferentially,to cell walls and sites of their formation in culturcd carrotcells. Both ³H-IAA and ³H-zatin (as in so-called Growth PromotingSystem II) contributed their radioactivity preferentially tothe nucleoli of the cultured cells. Some other conspicuous distributionsof radioactivity (e.g. from ³H-AF_1Aesc to plastids and from³H-IAA to the interstitial substance, i.e. middle lamella, whereenlarging cells separate) involved these tritiated moietieswithout regard to their counterparts in Growth Promoting SystemsI and II, respectively. The problems raised by such multiple effects due to differentgrowth factors acting singly and in combinations at differentcell sites are both recognized and discussed. growth factors, Aesculus woerlitzeensis, autoradiography, tritiation, cell sites, carrot, Daucus carota, coconut, electron microscopy     

Involvement of Cyclic AMP in ABA- and Ca2--Mediated Signal Transduction of Stomatal Regulation in Vicia faba

The focus of this study is to investigate possible involvementof cyclic AMP in regulation of Vicia stomatal movements. Thepresence of 0.1 mM 8-Br-cAMP, a membrane-permeable analogueof cAMP, alone in the incubation medium did not affect stomatalopening in the light in leaf epidermal peel experiments. However,addition of 0.1 mM 8-Br-cAMP completely reversed exogenous ABA-and C^a2+-induced inhibition of stomatal opening. Consistentwith these results, patch-clamping experiments showed that intracellularaddition of 0.5 mM or 1 mM cAMP significantly reversed the inhibitionof whole-cell inward K⁺ currents by internally supplied 13 µMCa²⁺ or 10 µM ABA in stomatal guard cell protoplasts,respectively. Furthermore, intracellular addition of either10 µM prostaglandin E₁ (PGE1, an adenylate cyclase activator)or 1 mM 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesteraseinhibitor) mimicked the effect of exogenous cAMP on the removalof ABA- or Ca²⁺ inhibition of inward K⁺-current. These resultssuggest that a cAMP signaling pathway is involved in signaltransduction in stomatal regulation by interacting with ABAand Ca²⁺ signaling cascades. A hypothetical mechanism by whichcAMP may regulate K⁺ in stomatal guard cells is also discussed. (Received May 6, 1999; Accepted August 27, 1999)     

Ionic Relations of Garden Orache, A triplex hortensis L.: Growth and Ion Distribution at Moderate Salinity and the Function of Bladder Hairs

The growth of garden orache, A triplex hortensis was studiedunder conditions of mild NaCl or Na₂SO₄ salinity. Growth, drymatter production and leaf size were substantially stimulatedat 10 mM and 50 mM Na⁺ salts. Increased growth, however, appearedto be due to a K⁺-sparing effect of Na⁺ rather than to salinityper se. The distribution of K⁺ and Na⁺ in the plant revealeda remarkable preference for K+ in the roots and the hypocotyl.In the shoot the K/Na ratio decreased strongly with leaf age.However, the inverse changes in K⁺ and Na⁺ content with leafage were dependent on the presence of bladder hairs, which removedalmost all of the Na⁺ from the young leaf lamina. Measurementsof net fluxes of K⁺ and Na⁺ into roots and shoots of growingAtriplex plants showed a higher K/Na selectivity of the netion flux to the root compared to the shoot. With increasingsalinity the selectivity ratio S_{K, Na}^* of net ion fluxes tothe roots and to the shoots was increased. The data suggestthat recirculation of K⁺ from leaves to roots is an importantlink in establishing the K/Na selectivity in A. hortensis plants.The importance of K⁺ recirculation and phloem transport forsalt tolerance is discussed. Key words: Atriplex hortensis, Salinity, Potassium, Sodium, K⁺ retranslocation, Bladder hairs, Growth stimulation     

Synthesis of Citrulline and Arginine in Chlorella pyrenoidosa

The distribution of radioactivity in Chlorella during dark ¹⁴CO₂fixation was investigated either (a) in normal cells with andwithout added ammonium chloride, or (b) in nitrogen-starvedcells supplied with intermediates of the Krebs-Henseleit ureacycle. In the control experiments almost all the activity was presentin compounds of or associated with, the tricarboxylic acid cycle. The amino-acids citrulline and arginine became radioactive onlyin the presence of ammonia or ornithine where initially theycomprised 40–60 per cent. of the total activity, reactionsof the Krebs–Henseleit urea cycle being implicated intheir formation. No evidence could be found for a complete ureacycle. Unidentified compounds deriving their radioactivity fromthe C₄ carbon of citrulline and/or arginine were detected andformed up to 40 per cent. of the total ¹⁴CO₂ incorporated after25 min.     

Purification and Characterization of Assimilatory Nitrate Reductase from the Cyanobacterium Plectonema boryanum

Assimilatory nitrate reductase (NR) was solubilized by acetonetreatment from Plectonema boryanum and was purified 7,700-foldby heat treatment, ammonium sulfate fractionation and chromatographyon DEAE-Sephacel and Sephadex G-150. Purified NR had a specificactivity of 85 µmol NO₂^– formed min^–1 mg^–1protein. The enzyme retained both ferredoxin (Fd)- and methylviologen (MV)-linked NR activities throughout the purificationprocedure. Molecular weight was 80,000. The pH optimum was 10.5in the MV-assay and 8.5 when assayed with enzymatically reducedFd as the electron donor. Apparent Km values for nitrate andMV were 700 µM and 2,500µM in the MVassay and 55µM and 75 µM for nitrate and Fd in the Fd-assay.The enzyme was inhibited by thiol reagents and metal-chelatingreagents. (Received October 1, 1982; Accepted March 8, 1983)     

The effects of valinomycin on respiration and volume changes in plant mitochondria

The effects of valinomycin on the respiration and volume changeshave been studied with isolated mitochondria from bean hypocotyl(Phaseolus vulgaris L.) and cauliflower bud (Brassica oleraceaL.). In the presence of 10 mM K salts of chloride, acetate,phosphate, and sulfate respiration is stimulated by valinomycinconcomitant with osmotic swelling. When swelling declines respirationwith organic acid substrates also declines. In the presenceof the K salts of acetate and PO₄ but not Cl^– the terminationof respiration leads to contraction. The contraction in K-PO₄is inhibited by addition to the external medium of between 65to 100 mM K-PO₄. The results are interpreted to suggest thatvalinomycin in the presence of KCl facilitated the movementof K down an electrical gradient, with the Cl anion followingand osmotic swelling resulting. However, in a medium containingacetate or PO₄ the anions are actively transported against anelectrical gradient at the expense of metabolic energy. Valinomycinfacilitates the influx of K⁺ with the actively transported anionand swelling follows. When respiration terminates the activelytransported anions move passively back down their electrochemicalgradient and osmotic contraction follows. ¹ Present address: Department of Biology, Fort Lewis College,Durango, Colorado 81301, U.S.A. (Received July 21, 1972; )     

Characterization of inorganic phosphate transport in osteoclast-like cells

Osteoclasts possess inorganic phosphate (P_i) transport systems to take up external P_i during bone resorption. In the present study, we characterized P_i transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-B ligand (RANKL). In undifferentiated RAW264.7 cells, P_i transport into the cells was Na⁺ dependent, but after treatment with RANKL, Na⁺-independent P_i transport was significantly increased. In addition, compared with neutral pH, the activity of the Na⁺-independent P_i transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na⁺-independent system consisted of two components with K_m of 0.35 mM and 7.5 mM. The inhibitors of P_i transport, phosphonoformic acid, and arsenate substantially decreased P_i transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K⁺ ionophore, valinomycin, significantly suppressed P_i transport activity. Analysis of BCECF fluorescence indicated that P_i transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K⁺ ion stimulated P_i transport, suggesting that membrane voltage is involved in the regulation of P_i transport activity. Finally, bone particles significantly increased Na⁺-independent P_i transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a P_i transport system with characteristics that are different from those of other Na⁺-dependent P_i transporters. We conclude that stimulation of P_i transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment. Na⁺-dependent phosphate cotransporter; RAW264.7; phosphate uptake     

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; )     

An Experimental Study of Calcium Acquisition and its Effects on the Calcifuge Moss Pleurozium schreberi

In a field experiment to investigate the sources and effectson growth of Ca in the calcifuge moss Pleurozium schreberi,significant quantities of Ca reached the growing shoot apicesfrom a CaCO₃ layer placed on the mineral soil surface Top applicationsof 0.5 and 5 mol m^–3 CaCl₂ raised the exchangeable andintracellular Ca concentrations and displaced natural exchangeableK and Mg The 5 mol m^–3 CaCl₂ treatment also caused a significantreduction in intracellular Mg indicating that Mg uptake is dependenton an initial exchange step No growth differences were notedbetween treatments, possibly because ionic changes had not reacheda detrimental level within the 28 weeks of the experiment ina second experiment, shoot apices of Pleurozium schreberi, Pseudoscleropodiumpurum and Calliergon cuspidatum were grown on nylon gauze underintermittent distilled-water mist At weekly intervals the shootswere saturated with CaCl₂ solutions providing factorial combinationsof Ca and pH Growth of C cuspidatum and P purum from chalk soilwas reduced at high (0.01) Ca concentration whereas Pleuroziumschreberi and Pseudoscleropodium purum from acidic clay wereunaffected The pH treatments did not significantly affect mossgrowth Initial tissue levels of K and Mg were lower in the mossesfrom chalk and it is suggested that the CaCl₂ treatments causednutrient deficiencies in these plants Mosses from acidic soilcontained less exchangeable Ca than the chalk plants and grewpoorly in the absence of CaCl₂, perhaps due to the developmentof Ca deficiency Bryophyte growth, calcium uptake, pH, mineral nutrition, Pleurozium schreberi, Pseudoscleropodium purum, Calliergon cuspidatum     

Induction of Glucose 6-Phosphate Transport Activity in Chloroplasts of Mesembryanthemum crystallinum by the C3-CAM Transition

To study possible changes in the transport metabolites betweenchloroplasts and cytoplasm during CAM induction of Mesembryanthemumcrystallinum, we compared substrate specificity of P1¹ translocator(s)in isolated chloroplasts from the C₃ and CAM-induced plants.The [¹⁴C]glu-cose 6-phosphate (G6P) transport activity was significantonly in the chloroplasts of CAM-mode plants and not detectablein those of C₃-mode, while a similar high rate of [³²P]P_i uptakewas observed with both types of chloroplasts. Kinetic analysisof G6P uptake in the CAM chloroplasts showed a high V_max [10.6µmol (mg Chl)^–1 h^–1] and a comparatively lowK_m value (0.41 mM); the latter was similar to K_i values of P_i,3-phosphoglycerate and phospho-enolpyruvate, 0.30, 0.34 and0.47 mM, respectively. On the other hand, [32P]Pi uptake inthe CAM chloroplasts was inhibited competitively by G6P witha K_i value (8.4 mM) 20-fold higher than the K_m value for G6Puptake, while that in C₃ chloroplasts was not inhibited at all.These results suggest that a new G6P/P_i, counterexchange mechanismis induced in the chloroplast envelope of CAM-induced M. crystallinumin addition to the ordinary type of P, translocator, that cannottransport G6P, already present in the C₃-type chloroplasts. (Received March 17, 1997; Accepted May 10, 1997)     

(Na+-K+)-stimulated ATPase in Leaves of C4 Plants: Possible Involvement in Active Transport of C4 Acids

Leaves of three C₄ plants, Setaria italica, Pennisetum typhoides,and Amaranthus paniculatus possessed five- to ten-fold higheractivities of a (Na⁺-K⁺)-dependent ATPase than those of twoC₃ plants, Oryza sativa and Rumex vesicarius. Na⁺-K⁺ ATPasefrom leaves of Amarathus exhibited an optimal pH of 7?5 andan optimal temperature of 35 ?C. It required 40 mM K⁺ and 80mM Na⁺ for maximal activity. Ouabain partially inhibited (Na⁺-K⁺)-dependentATPase activity in leaves of C₄ plants. Ouabain also blockedthe movement of label from initially formed C₄ acids into endproducts in leaves of only C₄ plants, Setaria and Amaranthusbut not in a C₃ plant, Rumex. We propose that Na⁺-K⁺ ATPasemay mediate transfer of energy during active transport of C₄acids from mesophyll into the bundle sheath.     

Protective role of extracellular chloride in fatigue of isolated mammalian skeletal muscle

A possible role of extracellular Cl^– concentration ([Cl^–]_o) in fatigue was investigated in isolated skeletal muscles of the mouse. When [Cl^–]_o was lowered from 128 to 10 mM, peak tetanic force was unchanged, fade was exacerbated (wire stimulation electrodes), and a hump appeared during tetanic relaxation in both nonfatigued slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles. Low [Cl^–]_o increased the rate of fatigue 1) with prolonged, continuous tetanic stimulation in soleus, 2) with repeated intermittent tetanic stimulation in soleus or EDL, and 3) to a greater extent with repeated tetanic stimulation when wire stimulation electrodes were used rather than plate stimulation electrodes in soleus. In nonfatigued soleus muscles, application of 9 mM K⁺ with low [Cl^–]_o caused more rapid and greater tetanic force depression, along with greater depolarization, than was evident at normal [Cl^–]_o. These effects of raised [K⁺]_o and low [Cl^–]_o were synergistic. From these data, we suggest that normal [Cl^–]_o provides protection against fatigue involving high-intensity contractions in both fast- and slow-twitch mammalian muscle. This phenomenon possibly involves attenuation of the depolarization caused by stimulation- or exercise-induced run-down of the transsarcolemmal K⁺ gradient. potassium; skeletal muscle contraction; membrane potential; myotonia     

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine