Enzymatic Conversion of Pheophorbide a to the Precursor of Pyropheophorbide a in Leaves of Chenopodium album

Soluble proteins extracted from leaves of Chenopodium albumcatalyzed the conversion of pheophorbide a to a precursor ofpyropheophorbide a, putatively identified as C-13²-carboxyl-pyropheophorbidea. The precursor was then decarboxylated non-enzymatically toyield pyropheophorbide a. Soluble proteins and pheophorbidea, as the substrate, were required for the formation of theprecursor, and boiled proteins were enzymatically inactive.The maximum rate of conversion of pheophorbide a to the precursoroccurred at pH 7.5. The K_m for pheophorbide a was 12.5 µMat pH 7.0. Both pheophorbide b and bacteriopheophorbide a couldserve as substrates, but protopheophorbide a could not. Formationof methanol was detected during the enzymatic reaction, an indicationthat the enzyme is an esterase. Among seven alcohol analogstested, only methanol inhibited the enzymatic activity uncompetitively,with a K₁ of 71.6 mM. Mass-spectrometric (MS) analysis of theprecursor yield a peak at m/z 579 that indicated the releaseof a methyl group from pheophorbide a. It appears thereforethat the enzyme catalyzes the demethylation of the carbomethoxygroup at C-13² of pheophorbide a by hydrolysis to yield methanoland the precursor, C-13²-carboxyl-pyropheophorbide a, whichis converted to pyropheophorbide a by spontaneous decarboxylation.We have tentatively designated the enzyme "pheophorbidase".The presence of the enzyme was dependent on plant species andit was expressed constitutively. ¹Present address: Faculty of Science, Shizuoka University, Ohya,Shizuoka, 422 Japan     

Cell Expansion and Wall pH in the Fern Regnellidium diphyllum, A Plant Lacking Acidinduced Growth

Petioles of the semi-aquatic fern Regnellidium diphyllum donot show acid growth but low wall pH is a necessary conditionfor maximum rates of IAA-induced cell expansion. Measurementsof wall pH by two indirect methods indicate an unusually lowvalue, in the range pH 4 to 5. This is one to two pH units belowthat estimated for petioles of the semi-aquatic dicotyledonNymphoides peltata, a species in which IAA aand ethylene causegrowth responses very similar to those in Regnellidium but whereacid growth occurs. Having shown previously that fusicoccinenhances proton secretion in both Regnellidium and Nymphoides,we now show that although it causes a reduction in the estimatedapoplast pH to below 4·0 in Regnellidium, cell expansionis not promoted. The FC-induced reduction in pH in Nymphoidesis less and occurs more slowly, but growth is promoted significantly;when IAA and fusicoccin are present together, growth promotionis approximately additive for Nymphoides A model is proposed for Regnellidium in which equilibrium wallpH is maintained at a low value that is optimal for acid growth,the availability of acid-labile sites in the wall being thechief limitation to cell extension. We suggest that this controlmechanism may be widespread for organs without a cuticle, includingroots and the gametophytes of lower plants growing in acidicconditions. Key words: Acid growth, wall pH, fusicoccin, Regnellidium diphyllum, Nymphoides peltata     

Studies in the Principles of Phytotoxicity: III. THE pH FACTOR AND THE TOXICITY OF 3:5-DINITRO-o-CRESOL, A WEAK ACID

It has previously been shown that the concentration of 3:5-dinitro-ocresolrequired in the external medium to halve (a) the growth rateof the mould Trichoderma viride or (b) the respiration rateof yeast increased same 75–100 times as the pH changedfrom 4 to 7. In the present investigation it has been foundthat when solutions or suspensions of 3:5-dinitro-o-cresol areapplied as sprays to seedlings of Brassica alba the concentrationneeded to kill half the plants changes by a factor of less than2 over the same pH range. On the other hand, when Lemma minor is grown in nutrient solutioncontaining the toxicant, the concentration required to halvethe rate of frond multiplication increases sharply as the pHis raised from 5.4 to 6.8. Moreover, when leaf disks of B. albaare vacuum infiltrated with buffered solutions of 3:5-dinitro-o-cresolthe concentration needed to halve the respiration rate rises50-fold between pH 4 and 8. It is considered that the essential condition which determinesthe similarity of the result for L. minor, the leaf disks, andmicro-organisms is that a relatively large volume of solutioncontaining the toxicant is in direct contact with the tissues.In contrast, under the conditions of spraying the volume ofspray droplets relative to that of plant tissue is small. Thusthe pH effect ie masked since the buffering capacity of thecells exceeds that of the droplets, with the result that 3:5-dinitro-o-cresollargely acts upon the cells at a pH determined by the tissues,a pH which may bear little relation to that of the originalsolution. Supporting evidence is produced that the pH factor operatesin the same way for other weak acids, such as the chlorophenoxyaceticacids.     

High pH Causes Disintegration of the Root Surface in Lupinus angustifolius L.

The effect of high pH on the morphology and anatomy of the rootsof lupin (Lupinus angustifolius L. cv. Yandee) and pea (Pisumsativum L. cv. Dundale) was examined in buffered solution. Themorphology and anatomy of lupin roots were markedly altered,and root growth was reduced by increasing solution pH from 5·2to 7·5, whereas pea roots were unaffected. In lupin roots,pH 7·5 caused disintegration of the root surface andimpaired root hair formation. Lupin roots grown at pH 7·5also had decreased cell lengths but increased cell diameterin both the epidermis and the cortex in comparison to rootsgrown at pH 5·2. High pH reduced cell volume greatlyin the epidermis, to a lesser extent in the outer cortex andnot at all in the inner cortex. It appears that in lupins, theprimary detrimental effects of growth at pH 7·5 is reducedlongitudinal growth of cells near the root surface with a consequentreduction in elongation of the cells in inner cortex.Copyright1993, 1999 Academic Press Lupinus angustifolius L., Pisum sativum L., high pH, root morphology, root anatomy     

Contractile Proteins of a Benthic Fish. III. Subunit Composition of Myosin

SYNOPSIS. Ultracentrifugal and electrophoretic experiments arereported on the subunit composition of myosin from skeletalmuscle of a benthic fish, Coryphaenoides species. Coryphaenoidesmyosin undergoes extensive association in concentrated KGI solutionsat neutral pH, but sedimentation equilibrium experiments indicatethe presence of a small fraction (3%) of monomeric myosin withmolecular weight approximately 440,000. At pH 11, some of theaggregated myosin is dissociated, and monomeric myosin is itselfdissociated into a heavy component (410,000 mol wt) and a lightcomponent (14,000 mol wt) that comprises 5–7% of the protein.The lialkali component of Coryphaenoides myosin yields a singlepredominant band on cellulose acetate electrophoresis and SDS-ureaelectrophoresis in 9% acrylamide gel. The stoichiometric evidenceindicates that Coryphaenoides myosin contains two heavy chains(205,000 mol wt) and two light chains (14,000 mol wt) that areequivalent with respect to net electrostatic charge and molecularweight. Preparations of myosin obtained by direct extractionfrom muscle mince and by dissociation of actomyosin extractedfrom muscle mince also contain 5% of a 47,000 mol wt componentpresumably actin), traces of 34–36,000 mol wt component,and about 5.7% of low molecular weight material (10,000–15,000)that probably represents contaminant protein, although the possibilityof denatured nivosin subunits cannot be excluded.     

Efflux of photosynthate and acid from developing seed coats of Phaseolus vulgaris L.: a chemiosmotic analysis of pump-driven efflux

Seed coats of Phaseolus vulgaris L. unload photosynthetic products,mineral ions and acid into the apoplastic space surroundingthe embryo. We report measurements, on detached seed coats,of the rates of unloading of photosynthates, ions and acid atdifferent external pH and in the presence of treatments intendedto alter the rate of proton pumping. We also report measurementsof membrane potential difference (PD) and of cytoplasmic pHunder the same conditions, measurements which have allowed usto validate the treatments we used and to investigate functionalrelationships between membrane processes. A chemiosmotic model of the seed-coat cell membrane is proposed,in which sucrose efflux and acid efflux are both driven by theproton pump. Sucrose efflux is proposed to occur by sucrose/protonantiport driven by the proton-motive force (PMF), and acid effluxto occur by pumped protons accompanied by a passive efflux ofanions. We use our measurements to estimate the net efflux ofsucrose on the antiporter and the total efflux of protons onthe pump. We have tested the model by using experimental treatments designedto manipulate the pump rate as the independent variable. Underthese conditions, and assuming the model is correct, the pumprate determines the cytoplasmic pH. Over the range covered byour experiments the net sucrose efflux is dependent on externaland cytoplasmic pH, the latter having the major role. The effluxof acid, under the same treatments, depends primarily on theproton pump rate, and was found to be well fitted by a quadraticfunction of pump rate. This means that, as pump rate increases,an increasing proportion of the pump output is used by acidefflux and a decreasing proportion by sucrose antiport. The membrane PD, although an important component of the PMF,does not appear to function in rate control of net sucrose orof acid efflux, since neither efflux is correlated with membranePD under our treatments which vary the pump rate. The PD correlateswell with external potassium concentration, and seems largelydetermined by the diffusion of potassium ions and anions. Key words: Phaseolus vulgaris L, photosynthate efflux, proton pump, sucrose/proton antiport, seed coat, membrane transport model     

Role for the Vacuolar H+-ATPase in Regulating the Cytoplasmic pH: An in Vivo Study Carried out in chl1, an Arabidopsis thaliana Mutant Impaired in NO-3 Transport

The effects of the growth in a medium containing NH₄NO₃ as nitrogensource were studied on cell sap pH, cytoplasmic pH and malatecontent in chl1, an Arabidopsis thaliana mutant impaired inchlorate and nitrate transport. In all the conditions testedthe pH of the cytoplasm in chl1 was more alkaline, and thatof the vacuole was more acidic as compared with those measuredin wt. Treatment with bafilomycin A1, a specific inhibitor ofthe vacuolar H⁺-ATPase, induced a small alkalinization of thevacuole, and a significant acidification of the cytoplasm, theseeffects being greater in chl1 than in wt. The greater responseof the mutant to bafilomycin Al suggests that, in the absenceof the inhibitor, the activity of the tonoplast H⁺-ATPase inchl1 is higher than in wt, this diversity being a possible reasonfor the differences in intracellular pH detected between thetwo strains. A possible role for the vacuolar H⁺-ATPase in regulatingthe cytoplasmic pH is discussed. (Received August 2, 1995; Accepted February 1, 1996)     

Regulation of Pyruvate Decarboxylase In Vitro and In Vivo

Results presented in this paper strongly support the view thatregulation of the key enzyme of alcoholic fermentation, pyruvatedecarboxylase (PDC), is achieved in a number of ways, all associatedwith possible lowering of the cytoplasmic pH during anoxia.These mechanisms include not only the well-known acid pH optimumof PDC, but also long-term, reversible changes in characteristicsof the enzyme established both in vitro and in vivo. Following transfer of desalted extracts from pH 6.0 to 7.4,maximal activity of PDC was decreased, while there was a considerableincrease in the lag before maximal activity was reached. Similarchanges in enzyme characteristics were observed when wheat (Triticumaestivum L. cv. Gamenya) roots and rice (Oryza sativa L. cv.Calrose) coleoptiles were transferred from anoxic to aerobicsolutions, provided PDC was assayed within 10 min of the startof maceration. All of the above changes were usually readilyreversible when extracts were returned to pH 6.0, or when plantswere returned to anoxic solutions. Additional regulation of PDC would be achieved by the S_0.5 forpyruvate which is 0.75 mol m^–3 at pH 6.0, 1.0 mol m^–3at pH 6.8, and 2.5 mol m^–3 at pH 7.4; the latter is wellabove estimates for pyruvate concentrations in the cytoplasmof aerated tissues. We assess that the combined effects of the acid pH optimum,the high S_0.5 at pH 7.4 and the long-term decreases in activityobserved during incubation at pH 7.4 would reduce PDC activityin aerobic cells to at most 7% of the activity in anoxic cells.Possible additional controls for the pathway of alcoholic fermentationare briefly considered. Key words: PDC, regulation, anoxia     

Methylation of Xenobiotic Thiols by Euglena gracilis: Characterization of a Cytoplasmic Thiol Methyltransferase

The green alga Euglena gracilis contains a thiol methyltransferasethat catalyzes the S-adenosylmethionine-dependent methylationof pentachlorobenzenethiol. The enzyme was localized in thecytoplasm and partially purified. The pH optimum for the enzymewas 6.5. The enzyme methylated a number of foreign thiols, butnot the cellular thiols, glutathione or cysteine. Phenols andanilines were not substrates. When pentachloro-benzenethiolwas the methyl acceptor the K_m was found to be 82 µM andthe corresponding K_m for S-adenosylmethionine was 140 µM.The molecular weight of the enzyme was 21,000, as determinedby gel filtration. A role for this enzyme in detoxifying xenobioticthiols is proposed. (Received September 28, 1984; Accepted April 25, 1985)     

Cytosolic pH regulates GCl through control of phosphorylation states of CFTR

Our objective inthis study was to determine the effect of changes in luminal andcytoplasmic pH on cystic fibrosis transmembrane regulator (CFTR)Cl conductance(G_Cl). Wemonitored CFTRG_Cl in the apicalmembranes of sweat ducts as reflected byCl diffusion potentials(V_Cl) andtransepithelial conductance(G_Cl). We foundthat luminal pH (5.0-8.5) had little effect on thecAMP/ATP-activated CFTRG_Cl, showing thatCFTR G_Cl ismaintained over a broad range of extracellular pH in which it functionsphysiologically. However, we found that phosphorylation activation ofCFTR G_Cl issensitive to intracellular pH. That is, in the presence of cAMP and ATP [adenosine5'-O-(3-thiotriphosphate)],CFTR could be phosphorylated at physiological pH (6.8) but not at lowpH (~5.5). On the other hand, basic pH prevented endogenousphosphatase(s) from dephosphorylating CFTR.After phosphorylationof CFTR with cAMP and ATP, CFTRG_Cl is normallydeactivated within 1 min after cAMP is removed, even in the presence of5 mM ATP. This deactivation was due to an increase in endogenousphosphatase activity relative to kinase activity, since it was reversedby the reapplication of ATP and cAMP. However, increasing cytoplasmicpH significantly delayed the deactivation of CFTRG_Cl in adose-dependent manner, indicating inhibition of dephosphorylation. Weconclude that CFTRG_Cl may beregulated via shifts in cytoplasmic pH that mediate reciprocal controlof endogenous kinase and phosphatase activities. Luminal pH probably has little direct effect on these mechanisms. This regulation of CFTRmay be important in shifting electrolyte transport in the duct fromconductive to nonconductive modes.

     

Studies in the Principles of Phytotoxicity: IV. THE EFFECTS OF THE DEGREE OF NITRATION ON THE TOXICITY OF PHENOL AND OTHER SUBSTITUTED BENZENES

1. The relative toxicity of phenol, o- and p-nitro, 2:4-dinitro-,and 2:4:6- trinitrophenol was measured by up to five techniques.Determinations were made of the concentrations required to reduceby half (i) the growth rate of Trichoderma viride at pH 3, (ii)the frond multiplication rate of Lemna minor at pH 5·1–5·4,(iii) the respiration rate of yeast at pH, (iv) the respirationrate of infiltrated leaf disks of Brassica alba at pH 3, and(v) the number of B. alba seedlings surviving after sprayingwith buffered solutions of the compounds. Whenever possibletoxicity was measured at a pH level below pK so as to eliminatethe effect of pH on toxicity, but in some of the measurementswith 2:4-dinitro- phenol (pK = 4·0) and with picric acid(pK = 0·8) this was not feasible. It was then necessaryto measure toxicity at a pH at which the compound was much dissociated;from the measured values of toxicity of dinitrophenol the toxicityat a lower pH was estimated from the generalized pH-activityrelationship of Simon and Beevers (5952). 2. The relative toxicity of the nitrophenols is very similarwhichever test procedure is employed. The first two nitrationsare accompanied by large increases in toxicity, but with thethird successive nitration there is a decrease. 3. The toxicity of the corresponding mono, di, and trinitroderivatives of anisole, phenetole, toluene, and nitrobenzenewas determined using T. viride. In each series the first andthird stages of nitration brought about large increases in toxicitybut the intermediate one had little effect. 4. All the p-nitro compounds were more toxic than their o- isomers. 5. On the basis of the concepts put forward by Ferguson (1939)and Albert (1951) it is concluded that the toxic action of thenitroanisoles and nitrotoluenes is ‘structurally non-specific’,i.e. the effect is not dependent on the presence or absenceof a particular chemical group in the molecule. With both thenitrobenzene and the phenol series the results suggest thatthe exact chemical structure is an important factor.     

H+ tolerance of Chara Internodal Cells and Apparent Net Influx of H+ in Weakly Acidic Solutions: Implication of the Net Flux of H+ as a Minor Component among the Total Net Flux of Ions across the Intact Cell Membrane of Chara

Precise measurements of the net flux of protons in Chara internodalcells were made with a recently designed high-resolution pH-meter.Survival of intact Chara internodal cells in artificial pondwater (APW) that contained HC1 at various concentrations wasalso examined. The apparent net flux of H⁺ was inward and muchsmaller than that reported so far. In APW at pH 4.005, a valuehigher than the extracellular pH expected from the values ofH⁺ efflux reported to date, all of the intact Chara internodalcells died within a day. With reference to the data on the circadianflow of ions in the pulvinus of Phaseolus [Kiyosawa (1979) PlantCell Physiol. 20: 1621–1634, Hosokawa and Kiyosawa (1983)Plant Cell Physiol. 24: 1065–1072] and ionic regulationin Chara L-cells [Kiyosawa and Okihara (1988) Plant Cell Physiol.29: 9–19], a discussion is presented of the prossiblyminor contribution of the net flux of H⁺ in the generation ofthe electrical membrane potential. Regulation of the net fluxof H⁺ in weakly acidic APW is also discussed. (Received September 4, 1989; Accepted January 25, 1990)     

Evidence of a Copper-tolerant Ecotype of the Hepatic Solenostoma crenulatum

Two populations of the hepatic Solenostoma crenulatum, fromcopper mine and lead mine spoil tips, were compared for theirresistance to supplied copper. Incubation in copper solutionsshowed copper uptake to be very similar in the two populations.On the basis of greater potassium leakage, indicative of membranedamage, and inhibition of photosynthesis, in the lead mine populationsupplied with copper, it is suggested that the copper mine populationis a copper-tolerant ecotype. Potassium loss was a specificresponse to copper and was not caused by low pH, osmotic shockor generalized cation induced damage. Solenostoma crenulatum, hepaticae, copper tolerant ecotype     

The light-dependent effect of external pH on the membrane potential of Nitella

In the light the membrane potential of Nitella flexilis andNitella axilliformis was hyperpolarized by raising the externalpH above pH 5.5, at the rate of 30–40 mV/pH below pH 8.This hyperpolarization was largely reduced in the dark. The membrane potential was sensitive to the external pH of mediawith a low potassium concentration, where it was relativelyinsensitive to potassium concentration. In media of a high concentrationwhere it was sensitive to the potassium concentration, the membranebecame insensitive to the external pH. The transition from apH-sensitive to a pH-insensitive state occurred rather abruptlyon increasing the external potassium concentration. (Received September 1, 1972; )     

Sodium Dependent Photosynthetic Oxygen Evolution in a Marine Diatom

Photosynthetic oxygen evolution in air-equilibrated culturesof Phaeodactylum tricornutum is dependent on the presence ofsodium, but not potassium; sodium cannot be replaced by eitherpotassium, lithium or ammonium. Respiration is not sodium dependent.At constant CO₂ concentrations the depression of oxygen evolutionin the absence of sodium is more pronounced at pH 8.0 than atpH 6.5 and it is concluded that sodium facilitates the utilizationof bicarbonate. Sodium increases the affinity of Phaeodactylumfor inorganic carbon as does growth at low inorganic carbonconcentrations. Key words: Sodium, Photosynthesis, Marine diatom, Phaeodactylum     

Apoplastic Sugar Concentration and pH in Barley Leaves Infected with Brown Rust

Soluble sugars were extracted by low speed centrifugation fromthe apoplast of leaves of barley (Hordeum distichum L.) infiltratedwith water. Infection of the leaf with the brown rust fungus(Puccinia hordeii) resulted in a reduction in the concentrationof sucrose, glucose and fructose in the apoplast. Sugars werepresent in an apoplastic space occupying 12 and 17 cm³ m^–2of leaf area in healthy and infected tissue, respectively. Uptakeof hexoses by intercellular hyphae is suggested as a cause ofthis reduction. The pH of apoplastic sap extracted from rust-infectedleaves was increased to pH 7·3 from pH 6·6 incontrols. The effect of a reduced apoplastic sugar pool andincreased pH on export from infected leaves is discussed. Key words: Apoplast, barley (Hordeum distichum L.), brown rust (Puccinia hordeii Otth.), pH, sucrose, hexose     

Temporal variations in carbon isotope ratio of phytoplankton in a eutrophic lake

Temporal variations in carbon isotope ratio of phytoplanktonand dissolved inorganic carbon (DIC) in Lake Suwa were reported.In summer, blooming of Microcystis spp. resulted in low concentrationsof DIC and high pH, and HCO₃^– was the prominent speciesof DIC. Chlorophyll-specific rates of photosynthesis were relativelyconstant irrespective of the algal biomass during summer. Carboxylationin photosynthesis of Microcystis spp. was mainly catalyzed byribulose bisphosphate carboxylase (RuBPCase). Carbon isotopediscrimination between ¹³C of phytoplankton and DIC was considerablysmall in early summer and appeared to be negatively correlatedto DIC concentration. We concluded that carbon fixation by phytoplanktonin Lake Suwa is controlled not by the switch of photosyntheticpathways, but by low DIC concentration and high pH, suggestingthat photosynthesis of Microcystis spp. in Lake Suwa is governedby uptake kinetics other than the carboxylation step.     

Carbonic Anhydrase of a Unicellular Red Alga Porphyridium cruentum R-l. I. Purification and Properties of the Enzyme

Cells of Porphyridium cruentum R-l, a unicellular red alga,grown under ordinary air (0.04% CO₂) showed much higher activityof carbonic anhydrase (CA) than those grown under CCvenrichedair (2% CO₂). CA activity was not detected in a suspension ofintact cells, and was detectable only after the cells had beenhomogenized, indicating that this enzyme was localized onlywithin the algal cells. After partial purification of Porphyridium CA, its mol wt wasestimated as 59 kDa by SDS-PAGE and 55 kDa by gelfiltration.This suggests that the native enzyme is a monomer. Its activitywas not affected by benzensulfonamides, potent inhibitors ofCAs isolated from Chlamydomonas and other organisms. Chloride(or bromide) ions was essential for CA activity. CA activitymarkedly decreased when the cell extract had been incubatedat pH lower than 7 before assay. Upon readjusting the pH ofthe preincubation medium to 9 or higher, the enzyme activitywas restored, indicating that the inactivation is reversible. (Received April 17, 1987; Accepted July 21, 1987)     

Response of predatory zooplankton populations to the experimental acidification of Little Rock Lake, Wisconsin

To assess the effects of lake acidification on large predatoryzooplankton, we monitored population levels of four limnetictaxa for 6 years in a lake with two basins, one of which wasexperimentally acidified (2 years at each of three levels: pH5.6, 5.2 and 4.7). Concentrations of phantom midge (Chaoborusspp.), the most abundant large predator, remained similar inthe treatment and reference basins until the fourth year (pH5.2) when they increased in the treatment basin. In contrast,Epischuru lacustris and Leptodora kindtii disappeared from limneticsamples, and water mites declined to near zero upon acidification.Treatment basin populations of E.Iacustris declined sharplyduring the second year of acidification. The nature of the declinesuggested sensitivity of an early life stage during the firstyear at pH 5.6. Leptodora kindtii showed no population responseat pH 5.6, but declined to essentially zero at pH 5.2. Treatmentbasin populations of water mites fluctuated until decliningin the fifth and sixth years (pH 4.7). These changes indicatea variety of direct and indirect responses to lake acidification.     

A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway

Vitamin B₆ is essential for cellular functions and growth due to its involvement in important metabolic reactions. Humans and other mammals cannot synthesize vitamin B₆ and thus must obtain this micronutrient from exogenous sources via intestinal absorption. The intestine, therefore, plays a central role in maintaining and regulating normal vitamin B₆ homeostasis. Due to the water-soluble nature of vitamin B₆ and the demonstration that transport of other water-soluble vitamins in intestinal epithelial cells involves specialized carrier-mediated mechanisms, we hypothesized that transport of vitamin B₆ in these cells is also carrier mediated in nature. To test this hypothesis, we examined pyridoxine transport in a model system for human enterocytes, the human-derived intestinal epithelial Caco-2 cells. The results showed pyridoxine uptake to be 1) linear with time for up to 10 min of incubation and to occur with minimal metabolic alteration in the transported substrate, 2) temperature and energy dependent but Na⁺ independent, 3) pH dependent with higher uptake at acidic compared with alkaline pHs, 4) saturable as a function of concentration (at buffer pH 5.5 but not 7.4) with an apparent Michaelis-Menten constant (K_m) of 11.99 ± 1.41 µM and a maximal velocity (V_max) of 67.63 ± 3.87 pmol · mg protein^-1 · 3 min^-1, 5) inhibited by pyridoxine structural analogs (at buffer pH 5.5 but not 7.4) but not by unrelated compounds, and 6) inhibited in a competitive manner by amiloride with an apparent inhibitor constant (K_i) of 0.39 mM. We also examined the possible regulation of pyridoxine uptake by specific intracellular regulatory pathways. The results showed that whereas modulators of PKC, Ca⁺²/calmodulin (CaM), and nitric oxide (NO)-mediated pathways had no effect on pyridoxine uptake, modulators of PKA-mediated pathway were found to cause significant reduction in pyridoxine uptake. This reduction was mediated via a significant inhibition in the V_max, but not the apparent K_m, of the pyridoxine uptake process. These results demonstrate, for the first time, the involvement of a specialized carrier-mediated mechanism for pyridoxine uptake by intestinal epithelial cells. This system is pH dependent and amiloride sensitive and appears to be under the regulation of an intracellular PKA-mediated pathway. vitamin B₆; intestinal transport; transport regulation; Caco-2 cell