Showed Saturation Kinetics Research Articles

In vitro modeling of liver membrane copper transport

The process of hepatobiliary copper (Cu) secretion is still poorly understood Cu secretion as a complex with glutathione and transport via a lysosomal pathway have been proposed. The recent cloning and sequencing of the gene for Wilson disease indicates that Cu transport in liver cells may be mediated by a Cu transporting Ptype ATPase. Biochemical evidence for ATP-dependent Cu transport in mammalian systems, however, has not been reported so far. We have investigated Cu transport in rat liver plasma membrane vesicles enriched in canalicular or basolateral membranes in the presence and absence of ATP (4 mM) and an ATP-regenerating system. The presence of ATP clearly stimulated uptake of radiolabeled Cu (64Cu, 10 μM) into canalicular plasma membrane vesicles and, to a lesser extent, also into basolateral plasma membrane vesicles. ATP-dependent Cu transport was dose-dependently inhibited by the P-type ATPase inhibitor vanadate, and showed saturation kinetics with an estimated K m of 8.6 μM and a V max of 6.9 nmol/min/mg protein. ATP-stimulated Cu uptake was similar in canalicular membrane vesicles of normal Wistar rats and those of mutant GY rats, expressing a congenital defect in the activity of the ATP-dependent canalicular glutathione-conjugate transporter (cMOAT). These studies demonstrate the presence of an ATP-dependent Cu transporting system in isolated plasma membrane fractions of rat liver distinct from cMOAT. (J. Clin. Invest. 1995.95:412-416.) Key words: copper · bile secretion · P-type ATPase · vesicle transport · Wilson disease. (Reproduced from The Journal of Clinical Investigation, 1995, vol. 95, pp. 412-416 by copyright permission of The Society for Clinical Investigation.).

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L.

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast.

Copper transport and kinetics in cultured C6 rat glioma cells.

C6 rat glioma cells accumulate and efflux 67Cu. Both processes showed saturation kinetics with increasing 67Cu concentration. The Michaelis constant (Km) for uptake was 0.63 +/- 0.14 microM; maximum velocity (Vmax) was 3.29 +/- 0.57 pmol Cu.mg protein-1.min-1. The Km for efflux was 0.15 +/- 0.06 microM; Vmax was 1.08 +/- 0.71 pmol Cu.mg protein-1.min-1. p-Chloromercuribenzoate (p-CMB) totally blocked 67Cu efflux but had no effect on Km or Vmax of uptake. Total 67Cu in the cell after 50 min was partitioned equally between particulate and soluble fractions. p-CMB-treated cells accumulated more 67Cu, but < 10% was bound to the particulate (membrane) fraction. Pb also increased 67Cu accumulation without affecting Km and Vmax of 67Cu uptake. These data suggest that carriers for import and export of Cu in C6 cells are distinct or operate in two different cellular environments. Efflux is a sulfhydryl-dependent process subject to inhibition by Pb. The data are consistent with a P-type ATPase in the efflux of Cu from cells and the potential for Pb to inhibit the efflux mechanism.

The purification of a cysteine-dependent NAD+ glycohydrolase activity from bovine erythrocytes and evidence that it exhibits a novel ADP-ribosyltransferase activity.

An NAD+:cysteine ADP-ribosyltransferase activity was purified from bovine erythrocytes on the assumption that, like pertussis toxin, the enzyme would exhibit a cysteine-dependent NAD+ glycohydrolase activity. A three-step purification procedure was developed involving (1) precipitation with 40% (NH4)2SO4, (2) binding to a cysteine-Sepharose affinity column, and (3) binding to an NAD+ affinity column. PAGE showed a single band of M(r) 45,000. The enzyme had been purified 47,000-fold and had a specific activity of 1900 nmol nicotinamide released/min per mg. A study of the kinetic properties of this enzyme showed saturation kinetics for cysteine (Km = 4.0 mM). The ability of this enzyme to ADP-ribosylate protein was investigated using re-sealed inverted bovine erythrocyte ghosts. Incubation of the purified enzyme with erythrocyte ghosts and [adenylate-32P]NAD+ led to the enhanced dose-dependent labelling of several proteins, a doublet of high M(r) and proteins of M(r) 60,000, 55,000 and 29,000, identified by autoradiography of separated proteins on SDS/PAGE. The enzyme-catalysed labelling of the major component at M(r) 55,000 was blocked by pre-treatment of the erythrocyte ghosts with N-ethymaleimide, a sulphydryl alkylating agent, and the label was released by mercuric ion, but not by hydroxylamine. These experiments suggested that a cysteine residue on the target protein had been mono-ADP-ribosylated. This supposition was further supported by identification of the mercf1p4ion-released radiolabelled product as ADP-ribose by HPLC, and the observation that free ADP-ribose was unable to modify the membrane target protein directly.

Calcium transport mechanism in human colonic apical membrane vesicles

Background & Aims : Recent studies have shown the role of human colon in the absorption of calcium, especially in the presence of severe disease or resection of the small bowel. The aim of the current study was to explore the mechanism(s) of calcium uptake by the purified human colonic apical membrane vesicles. Methods : Apical membrane vesicles were purified from mucosal scrapings of colons from organ donors, and 45Ca uptake was measured using a rapid filtration technique. Results : The majority of the 45Ca associated with vesicles represented uptake into closed intravesicular space, whereas the remaining 45Ca uptake represented binding to the vesicles. Ca 2+ uptake was found to be dependent on time, pH, temperature, and ionic strength of the incubation medium and inhibitable by ruthenium red, La 3+, and ethylene glycol-bis(β-amino-ethyl ether)- N,N,N′,N′-tetraacetic acid. Experiments of the effects of membrane potential generated by K +/valinomycin or anion substitutions on Ca 2+ uptake showed that the Ca 2+ uptake process was potential insensitive. Calcium uptake was unaffected by outwardly directed H +, K +, and Na + gradients. Ca 2+ uptake showed saturation kinetics with no significant differences in Michaelis constant and maximum velocity values of this transporter between proximal and distal colonic segments. Conclusions : The uptake of Ca 2+ by human colonic apical membranes involves predominantly a carrier-mediated transport mechanism.

Cucumber cotyledon lipoxygenase oxygenizes trilinolein at the lipid/water interface.

The reactivity of cucumber cotyledon lipoxygenase with trilinolein was examined. The activity of the enzyme against linoleic acid rapidly decreased with increasing pH of the assay solution, and essentially no activity could be detected above pH 8.5. The rapid decrease in activity was not the result of an inactiveness of the enzyme at alkaline pH, because with trilinolein, the enzyme showed a broad pH-activity profile, and substantial activity could be detected even at pH 9.0. Rather, the decrease in activity was due to the dissociation of the linoleic acid emulsion into acid-soap aggregates and/or the monomeric form, depending on the ionization of the terminal carboxylic group. This suggests that cucumber cotyledon lipoxygenase acts only on an insoluble substrate at the lipid/water interface but not on a soluble one. High-performance liquid chromatography analyses of the products formed from trilinolein revealed that the enzyme inserted oxygen into the acyl moiety of trilinolein without hydrolysis of the ester bonds. Preincubation of the enzyme with triolein emulsions effectively abolished its activity against trilinolein added afterward. Furthermore, the enzyme was adsorbed on the trilinolein or triolein emulsion droplets in an essentially irreversible manner. A reaction velocity curve of the enzyme with trilinolein showed saturation kinetics. This is thought to be due to a regional substrate deficiency as the reaction proceeds. These lines of evidence indicate that the enzyme, once bound to the lipid/water interface, is unable to break free and bind to other emulsions.

Development of an assay for Mg‐dechelatase of oilseed rape cotyledons, using chlorophyllin as the substrate

Chlorophyllin (Chlin), the Mg‐chlorin obtained from chlorophyll (Chl) was employed as substrate of Mg‐dechelatase. The release of Mg2+ was associated with a shift of absorption from 644 to 687 nm. Changes of absorption at 687 nm were taken as a measure of Mg‐dechelatase activity present in preparations of oilseed rape thylakoids. Absorption changes were correlated linearly with enzyme dose. The pH optimum of Mg release from Chlin was ca 9 with a broad flank down to pH 7. The reaction showed saturation kinetics with an apparent Km value of ca 17 nM. The activity was inhibited in the presence of cysteamine or reduced glutathion. There was no effect of the thiol reagent N‐ethyl maleimide. The bulk of dechelatase activity was associated with the chloroplast membranes. The enzyme is partially latent and the appearance of full activity requires the solubilization of thylakoids with detergent. The highest activities were detected in mature green rape cotyledons. During dark‐induced senescence the activity declined at roughly the same rate as Chl was lost in the leaf tissue.

Manganese kinetics in the sea star Asterias rubens (L.) exposed via food or water

Accumulation and elimination of manganese from contaminated food and seawater in the tissues of the sea star, Asterias rubens, were investigated in the laboratory. Dissolved 54Mn was accumulated linearly with time and the concentration factor reached 19 after 23 days. Pyloric caeca, stomach and tube feet reached concentration factors of 14, 13 and 61, respectively. Tube feet, pyloric caeca and remaining tissues contained approximately 10–20, 5 and 70%, respectively, of the total body burden. 54Mn accumulated from seawater was eliminated according to 1st order kinetics with a half-life of 36 days. Sea stars fed homogenates of Mytilus edulis assimilated 69–83% of the 54Mn ingested. Elimination of 54Mn accumulated from food could be described as a two compartment system, with half of the body burden of 54Mn lost with a half-life of 25 days and the other half with a half-life of 1.8 days. Pyloric caeca, tube feet and remaining tissues contained approximately 30–40, 20–25 and 30%, respectively, of the total body burden of 54Mn accumulated from food. The highest concentration was found in the pyloric caeca. In sea stars exposed to 0.4 mg stable Mn ++ l −1, manganese accumulated linearly with time in the tissues examined, the highest concentrations being reached in the tube feet. At exposure to 1.0 mg Mn ++ l −1, manganese accumulated linearly with time in the stomach, whereas pyloric caeca and tube feet showed saturation kinetics. Sea stars showed no mortality at exposure to 10 and 25 mg Mn ++ l −1. Sea stars exposed to 50, 100 and 200 mg Mn ++ l −1 had median survival times of 72, 18 and 14.4 h, respectively.

Adenosine triphosphate-dependent copper transport in isolated rat liver plasma membranes.

The process of hepatobiliary copper (Cu) secretion is still poorly understood: Cu secretion as a complex with glutathione and transport via a lysosomal pathway have been proposed. The recent cloning and sequencing of the gene for Wilson disease indicates that Cu transport in liver cells may be mediated by a Cu transporting P-type ATPase. Biochemical evidence for ATP-dependent Cu transport in mammalian systems, however, has not been reported so far. We have investigated Cu transport in rat liver plasma membrane vesicles enriched in canalicular or basolateral membranes in the presence and absence of ATP (4 mM) and an ATP-regenerating system. The presence of ATP clearly stimulated uptake of radiolabeled Cu (64Cu, 10 microM) into canalicular plasma membrane vesicles and, to a lesser extent, also into basolateral plasma membrane vesicles. ATP-dependent Cu transport was dose-dependently inhibited by the P-type ATPase inhibitor vanadate, and showed saturation kinetics with an estimated Km of 8.6 microM and a Vmax of 6.9 nmol/min/mg protein. ATP-stimulated Cu uptake was similar in canalicular membrane vesicles of normal Wistar rats and those of mutant GY rats, expressing a congenital defect in the activity of the ATP-dependent canalicular glutathione-conjugate transporter (cMOAT). These studies demonstrate the presence of an ATP-dependent Cu transporting system in isolated plasma membrane fractions of rat liver distinct from cMOAT.

The cleavage of 1- and 2-naphthyl acetates by cyclodextrins in basic aqueous solution

The reactions of 1- and 2-naphthyl acetate (1-NA and 2-NA) with four cyclodextrins (CDs): α-CD, β-CD, γ-CD, and ‘hydroxypropyl-β-CD’(Hp-β-CD), in basic aqueous solution, all show saturation kinetics. Even though the strength of substrate binding varies appreciably, the limiting accelerations at high [CD] are all relatively modest (3 to 30-fold). The effects on the two isomeric esters are generally similar but there are significant differences between the CDs. These differences are discussed in terms of the relative importance of transition-state and initial-state binding, and in relation to structural variations in the four cyclodextrins. Except for one case, the second-order rate constants (k2) for the reactions of 1-NA and 2-NA with the CDs are appreciably greater than those for reaction with trifluoroethanol (TFE), as expected for stabilization of the transition state by inclusion of part of the ester in the CD cavity. For 2-NA reacting with a-CD, k2 is virtually the same as that for reaction with TFE, suggesting that inclusion is not a significant factor in this particular case. Consistent with this suggestion, the cleavage of 2-NA by α-CD is not competitively inhibited by alcohols, it is mediated by them. The cleavage of 1-NA by β-CD can also be mediated by alcohols and by alkanesulfonate ions, but the analogous reaction of 2-NA is not as susceptible.

Uptake of high‐density lipoprotein by Y‐organs of the crab, Cancer antennarius. I. characterization in vitro and effects of stimulators and inhibitors

AbstractCholesterol is the obligate precursor for ecdysteroid hormone synthesis by the ecdysial glands (Y‐organs) in crustaceans, and all cholesterol in the hemolymph is bound to high‐density lipoprotein (HDL). The mechanism was studied of how Y‐organ cells acquire cholesterol. Y‐organ segments were incubated with HDL isolated from hemolymph and labeled with 125I. After incubation, tissue was homogenized in acid to determine radioactivity in acid‐precipitable (cell associated, intact) HDL and in acid‐soluble (degraded) HDL. Both HDL uptake and degradation showed saturation kinetics. At saturation most of the total counts represented degraded HDL; by 3 h, degradation was 80%. Rates of HDL uptake and breakdown were higher in Y‐organs from de‐eyestalked crabs (deprived thereby of molt‐inhibiting hormone, MIH) than in glands from intact crabs. Both parameters were depressed by inhibitors of glycolysis and oxidative phosphorylation dose dependently and by low temperature. HDL uptake also was depressed by cAMP added to the medium experimentally or through efflux from the tissue during incubation. These results indicate a mechanism for HDL uptake that entails receptor‐mediated, energy‐dependent endocytosis of the entire HDL‐cholesterol complex. Also the results suggest that HDL uptake and degradation are mediated by cAMP and depressed by an eyestalk factor, presumably MIH. © 1995 Wiley‐Liss, Inc.

Kinetic study of the epoxidation of alkenes by iodosylbenzene catalysed by iron(III) tetra(4-N-methylpyridyl)porphyrin in methanol

A kinetic analysis of the epoxidation of alkenes by iodosylbenzene catalysed by iron(III) tetra(4-N-methylpyridyl)porphyrin (FeIIIT4MPyP) in methanol solution is presented. Using the initial rate method, the rates of epoxidation are found to be first-order in FeIIIT4MPyP and to show saturation kinetics in both the oxidant and the substrate. Interestingly, for the two most reactive styrenes, 4-methoxy- and 4-methylstyrene, the rate of epoxidation is first-order in the substrate: saturation kinetics in substrate are not observed. A Michaelis–Menten-like rate equation is derived for the system, under conditions where the catalyst is saturated in oxidant, and is used to compare the rates of epoxidation of a selection of aliphatic alkenes and styrenes.The reactivity of an alkene is found to be determined by the influence of the substituent on the formation of a complex with the active oxidant and on the subsequent breakdown of this intermediate to give products. The results from the less reactive styrenes are analysed using the Hammett equation and the mechanism of the epoxidation is discussed.

Mutants affecting nucleotide recognition by T7 DNA polymerase.

Analysis of two mutations affecting nucleotide selection by the DNA polymerase from bacteriophage T7 is reported here. Two conserved residues (Glu480 and Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA polymerase were mutated using site-directed mutagenesis (Glu480-Asp and Tyr530-Phe). The kinetic and equilibrium constants governing DNA binding, nucleotide incorporation, and pyrophosphorolysis were measured with the mutants E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical quench-flow methods. Both mutants have slightly lower Kd values for DNA binding compared to that of wild-type(exo-). With Y530F(exo-) the ground state nucleotide binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower for dATP and 8-10-fold lower for dTTP binding. With E480D(exo-), the binding constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP binding compared to those constants for wild-type(exo-). The significance of a specific destabilization of dTTP binding by these amino acids was examined using a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number of hydrogen bonds of an A:T base pair. The Kd for dCTP opposite inosine was unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454 microM) and with E480D(exo-) (1 mM). The Kd for dITP was the same with wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly higher with E480D(exo-) (3.2 mM). These data support the suggestion that E480 selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding to the unbonded carbonyl. Data on the incorporation of dideoxynucleotide analogs were consistent with the observation of a selective stabilization of dTTP by both residues. Pyrophosphorolysis experiments revealed that neither mutation had a significant effect on the chemistry of polymerization. The fidelity of the mutants were examined in misincorporation assays. Both E480D(exo-) and Y530F(exo-) showed saturation kinetics with the wrong nucleotide, with binding constants of 1-3 mM compared to the estimated binding affinity of 6-8 mM with wild-type(exo-). Accordingly, both mutants showed slightly lower selectivity against misincorporation. Taken together, these results indicate that E480 and Y530 each contribute to ground state nucleotide binding and suggest that the E480 may serve to specifically stabilize the incoming dTTP of A:T base pairs to compensate for the fewer hydrogen bonds compared to G:C base pairs.

Hepatic uptake and intestinal absorption of bile acids in the rabbit

The existence of transporters for bile acids (BA) in liver and intestine has been well documented, but information is still needed as to their respective transport capacity. In the present investigation, we compared the hepatic and intestinal transport rates for BA, using perfused livers and intestines. The livers and intestines were separately perfused and dose-response curves (0.25-10 mM) for tauroursodeoxycholate, taurocholate and taurodeoxycholate were obtained. The intestinal and mesenteric concentration and bile acid pattern were also evaluated in six non-fasting rabbits. Taurocholic, tauroursodeoxycholic and taurodeoxycholic acid ileal absorption showed saturation kinetics in the intestine as in the liver; the maximal uptake velocity for each bile acid in the liver was tenfold higher than the respective maximal transport velocity in the intestine; the Km values obtained in the liver were of the same order of magnitude, i.e. in the millimolar range. Taurocholic, tauroursodeoxycholic and taurodeoxycholic acid transport differences in the liver paralleled those in the intestine. Although the intestine was not homogeneously filled, the bile acid concentration in the ileal content fell into the range of the Km for the three studied bile acids, while the portal blood total bile acid concentration was inferior to the observed Kms of liver uptake. Therefore, both the hepatic and intestinal systems do not operate at their maximal transport rates at the prevailing concentrations in portal blood and luminal content, and the hepatic transport occurs at its highest efficiency (below the Km values) in physiological conditions.

Transport of Ascorbic and Dehydroascorbic Acids across Protoplast and Vacuole Membranes Isolated from Barley (Hordeum vulgare L. cv Gerbel) Leaves.

Protoplasts, vacuoles, and chloroplasts were isolated from leaves of 8-d-old barley (Hordeum vulgare L. cv Gerbel) seedlings. Transport of ascorbate and dehydroascorbate into protoplasts and vacuoles was investigated. Contents of ascorbic acid, glutathione, and [alpha]-tocopherol and ascorbate peroxidase activity and glutathione reductase activity were analyzed in protoplasts, vacuoles, and chloroplasts. Uptake of ascorbate and dehydroascorbate by protoplasts showed saturation kinetics (Km = 90 [mu]M reduced ascorbic acid, 20 [mu]M dyhydroascorbic acid). Effects of various membrane transport inhibitors suggested that transport was carrier mediated and driven by a proton electrochemical gradient. Translocation of ascorbate and dehydroascorbate into vacuoles did not show saturation kinetics. Neither was it influenced by effectors or by ATP but only by Mg2+, suggesting that translocation did not occur by carrier. Ascorbic acid was predominantly localized in the cytosol. Contents in the chloroplasts and vacuoles were low. The results are consistent with the view that ascorbate is synthesized in the cytosol and released to chloroplasts, apoplast, and vacuole following a concentration gradient. Translocation from the apoplast into the cytosol is against a steep gradient and appears to control the concentration of ascorbic acid in the apoplast. In its function as an antioxidant, ascorbate in the apoplast may be oxidized to dehydroascorbate, which can be efficiently transported back into the cytosol for regeneration to ascorbate.

Trp89 in the lid of Humicola lanuginosa lipase is important for efficient hydrolysis of tributyrin.

To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) from Humicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a Vmax = 6000 +/- 300 mmol.min-1.g-1 and an apparent Km = 16 +/- 2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent Km for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a glutamine/proton cotransporter from Ricinus comnmnis roots using isolated plasma membrane vesicles

The mechanism of glutamine transport at the plasma membrane of sink tissue cells was investigated using isolated plasma membrane vesicles from roots of Ricinus communis L. var. sanguineous. Glutamine transport was found to be driven by both the pH gradient (ΔpH) and a membrane potential (ΔΨ) (alkaline and negative internal), which were created artificially across the plasma membrane. Glutamine wus accumulated 15–20‐fold in the presence of both a ΔpH and ΔΨ. There appeared to be a direct pH effect on ΔPS‐driven transport, as a higher rate of transport was observed at pH 5.5 than at pH 7.5. The ΔpH +ΔΨ ‐driven transport showed saturation kinetics with a Km of 287 μM. Altering the membrane potential changed the Vmax but had no effect on the Km of glutamine transport. These results are consistent with the presence of a proton‐coupled, carrier‐mediated system for glutamine uptake in Ricinus roots. A range of protein modifiers and transport inhibitors had limited effects on glutamine transport: highest inhibition uas observed with cytochalasin D. When glutamine transport was compared in plasma membrane vesicles isolated from the root lips of Ricinus and from the remainder of the root tissue a lower level of transport was observed in the root tips. A method for the solubilization and reconstitution of glutamine transport activity using the detergent CHAPS is also described.

Putrescine and paraquat uptake in human lung slices and isolated type II pneumocytes

Paraquat is accumulated into the lungs of various species by an active uptake system which also appears to mediate the uptake of endogenous polyamines, such as putrescine. The accumulation of putrescine in the human lung has been previously shown to be mainly located in the type II cells. In the present study, we have studied the mutually competitive inhibition of putrescine and paraquat in human lung slices and the inhibition of putrescine by paraquat or cystamine in isolated human type II pneumocytes. Peripheral lung tissue taken from patients undergoing pneumectomy or lobectomy was used. The initial steps of the cell isolation procedure differed from the literature in that the tissue was first sliced in 0.7 mm thick slices, which were washed in phosphate buffered saline without calcium and magnesium (PBS −), followed by incubations with trypsin. The type II cells were purified and isolated by differential adherence on plastic followed by Percoll gradient centrifugation. Uptake was determined 48 hr after cell isolation. The accumulation of radiolabelled putrescine showed saturation kinetics, with the following apparent kinetic parameters: k n 6.7 and 6.2–7.6μM and V max2.7 and 3.0–3.4μmol/g prot/hr for slices and isolated cells, respectively. In the presence of paraquat, putrescine uptake was reduced, in both systems, in a manner compatible with competitive inhibition, with calculated inhibition constants ( k i) of 549–614 and 659–895 μM paraquat for slices and isolated cells, respectively. The accumulation of putrescine in isolated human pneumocytes was strongly reduced in the presence of cystamine, with calculated k i of 3.7 μM cystamine. These data indicate that putrescine, paraquat and cystamine accumulate in the human lung by the same uptake system, but that the affinities for the three substrates differ. The presence of an uptake system for putrescine in cultured human pulmonary type II is probably useful as a functional viability test.

Phosphatidate Kinase, A Novel Enzyme in Phospholipid Metabolism (Characterization of the Enzyme from Suspension-Cultured Catharanthus roseus Cells).

Phosphatidate kinase (adenosine 5[prime]-triphosphate:phosphatidic acid phosphotransferase), a novel enzyme of phospholipid metabolism, was detected recently in the plasma membranes of suspension-cultured Catharanthus roseus cells and purified (J.B. Wissing, H. Behrbohm [1993] Plant Physiol 102: 1243-1249). In the present work the properties of phosphatidate kinase are described. The enzyme showed a pH optimum of 6.1 and an isoelectric point of 4.8, and was rather stable in the presence of its substrates. Although the kinase accepted both ATP and GTP, with Km values of about 12 and 18 [mu]M, respectively, the only lipid substrate was phosphatidic acid; neither lysophosphatidic acid nor any other lipid tested was phosphorylated. With 32P- and 14C-labeled diacylglycerol pyrophosphate, the product of the enzyme, it was shown that the kinase catalyzes a reversible reaction. The activity of the extracted enzyme depended on the presence of surfactants such as Triton X-100 or [beta]-octylglucoside, whereas deoxycholate was strongly inhibitory. Kinetic analysis with Triton X-100/phosphatidate mixed micelles performed according to the "surface dilution" kinetic model showed saturation kinetics with respect to both bulk and surface concentration of phosphatidate. The interfacial Michaelis constant for phosphatidate was determined as 0.6 mol %.

Production of L(+)-lactic acid using immobilized rhizopus oryzae reactor performance based on kinetic model and simulation

The production of L(+)-lactic acid using alginate immobilizedRhizopus oryzae in tapered-column fluidized-bed batch reactor was tested and simulated using the kinetic data taken independently in shake-flask cultures. The data show saturation kinetics with substrate and product inhibitions in linear form. Analysis of the kinetic data gave kinetic constants:V m, 11.04 g lactic acid/(L-bead. h);K m, 20.9 g glucose/L; andK i, 365 g glucose/L for lactic acid production. The product inhibition constant,K p, was found to be 316 g lactic acid/L. The simulation results showed a good agreement with the experimental results when the initial lag phase was taken into account in the simulation model. Without the adjustment for the initial lag period, the kinetic model showed higher conversion. Starting with a glucose concentration of 150 g/L, it was possible to produce 73 g/L of L(+)-lactic acid in 44.5 h. The lactic acid yield was 64.8% by weight based on the amount of glucose consumed.