Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway.

The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl-glucose-M63 plates, demonstrating the utilization of these compounds as osmoprotectants. Moreover, betaine and choline stimulated the growth of H. elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salinities below and above the optimum. We found that H. elongata DSM 3043 has at least one high-affinity transport system for betaine (K(m) = 3.06 microM and Vmax = 9.96 nmol of betaine min(-1) mg of protein(-1)). Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease. Finally, thin-layer chromatography and 13C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H. elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium.     

Choline-O-Sulfate Biosynthesis in Plants (Identification and Partial Characterization of a Salinity-Inducible Choline Sulfotransferase from Species of Limonium (Plumbaginaceae)

Choline-O-sulfate is a compatible osmolyte accumulated under saline conditions by members of the halophytic genus Limonium and other Plumbaginaceae. A choline sulfotransferase (EC 2.8.2.6) responsible for the formation of choline-O-sulfate was characterized in Limonium species. A simple radiometric assay was developed in which [14C]choline was used as substrate, and the h [14C]choline-O-sulfate product was isolated by ion-exchange chromatography. The choline sulfotransferase activity was soluble, required 3[prime]-phosphoadenosine-5[prime]-phosphosulfate as the sulfate donor, and showed a pH optimum at 9.0. Apparent Km values were 25 [mu]M for choline and 5.5 [mu]M for 3[prime]-phosphoadenosine-5[prime]-phosphosulfate. Choline sulfotransferase activity was detected in various Limonium species but was very low or absent from species that do not accumulate choline-O-sulfate. In roots and leaves of Limonium perezii, the activity was increased at least 4-fold by salinization with 40% (v/v) artificial sea water. Choline sulfotransferase activity was also induced in cell cultures of L. perezii following salt shock with 20% (v/v) artificial sea water or osmotic shock with 19% (w/v) polyethylene glycol 6000. Labeling experiments with [14C]choline confirmed that the enzyme induced in cell cultures was active in vivo.     

Regulation of sulfate transport in filamentous fungi

Inorganic sulfate enters the mycelia of Aspergillus nidulans, Penicillium chrysogenum, and Penicillium notatum by a temperature-, energy-, pH-, ionic strength-, and concentration-dependent transport system (“permease”). Transport is unidirectional. In the presence of excess external sulfate, ATP sulfurylase-negative mutants will accumulate inorganic sulfate intracellularly to a level of about 0.04 m. The intracellular sulfate can be retained against a concentration gradient. Retention is not energy-dependent, nor is there any exchange between intracellular (accumulated) and extracellular sulfate. The sulfate permease is under metabolic control. Sulfur starvation of high methionine-grown mycelia results in about a 1000-fold increase in the specific sulfate transport activity at low external sulfate concentrations. l-Methionine is a metabolic repressor of the sulfate permease, while intracellular sulfate and possibly l-cysteine (or a derivative of l-cysteine) are feedback inhibitors. Sulfate transport follows hyperbolic saturation kinetics with a Michaelis constant (Km) value of 6 × 10⁻⁵ to 10⁻⁴m and a V_max (for maximally sulfurstarved mycelia) of about 5 micromoles per gram per minute. Refeeding sulfur-starved mycelia with sulfate or cysteine results in about a 10-fold decrease in the V_max value with no marked change in the Km. Azide and dinitrophenol also reduce the V_max.     

Genetic and metabolic control of sulfate metabolism in Neurospora crassa: A specific permease for choline-O-sulfate

Neurospora crassa can use choline-O-sulfate as its sole sulfur source; the utilization of this compound involves its entry followed by intracellular hydrolysis. Neurospora possesses a transport system for the uptake of choline-O-sulfate which is specific for the sulfate ester and does not transport, nor is it inhibited by, either choline or inorganic sulfate. Mutant strains of Neurospora that are unable to transport or grow on inorganic sulfate can, nevertheless, utilize choline-O-sulfate for growth and transport the intact organic sulfate at a normal rate. Methionine, which represses a number of enzymes of sulfur anabolism, also represses the synthesis of the specific permease for choline-O-sulfate. A regulatory gene, cys-3, which controls the synthesis of choline sulfatase, aryl sulfatase, and several other related enzymes, also regulates the synthesis of the choline sulfate permease. Evidence is presented that the activity of choline sulfate permease is also regulated by a turnover process, the transport system having a functional half-life of approximately 3 hr.This investigation was supported by Public Health Service Grant 1 RO1 GM-18642 from the National Institute of General Medical Services.     

Regulation of choline sulphatase synthesis and activity in Aspergillus nidulans

1. Choline O-sulphate is taken up from the growth medium to the same extent by sulphur-deficient and sulphur-sufficient mycelia of Aspergillus nidulans, but hydrolysis of the transported sulphate ester in vivo only occurs in the sulphur-deficient mycelia. 2. Choline sulphatase activity could not be detected in vitro in sulphur-sufficient mycelia of wild-type and sulphur mutants of A. nidulans, but after sulphur starvation all strains showed appreciable activity of this enzyme. 3. Optimum activity of choline sulphatase in an ultrasonically treated preparation of sulphur-deficient mycelia was at pH7.5. The optimum substrate concentration was in excess of 25mm and K(m) was 0.035m. The enzyme was completely inhibited by 10mm-SO(3) (2-), PO(4) (3-), CN(-) and cysteine. 4. Growth of sulphur-deficient mycelia on various sulphur sources resulted in a decrease of choline sulphatase activity in vitro. The decrease appeared to be due to a repression of choline sulphatase synthesis rather than to inhibition of activity. De-repression by growth on a sulphur-deficient medium was prevented by cycloheximide. Unlike the choline sulphatase of bacteria the fungal enzyme did not need to be substrate-induced. 5. By using sulphur mutants the identity of the co-repressor was limited to S(2)O(3) (2-), cysteine-S-sulphonate, cysteine or compounds derived directly from them. Circumstantial evidence suggests that the co-repressor is cysteine. 6. Inhibition of choline sulphatase activity in vivo was demonstrated with cysteine as the sulphur source for growth.     

Characteristics of intermittent mitochondrial transport in guinea pig enteric nerve fibers

Enteric neurons controlling various gut functions are prone to oxidative insults that might damage mitochondria (e.g., intestinal inflammation). To resume local energy supply, mitochondria need to be transported. We used MitoTracker dyes and confocal microscopy to investigate basic characteristics of mitochondrial transport in guinea pig myenteric neurites. During a 10-s observation of 1 mm nerve fiber, on average, three mitochondria were transported at an average speed of 0.41 +/- 0.02 microm/s. Movement patterns were clearly erratic, and velocities were independent of mitochondrial size. The velocity oscillated periodically ( approximately 6 s) but was not consistently affected by structures such as en route boutons, bifurcations, or stationary mitochondria. Also, mitochondria transported in opposite directions did not necessarily affect each others' mobility. Transport was blocked by microtubule disruption (100 microM colchicine), and destabilization (1 microM cytochalasin-D) or stabilization (10 microM phalloidin) of actin filaments, respectively, decreased (0.22 +/- 0.02 microm/s, P < 0.05) or increased (0.53 +/- 0.02 microm/s, P < 0.05) transport speed. Transport was inhibited by TTX (1 microM), and removal of extracellular Ca(2+) (plus 2 mM EGTA) had no effect. However, depletion of intracellular stores (thapsigargin) reduced (to 33%) and slowed the transport significantly (0.18 +/- 0.02 microm/s, P < 0.05), suggesting an important role for stored Ca(2+) in mitochondrial transport. Transport was also reduced (to 21%) by the mitochondrial uncoupler FCCP (1 microM) in a time-dependent fashion and slowed by oligomycin (10 microM). We conclude that mitochondrial transport is remarkably independent of structural nerve fiber properties. We also show that mitochondrial transport is TTX sensitive and speeds up by stabilizing actin and that functional Ca(2+) stores are required for efficient transport.     

Regulation of sulfate metabolism in Neurospora crassa: Transport and accumulation of glucose 6-sulfate

Neurospora crassa can utilize glucose 6-sulfate as its sole sulfur source, although this compound cannot serve as a carbon source for this organism. Neurospora possesses a transport system capable of glucose 6-sulfate uptake; the system is energy dependent, is inhibited by extracellular sulfate, and is clearly distinct from the permeases responsible for the uptake of glucose and those for sulfate transport. The metabolism of glucose 6-sulfate apparently involves its transport as an intact molecule, followed by a slow intracellular hydrolysis. Methionine, which represses the synthesis of a number of enzymes of sulfur anabolism, also represses the synthesis of the transport system responsible for glucose 6-sulfate uptake. A regulatory gene, cys-3, which controls the synthesis of aryl sulfatase, choline sulfatase, choline-O-sulfate permease, and two distinct permease species, also regulates the permease for glucose 6-sulfate.     

Choline metabolism and membrane formation in rat hepatoma cells grown in suspension culture. 3. Choline transport and uptake by simple diffusion and lack of direct exchange with phosphatidylcholine

The initial rate of incorporation of methyl-labeled choline into the acid-soluble pool (phosphorylcholine) of Novikoff hepatoma cells growing in suspension culture was investigated as a function of the choline concentration in the medium. Below, but not above, 20 micro m, choline incorporation followed simple Michaelis-Menten kinetics at 24, 33, or 37 degrees C with an apparent K(m) of 4-7 micro m, and the V(max) values decreased with a Q(10) of about 2.3 with a decrease in temperature. Between 20 and 500 micro m, on the other hand, the rate of incorporation increased linearly with an increase in choline concentration in the medium, and the increase in incorporation rate with increase in choline concentration was about the same at all temperatures tested. The data suggest that at low concentrations choline is taken up mainly by a transport reaction, whereas at concentrations above 20 micro m, simple diffusion becomes the principal mode of uptake. The energy of activation for choline transport was estimated from an Arrhenius plot of the V(max) values as 67,000 J (16 kcal)/mole. At concentrations below 20 micro m, choline incorporation into membrane phosphatidylcholine also followed simple Michaelis-Menten kinetics, and the apparent K(m) was about the same as that for choline transport. The data support the conclusion that the transport of choline into the cell is the rate-limiting step in the conversion of choline to phosphorylcholine and its incorporation into phosphatidylcholine. At concentrations above 100 micro m, on the other hand, the ultimate rate of choline incorporation into phosphatidylcholine was independent of the choline concentration in the medium or the intracellular level of phosphorylcholine. Further, the rate of turnover of the choline moiety of phosphatidylcholine (half-life, 20-24 hr) either in whole cells or during incubation of isolated membrane fractions was unaffected by the presence of an excess of choline in the medium. The overall results indicate that a direct exchange between free choline and the choline moiety of phosphatidylcholine does not play a significant role in the incorporation of choline into phosphatidylcholine by Novikoff cells or in the turnover of the choline moiety of phosphatidylcholine, and that labeled choline therefore is a useful precursor in studying the synthesis and turnover of membrane phosphatidylcholine in these cells.     

Control of the general amino acid permease of Penicillium chrysogenum by transinhibition and turnover

When nitrogen-starved mycelium of Penicillium chrysogenum is incubated with relatively high concentrations of labeled hydrophobic amino acids, influx is followed by efflux of the corresponding labeled α-ketoacid. In spite of the efflux, further transport activity is suppressed. Cell-free extracts contain a transaminase that accepts all those amino acids exhibiting α-ketoacid efflux. Transaminase activity is constitutive but is induced to a 2- to 3-fold higher level during a 2-hr preincubation period with a hydrophobic amino acid. Cycloheximide prevents efflux and also the induction of the transaminase. Cycloheximide itself stimulates a partial decay in transport activity but mycelium preincubated with l-leucine and cycloheximide together retain a greater fraction of the original transport activity than mycelium preincubated with l-leucine alone. The results suggest that transport is regulated partially by transinhibition but a significant part of the substrate-induced decay of transport activity is caused by either (a) the degradation of a permease component (perhaps facilitated by transinhibition), or (b) the induction by the substrate of a regulator protein (perhaps the transaminase).The uptake of labeled substrates by nutrient sufficient mycelium correlates well with lipid solubility of the substrates. This suggests that the nonsaturable uptake observed in these mycelia results from free diffusion of the uncharged species.     

Low level transport of IgA to bile via the asialoglycoprotein receptor

The rat and rabbit transport IgA from blood to bile by a highly efficient transcellular pathway mediated by secretory component (SC). Other mammals do not express SC on liver hepatocytes, but they do transport a small amount of IgA to bile. In the first part of this study, human polymeric IgA was radiolabeled and depleted of SC binding activity by successive affinity adsorption. Transport of this preparation intact to rat bile was 4%, but was reduced to 2% when 50 mg unlabeled asialoglycoprotein was preadministered. The 2% decline corresponds to the percent of asialo-orosomucoid diverted to bile from the lysosomal pathway. In guinea-pigs, missorting of asialo-orosomucoid intact to bile was 10% of the injected dose. Transport of normal human IgA to bile was 1-2%, even though guinea-pigs do not express SC in the liver. Excess unlabeled asialofetuin reduced the transport of asialo-orosomucoid by 10-fold and IgA by 6-fold. This demonstrates that the asialoglycoprotein receptor can mediate transport of IgA to bile in small amounts, but that this transport may be only a biological artifact resulting from limited fidelity of intracellular protein sorting.     

Positive and Negative Effects of Tacrine (Tetrahydroaminoacridine) and Methoxytacrine on the Metabolism of Acetylcholine in Brain Cortical Prisms Incubated Under "Resting"Conditions

The effects of tacrine (1,2,3,4-tetrahydro-9-aminoacridine) and 7-methoxytacrine on the metabolism of acetylcholine were investigated in experiments on prisms of rat cerebral cortex incubated in vitro in low-potassium (3 mmol/L K+) media; cholinesterases were inactivated by paraoxon to avoid any action of tacrine and methoxytacrine via their inhibition. Under "resting" conditions, tacrine and methoxytacrine increased the synthesis of unlabeled acetylcholine in the prisms; at the same time, they inhibited the uptake of [14C]choline from the medium and the synthesis of [14C]acetylcholine. The concentration of free choline was not increased by tacrine or methoxytacrine in either the tissue or the medium. The contradiction between the increased synthesis of unlabeled and the diminished synthesis of labeled acetylcholine indicates that the utilization of intracellular choline (which is presumably mobilized from intracellular choline esters) for the synthesis of acetylcholine is increased by tacrine and methoxytacrine. This conclusion is supported by the observation that the inhibition of acetylcholine synthesis during incubation with hemicholinium-3 (an inhibitor of choline transport into cholinergic nerve terminals) was overcome when tacrine was present simultaneously with hemicholinium-3. When the prisms were preincubated with [14C]choline and incubated with tacrine or methoxytacrine only after this, the amount of [14C]acetylcholine recovered in the tissue plus the medium was higher at the end of incubation with tacrine or methoxytacrine than without them, again suggesting that the drugs were able to increase the utilization of intracellular [14C]choline or its esters for acetylcholine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A purine permease in Candida glabrata

Abstract Competition experiments revealed that adenine and guanine were transported by a purine permease in both Candida glabrata 4 and a C. glabrata 4 cytosine permease negative mutant. The C. glabrata 4 cytosine permease negative mutant was isolated using 5-fluorocytosine selection. This mutant no longer transported cytosine, but transported adenine and guanine. A transport system for hypoxanthine was not detected. Hence, in addition to the cytosine permease, a purine permease exists in C. glabrata . This differs from the purine cytosine permeases in Saccharomyces cereuisiae and Candida albicans which transport adenine, cytosine, guanine and hypoxanthine.     

Changes in properties of glutamate transport in Trichoderma viride vegetative mycelia upon adaptation to glutamate as carbon source

The U-(14)C-labelled glutamate uptake was measured in both sucrose- and glutamate-grown mycelia of Trichoderma viride. The biomass yield was five-fold lower with glutamate as a sole carbon source. The rate of glutamate transport measured at a glutamate concentration of 1 mM remained unchanged in glutamate-grown mycelia whereas the properties of the glutamate transport were substantially changed compared to sucrose-grown mycelia. The glutamate uptake in both sucrose- and glutamate-grown mycelia was inhibited by an uncoupler (3,3',4',5-tetrachlorosalicylanilide) but the inhibitory efficiency was higher in the latter. The affinity of the permease to glutamate increased approximately five-fold in the glutamate-grown mycelia (about 76 microM compared to about 16 microM). The pH optimum for glutamate uptake was 4 in sucrose-grown mycelia but the glutamate-grown mycelia had two pH optima, one at pH 4 and the second between pH 6 and 7. The inhibition of glutamate uptake by other amino acids yielded different inhibitory patterns in the two mycelia under study. The glutamate uptake in mycelia of different ages also showed differences in both transport rate and temporal pattern. The results show that the growth of mycelia on glutamate led to the appearance of an additional permease with different properties and suggest that only this permease is operating in mycelia grown on glutamate.     

Characterization of L-asparagine transport systems in Stemphylium botryosum.

L-Asparagine uptake by Stemphylium botryosum is mediated by two distinct energy- and temperature-dependent transport systems. One permease is relatively specific for L-asparagine and L-glutamine and is present in nutrient-sufficient mycelium. The specific permease shows an optimum pH at 5.2, saturation kinetics (Km = 4.4 x 10(-4) M, Vmax = 1.1 mumol/g per min), competitive gradient of L-asparagine, and higher affinity towards the L-isomer of asparagine. Amide derivatives of L-asparagine (5-diazo-4-oxo-L-norvaline or L-aspartyl hydroxamate) are the most effective competitors, alpha-amino derivative (N-acetyl asparagine) is a moderate competitor, and alpha-carboxyl derivative (L-asparagine-t-butylester) shows only slight inhibition of the specific permease. Derivatives of L-glutamine are significantly less effective competitors than those of L-asparatine. The level of the specific permease is affected by nitrogen sources and increases approximately threefold upon starvation. The nonspecific permease possesses an optimum pH at 6.8, saturation kinetics (Km = 7 x 10(-5) M, Vmax = 5 mumol/g per min, Kt = 7.4 x 10(-5) M for L-leucine), and high affinity towards various types of amino acids.     

Dehydroascorbic acid transport by GLUT4 in Xenopus oocytes and isolated rat adipocytes

Dehydroascorbic acid (DHA), the first stable oxidation product of vitamin C, was transported by GLUT1 and GLUT3 in Xenopus laevis oocytes with transport rates similar to that of 2-deoxyglucose (2-DG), but due to inherent difficulties with GLUT4 expression in oocytes it was uncertain whether GLUT4 transported DHA (Rumsey, S. C. , Kwon, O., Xu, G. W., Burant, C. F., Simpson, I., and Levine, M. (1997) J. Biol. Chem. 272, 18982-18989). We therefore studied DHA and 2-DG transport in rat adipocytes, which express GLUT4. Without insulin, rat adipocytes transported 2-DG 2-3-fold faster than DHA. Preincubation with insulin (0.67 micrometer) increased transport of each substrate similarly: 7-10-fold for 2-DG and 6-8-fold for DHA. Because intracellular reduction of DHA in adipocytes was complete before and after insulin stimulation, increased transport of DHA was not explained by increased internal reduction of DHA to ascorbate. To determine apparent transport kinetics of GLUT4 for DHA, GLUT4 expression in Xenopus oocytes was reexamined. Preincubation of oocytes for >4 h with insulin (1 micrometer) augmented GLUT4 transport of 2-DG and DHA by up to 5-fold. Transport of both substrates was inhibited by cytochalasin B and displayed saturable kinetics. GLUT4 had a higher apparent transport affinity (K(m) of 0.98 versus 5.2 mm) and lower maximal transport rate (V(max) of 66 versus 880 pmol/oocyte/10 min) for DHA compared with 2-DG. The lower transport rate for DHA could not be explained by binding differences at the outer membrane face, as shown by inhibition with ethylidene glucose, or by transporter trans-activation and therefore was probably due to substrate-specific differences in transporter/substrate translocation or release. These novel data indicate that the insulin-sensitive transporter GLUT4 transports DHA in both rat adipocytes and Xenopus oocytes. Alterations of this mechanism in diabetes could have clinical implications for ascorbate utilization.     

L-valine transport by the actinomycete Actinomyces species 26-115, the producer of actinomycin C]

Transport of L-valine by Actinomyces species 26-115, an organism producing actinomycin C depended on L-valine concentration in the medium and temperature and required a source of intrinsic energy. Km for L-valine transport was 3.5.10(-6)--6.0.10(-6) M. It somewhat differed from experiment to experiment. The above system transported also other neutral amino acids. L-isoleucine was a competing inhibitor of L-valine transport. The transport of L-valine was stereospecific. The activity of the transport system was regulated by the intracellular content of L-valine. Probably because of this the amino acid transport depended on the culture age, so far as the level of free valine in the mycelium at various stages of development was different.     

Transport and metabolism of folates by bacteria.

Transport of labeled folic acid (PteGlu), pteroylpolyglutamates (PteGlu3-5), 5-methyl-tetrahydrofolate (5-methyl-H4PteGlu), and methotrexate in late-log phase cells of Lactobacillus casei was active, and subject to inhibition by unlabeled pteroylmonoglutamates, pteroylpolyglutamates, and iodoacetate, but not glutamate or glutamate dipeptides. Pteroylpolyglutamates were transported without prior hydrolysis and shared a common uptake system with pteroylmonoglutamates. The affinity and maximum velocity of PteGlun uptake decreased with increasing glutamate chin length (Km:PteGlu1, 0.03 mum; PteGlu3, 0.32 mum; PteGlu4, 1.9 mum; PteGlu5, 3.7 mum) and comparisons with growth response curves suggested that polyglutamates were more effectively utilized by L. casei, once transported, than monoglutamate. No concentration of 5-methyl-H4PteGlu3-8 inside the cells was observed. The major folate metabolites found in L. casei preloaded with high levels of [3H]PteGlu (0.5 mum) were 10-formyl-H4PteGlu2 and 10-formyl-PteGlu. Both compounds were released, the monoglutamate more rapidly. Pteroyltriglutamate formation appeared to be a rate-limiting step in intracellular metabolism. No 10-formyl-Pte-Glu was found in iodoacetate-treated cells and efflux was inhibited. Cells preloaded with low levels of [3H]PteGlu (7 nm) metabolized the vitamin to polyglutamate forms, the major derivatives being H4PteGlun. First order exit rates of labeled folate from preloaded L. casei indicated an inhibition of PteGlu uptake with time. Exit rates dropped from 0.05 min-1 to greater than 0.002 min-1 as intracellular folate was metabolized from monoglutamate to polyglutamate derivatives (n larger than or equal to 3). In the latter case, materials lost by efflux were breakdown products and no folate of glutamate chain length greater than two was released. Pediococcus cerevisiae actively transported 5-methyl-H4PteGlu but did not take up to 5-methyl-H4PTeGlu3-8. No active accumulation of 5-methyl-H4PteGlu was observed in Streptococcus faecalis.     

Receptor-mediated vectorial transcytosis of epidermal growth factor by Madin-Darby canine kidney cells

Transcellular transport of a variety of ligands may be an important mechanism by which regulatory substances reach their site of action. We have studied the transcellular transport of two 6,000-mol-wt proteins, epidermal growth factor (EGF) and insulin, across polarized Madin-Darby canine kidney (MDCK) cells grown on dual-sided chambers on a nitrocellulose filter substrate. When grown on these chambers, MDCK cells are polarized and express distinct basal and apical surfaces. MDCK cells are capable of unidirectional transport of EGF from the basal-to-apical direction, 50% of bound EGF transported in 2 h. Transport was inhibited by the addition of unlabeled EGF in a dose-dependent manner. Anti-EGF receptor Ab, which inhibited binding, also inhibited transport. No transport in the apical-to-basal direction is noted. Insulin transport is not observed in either direction. Transport correlates with the presence of ligand-specific receptors on the cell surface. Hence, EGF receptors (Ro = 48,000, Kd = 3.5 X 10(-10) M) are found only on the basal surface of the MDCK cells and neither surface expresses insulin receptors. Characterization of the EGF receptors on MDCK cells, as assessed by affinity, molecular mass, and anti-receptor antibody binding reveals that this receptor has similar characteristics to EGF receptors previously described on a variety of cells. Hence, the EGF receptor can function as a transporter of EGF across an epithelial cell barrier.     

Characterization of the biotin transport system in Saccharomyces cerevisiae

The characteristics of the biotin transport mechanism of Saccharomyces cerevisiae were investigated in nonproliferating cells. Microbiological and radioisotope assays were employed to measure biotin uptake. The vitamin existed intracellularly in both free and bound forms. Free biotin was extracted by boiling water. Chromatography of the free extract showed it to consist entirely of d-biotin. Cellular bound biotin was released by treating cells with 6 n H(2)SO(4). The rate of biotin uptake was linear with time for 10 min, reaching a maximum at about 20 min followed by a gradual loss of accumulated free vitamin from the cells. Biotin was not degraded or converted to vitamers during uptake. Transport was temperature- and pH-dependent, optimum conditions for uptake being 30 C and pH 4.0. Glucose markedly stimulated biotin transport. In its presence, large intracellular free-biotin concentration gradients were established. Iodoacetate inhibited the glucose stimulation of biotin uptake. The rate of vitamin transport increased in a linear fashion with increasing cell mass. The transport system was saturated with increasing concentrations of the vitamin. The apparent K(m) for uptake was 3.23 x 10(-7)m. Uptake of radioactive biotin was inhibited by unlabeled biotin and a number of analogues including homobiotin, desthiobiotin, oxybiotin, norbiotin, and biotin sulfone. Proline, hydroxyproline, and 7,8-diaminopelargonic acid did not inhibit uptake. Unlabeled biotin and desthiobiotin exchanged with accumulated intracellular (14)C-biotin, whereas hydroxyproline did not.