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1.
Abstract: Data are presented in support of the transport of (-)- d -3-hydroxybutyrate across the blood-brain barrier (BBB) being a carrier-mediated process. The kinetic parameters in 21-day-old pentobarbital-anaesthetized rats were Vmax 2.0 μmol.g−1.min−1, K m 29 m M , and K D 0.024 ml.g−1.min−1. The value for Vmax was the same as that for l -lactate and pyruvate transport in animals of the same age. The transport of all three substrates was sensitive to inhibition by low concentrations of either 2-oxo-3-methylbutanoate or 2-0x0-4-methylpentanoate, the 2-oxo acids that can accumulate in patients with maple-syrup-urine disease. The K m values for the 2-oxo acids were severalfold lower than the respective K m values. 2-oxo-3-phenylpropionate was a poor inhibitor. The relative affinities of the various monocarboxylic acids for the transport system of the BBB distinguished it from similar systems described in brain, heart, and liver mitochondria; human erythrocytes; and Ehrlich ascites-tumour cells.  相似文献   

2.
Abstract: Since protein synthesis in the developing brain may, under certain conditions, be limited by amino acid availability, the present studies were undertaken to characterize the kinetics of large neutral amino acid transport through the blood-brain barrier (BBB) of the newborn rabbit. The Km, Vmax, and KD of the transport of eight amino acids were determined by a nonlinear regression analysis of data obtained with the carotid injection technique. Compared with kinetic parameters observed for the adult rat, the Km, Vmax, and KD of amino acid transport were all two- to threefold higher in the newborn. Albumin was found to bind tryptophan actively in vitro , but had no inhibitory effect on tryptophan transport through the newborn BBB. Glutamine was transported through the BBB of the newborn at rates severalfold higher than are seen in the adult rat. However, glutamine transport was not inhibited by high concentrations of N -methylaminoisobutyric acid (NMAIB), a model amino acid that is specific for the alanine-preferring or A-system present in peripheral tissues. In conclusion, these studies show that the BBB neutral amino acid transport system of the newborn rabbit has a lower affinity and higher capacity than does the BBB of the adult rat. Under conditions of high plasma amino acids, the increased capacity of the newborn transport system allows for a higher rate of amino acid transport into brain than would occur via the lower capacity system present in the adult rat brain.  相似文献   

3.
Abstract: (RS)-Nipecotic acid is taken up into cultured astrocytes by a saturable high-affinity transport system with a Km, of 28.8 ± 2.8 μM and a Vmax of 0.294 ± 0.022 nmol × min−1× [mg cell protein]−1. The uptake which represents a net inward transport was sodium-dependent, requiring translocation of one sodium ion for each molecule of nipecotic acid taken up. The most potent inhibitors of GABA uptake into astrocytes (GABA, (R)-nipecotic acid, (3RS,4SR)-4-hydroxynipecotic acid, and guvacine) were shown to be potent inhibitors of nipecotic acid uptake (IC50) 20, 25, 25, and 50 μm respectively), GABA being a competitive inhibitor. (S)-2,4-Diaminobutyric acid was a more efficient inhibitor than β-alanine of glial uptake of (RS)-nipecotic acid. It is concluded that astroglial uptake of (RS)-nipecotic acid and GABA is mediated by the same transport system.  相似文献   

4.
High- and Low-Affinity Transport of D-Glucose from Blood to Brain   总被引:21,自引:19,他引:2  
Abstract: Measurements of the unidirectional blood-brain glucose flux in rat were incompatible with a single set of kinetic constants for transendothelial transport. At least two transfer mechanisms were present: a high-affinity, low-capacity system, and a low-affinity, high-capacity system. The low-affinity system did not represent passive diffusion because it distinguished between D-and L-glucose. The Tmax and K m, for the high-affinity system were 0.16 mmol 100 g−1 min−1 and 1 mM; for the low-affinity system, ∼ 5 mmol 100 g−1 min−1 and ∼ 1 M. With these values, physiological glucose concentrations were not sufficient to saturate the low-affinity system. In normoglycemia, therefore, three independent pathways of glucose transport from blood to brain appear to exist: a high-affinity facilitated diffusion pathway of apparent permeability 235·10−7 cm s−1, a specific but nonsaturable diffusion pathway of permeability 85·10−7 cm s−l, and a nonspecifc passive diffusion pathway of permeability 2·10−7 cm s−1.  相似文献   

5.
Abstract: The mechanism of unidirectional transport of sodium from blood to brain in pentobarbital-anesthetized rats was examined using in situ perfusion. Sodium transport followed Michaelis-Menten saturation kinetics with a V max of 50.1 nmol/g/min and a K m of 17.7 m M in the left frontal cortex. The kinetic analysis indicated that, at a physiologic sodium concentration, ∼26% of sodium transport at the blood-brain barrier (BBB) was carrier mediated. Dimethylamiloride (25 µ M ), an inhibitor of Na+/H+ exchange, reduced sodium transport by 28%, whereas phenamil (25 µ M ), a sodium channel inhibitor, reduced the transfer constant for sodium by 22%. Bumetanide (250 µ M ) and hydrochlorothiazide (1.5 m M ), inhibitors of Na+-K+-2Cl/NaCl symport, were ineffective in reducing blood to brain sodium transport. Acetazolamide (0.25 m M ), an inhibitor of carbonic anhydrase, did not change sodium transport at the BBB. Finally, a perfusate pH of 7.0 or 7.8 or a perfusate P co 2 of 86 mm Hg failed to change sodium transport. These results indicate that 50% of transcellular transport of sodium from blood to brain occurs through Na+/H+ exchange and a sodium channel in the luminal membrane of the BBB. We propose that the sodium transport systems at the luminal membrane of the BBB, in conjunction with Cl/HCO3 exchange, lead to net NaCl secretion and obligate water transport into the brain.  相似文献   

6.
CHARACTERISTICS OF D-GLUCOSAMINE UPTAKE BY RAT BRAIN SYNAPTOSOMES   总被引:1,自引:1,他引:0  
Abstract— The uptake of D-glucosamine by rat brain synaptosomes is studied as a function of time, temperature and synaptosomal protein and substrate concentrations. The rate of D-glucosamine uptake, after correcting for simple diffusion, obeys Michaelis-Menten kinetics. The apparent kinetic constants for the uptake process are Km = 2.5 0.8 m m , Vmax = 3.7 ± 1.2 nmol/mg protein/min. D-Glucose, D-mannose, 2-deoxy-D-glucose and 3-0-methyl-o-glucose are potent inhibitors of D-glucosamine uptake. 2-Deoxy-D-glucose and D-glucosamine inhibit the uptake of one another in a simple competitive manner, indicating their sharing of a common transport system. Cytochalasin B, phloretin and phloridzin are powerful competitive inhibitors of D-glucosamine uptake with apparent inhibitor constants ( K1 ) of 7.0 × 10-5, 2.3 × 10-3 and 0.4 mM, respectively. The uptake is unaffected by Na+, Li+ and Mg2+, partially inhibited by NH4+, Mn2+ and Ca2+, and slightly stimulated by PO4-ions. D-Glucosamine uptake is also sensitive to inhibition by several sulfhydryl reagents, thus implying the involvement of sulfhydryl groups in the transport process. The apparent affinity constants for synaptosomal transport for both D-glucosamine and 2-deoxy-D-glucose are about 4 times greater in 7-day-old than in the adult rat brains.  相似文献   

7.
Abstract: The kinetic parameters, Km and Vmax, for the acetylation of choline and several close analogues were determined by using (a) purified choline acetyltransferase and (b) a hypotonically lysed synaptosomal extract prepared from the electric organ of Torpedo marmorata. Whereas the Km for choline was similar in both cases (0.51 and 0.42 m m ), the crude enzyme showed a three- to fivefold greater affinity for its analogues than the purified enzyme, the activity decreasing rapidly with increased N -alkyl substitution. Homocholine was a poor substrate, but was clearly acetylated by both preparations. The effect of salt on analogue acetylation by the crude enzyme was studied by increasing NaCl concentration from zero to 150 m m . There was an increase in both Km and Vmax for all substrates; choline, N,N,N -dimethylmonoethylaminoethanol, -monomethyldiethylaminoethanol and -dimethylmonobutylaminoethanol showed the greatest changes, whilst N,N,N -triethylaminoethanol and -dimethylmonopropylaminoethanol and homocholine were much less affected. However, in all cases, the kinetic parameter Vmax / Km remained unchanged. The maximal velocities of the different substrates varied more under conditions of high than of low salt. Sodium chloride up to 300 m m had no effect on the amount of enzyme which was bound to membranes in the synaptosomal extract. It is concluded that choline acetyltransferase has a high degree of substrate specificity, which is slightly altered by purification. The effects of salt cannot be explained as a consequence of nonspecific ionic association with membranes.  相似文献   

8.
Abstract— Uptake kinetics of l -glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic-oxaloacetate-transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min−1. mg−1 cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics with a Km of 220 μ m and a V max of 7.9 nmol. min−1. mg−1 cell protein. The saturable glutamate uptake was inhibited by d -glutamate, l -aspartate and α-aminoadipate whereas l -glutamine, GABA and glutarate had no effect. The uptake which was Ca2+-independent had a Km for sodium of 18m m and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 m m. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.  相似文献   

9.
D-GALACTOSE TRANSPORT BY SYNAPTOSOMES ISOLATED FROM RAT BRAIN   总被引:5,自引:3,他引:2  
Abstract— Synaptosomes prepared by differential and Ficoll density gradient centrifugation took up d -galactose by two saturable transport systems: one. a high affinity system with a K m of 0-25 mn and Vmax of 075 nmol/mg protein 3 min, the other, a low affinity system with a Km of 47 mM and a Vmax of 135 nmol/mg protein/3 min. The high affinity system was inhibited by 1-5 mM phlorizin but was unaffected by the absence of sodium ion or the presence of 1 mM ouabain. The low affinity system was unaffected by phlorizin or ouabain. Both systems were inhibited by high concentrations of glucose. 2-deoxyga-lactose. and inositol, and by 2.4-dinitrophcnol. Galactose was rapidly converted in synaptosomes to phos-phorylatcd intermediates and was more slowly oxidized to 14CO2  相似文献   

10.
Abstract: Cerebral capillary sequestration and blood-brain barrier (BBB) permeability to apolipoproteins E2 (apoE2), E3 (apoE3), and E4 (apoE4) and to their complexes with sAβ1–40, a peptide homologous to the major form of soluble Alzheimer's amyloid β, were studied in perfused guinea pig brain. Cerebrovascular uptake of three apoE isoforms was low, their blood-to-brain transport undetectable, but uptake by the choroid plexus significant. Binding of all three isoforms to sAβ1–40 in vitro was similar with a K D between 11.8 and 12.9 n M . Transport into brain parenchyma and sequestration by BBB and choroid plexus were negligible for sAβ1–40-apoE2 and sAβ1–40-apoE3, but significant for sAβ1–40-apoE4. After 10 min, 85% of sAβ1–40-apoE4 taken up at the BBB remained as intact complex, whereas free sAβ1–40 was 51% degraded. Circulating apoE isoforms have contrasting effects on cerebral capillary uptake of and BBB permeability of sAβ. ApoE2 and apoE3 completely prevent cerebral capillary sequestration and blood-to-brain transport of sAβ1–40. Conversely, apoE4, by entering brain microvessels and parenchyma as a stable complex with sAβ, reduces peptide degradation and may predispose to cerebrovascular and possibly enhance parenchymal amyloid formation under pathological conditions.  相似文献   

11.
NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)−1 min−1 at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH2-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent Km for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent Km for nitrate as well as Vmax whereas it did not alter the Km for NADH.  相似文献   

12.
Abstract: Crude brain homogenates and cerebral tissue slices from rats with cobalt metal implanted in right and left cerebral cortices were used to examine high- and low-affinity GABA transport. High-affinity GABA transport was maximally reduced to 34% of controls 7 days after cobalt implantation, a time that coincides with peak seizure activity in this model. Kinetic analysis of high-affinity GABA transport, using brain homogenates, revealed a significant change in Vmax 7 days after cobalt implantation. (Vmax= 446.4 ± 26.2 pmol/mg prot./min, cobalt, versus 787.8 ± 67.3, control). An analysis of the low-affinity system revealed no depression of Km , or Vmax parameters. Administration of valproic acid at a concentration as high as 1 mM in vitro or a dose of 300 mg/kg in vivo had no effect on high- or low-affinity GABA transport. The results obtained from cobalt-treated rats provide additional evidence for an involvement of GABA in experimental epilepsy.  相似文献   

13.
Abstract: Binding of [3H]LY278584, which has been previously shown to label 5-hydroxytryptamine3 (5-HT3) receptors in rat cortex, was studied in human brain. Saturation experiments revealed a homogeneous population of saturable binding sites in amygdala ( K D= 3.08 ± 0.67 n M, B max= 11.86 ± 1.87 fmol/mg of protein) as well as in hippocampus, caudate, and putamen. Specific binding was also high in nucleus accumbens and entorhinal cortex. Specific binding was negligible in neocortical areas. Kinetic studies conducted in human hippocampus revealed a K on of 0.025 ± 0.009 n M −1 min−1 and a K off of 0.010 ± 0.002 min−1. The kinetics of [3H]LY278584 binding were similar in the caudate. Pharmacological characterization of [3H]LY278584 specific binding in caudate and amygdala indicated the compound was binding to 5-HT3 receptors. We conclude that 5-HT3 receptors labeled by [3H]LY278584 are present in both limbic and striatal areas in human brain, suggesting that 5-HT3 receptor antagonists may be able to influence the dopamine system in humans, similarly to their effects in rodent studies.  相似文献   

14.
Cadmium accumulation in the chloroplast of Euglena gracilis   总被引:5,自引:0,他引:5  
Intracellular distribution of Cd, cysteine, glutathione, and Cd-induced thiol peptides in Euglena gracilis cultured under photoheterotrophic conditions was studied. After 3 days of culture with 0.2 m M CdCl2, 62% of the Cd accumulated by cells was equally distributed between the cytosolic and chloroplastic fractions. However, after 8 days, metal content increased in the crude chloroplastic fraction to 40% of total and decreased to 19% in the cytosol; in Percoll-purified chloroplasts the estimated content of Cd raised to 62%. Accumulation of Cd in chloroplasts could be mediated by a transporter of free Cd2+, since uptake of added CdCl2 in isolated chloroplasts exhibited a hyperbolic type of kinetics with a Km of 57 µ M and Vmax of 3.7 nmol (mg protein)−1 min−1. The contents of cysteine and glutathione markedly increased in both chloroplasts (7–19 times) and cytosol (4–9 times) by exposure to Cd2+, although they were always higher in the cytosol. Thiol-containing peptides induced by Cd were mainly located in the cytosol after 3 days, and in the chloroplasts after 8 days of culture. The data suggested that Cd was compartmentalized into chloroplasts in a process that may involve the transport of free Cd and the participation of thiol-peptides.  相似文献   

15.
Abstract: Understanding the mechanism of brain glucose transport across the blood-brain barrier is of importance to understanding brain energy metabolism. The specific kinetics of glucose transport have been generally described using standard Michaelis-Menten kinetics. These models predict that the steady-state glucose concentration approaches an upper limit in the human brain when the plasma glucose level is well above the Michaelis-Menten constant for half-maximal transport, K t. In experiments where steady-state plasma glucose content was varied from 4 to 30 m M , the brain glucose level was a linear function of plasma glucose concentration. At plasma concentrations nearing 30 m M , the brain glucose level approached 9 m M , which was significantly higher than predicted from the previously reported K t of ∼4 m M ( p < 0.05). The high brain glucose concentration measured in the human brain suggests that ablumenal brain glucose may compete with lumenal glucose for transport. We developed a model based on a reversible Michaelis-Menten kinetic formulation of unidirectional transport rates. Fitting this model to brain glucose level as a function of plasma glucose level gave a substantially lower K t of 0.6 ± 2.0 m M , which was consistent with the previously reported millimolar K m of GLUT-1 in erythrocyte model systems. Previously reported and reanalyzed quantification provided consistent kinetic parameters. We conclude that cerebral glucose transport is most consistently described when using reversible Michaelis-Menten kinetics.  相似文献   

16.
Absorption of ammonium from solutions of ammonium chloride by maize ( Zea mays L. cv. GS-2) tissue was studied. In contrast to an initial rapid phase of absorption in root tissue, a one hour lag period was recorded in leaf tissue. The maximum rate of uptake was observed at 5–10 m M NH4Cl in both tissues. Roots had a Km value of 1.0 m M and Vmax of 24.3 μmol ammonium (g fresh weight)−1 h−1, whereas the leaf tissue had a higher Km (4.1 m M ) and a lower Vmax (8.7 μmol). There was a concentration dependent increase in ethanol soluble and insoluble fractions of organic nitrogen during ammonium supply. The optimum pH for ammonium absorption for both tissues was 7.4. The optimal concentration of CaCl2 for ammonium absorption was 5 m M whereas that of KCl was only 1 m M . In both tissues, the absorption was inhibited substantially by DCMU, DNP, cycloheximide, lincomycin, sodium tungstate, sodium arsenate and to some extent also by the anions nitrate and sulfate. It is suggested that a carrier is involved in an active uptake of ammonium in the leaf tissues.  相似文献   

17.
Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants ( Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K+, Mg2+-ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 m M KCl and 3 m M ATP with apparent Km (ATP) and Vmax of 0.75 m M and 79 μmol (mg protein)−1 h−1, respectively. With 50 m M KCl and 7 m M ATP a pH optimum of 6.5, an apparent Km (ATP) of 6.3 m M and Vmax of 159 μmol (mg protein)−1 h−1 were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca2+ but insensitive to azide and nitrate.  相似文献   

18.
The thiol tripeptide glutathione (GSH; γ Glu-Cys-Gly) is very abundant in legume nodules where it performs multiple functions that are critical for optimal nitrogen fixation. Some legume nodules contain another tripeptide, homoglutathione (hGSH; γ Glu-Cys- β Ala), in addition to or instead of GSH. We have isolated from a pea ( Pisum sativum L.) nodule library a cDNA, GSHS2 , that is expressed in nodules but not in leaves. This cDNA was overexpressed in insect cells and its protein product was identified as a highly active and specific hGSH synthetase. The enzyme, the first of this type to be completely purified, is predicted to be a homodimeric cytosolic protein. It shows a specific activity of 3400 nmol hGSH min−1 mg−1 protein with a standard substrate concentration (5 m M β -alanine) and Km values of 1.9 m M for β -alanine and 104 m M for glycine. The specificity constant (Vmax/Km) shows that the pure enzyme is 57.3-fold more specific for β -alanine than for glycine. Southern blot analysis revealed that the gene is present as a single copy in the pea genome and that there are homologous genes in other legumes. We conclude that the synthesis of hGSH in pea nodules is catalysed by a specific hGSH synthetase and not by a GSH synthetase with broad substrate specificity.  相似文献   

19.
In the present paper, we confirmed that alkaline phosphatase (ALP) is the main phosphatase present in ascocarps of the edible mycorrhizal fungus Terfezia claveryi. The enzyme was partially purified by precipitation with polyethylene glycol. The purification achieved from a crude extract was fivefold, with 53% of the activity recovered, and acid phosphatase, most of the lipids and phenolic compounds were eliminated. Alkaline phosphatase was kinetically characterised at pH 10.0, the optimum for this enzyme, using p -nitrophenyl phosphate as substrate. The Vmax and Km values were 0.3 μmol·min−1·mg−1 protein and 9.0 m m , respectively. Orthovanadate was a competitive inhibitor of ALP, with a Ki of 42.5 μ m . The enzyme was histochemically localised in the peridium, the hypothecium and in the ascogenic hyphae of the gleba using both colour and fluorescent reactions. The results presented suggest that the ascocarp of T. claveryi, at some stages of its development, may become nutritionally autonomous and independent of the host plant.  相似文献   

20.
Atlantic salmon ( Salmo salar L.) fry hatched from eggs transferred from high-Na to low-Na water during the eyed stage of development had a significantly higher Vmax and lower Km (P <0.01) of the sodium uptake mechanism than fry hatched from eggs incubated entirely in low-Na or high-Na water.
Fry hatched from eggs transferred to acid, high aluminium water during the eyed stage of development had a similar Vmax and Km to fry hatched from eggs incubated entirely in high- or low-Na water. Eggs incubated continuously in acid, high aluminium (low-Na) water produced fry with significantly lower Km and Vmax values than fry hatched from eggs incubated continuously in low-Na water. Eggs and fry in acid, high aluminium water continually lost sodium and mortality was 100% at 5 5 M O degree-days (2–3 weeks after hatching).
The results are discussed with respect to the influence of perivitelline fluid ion activities in eggs in acid, high aluminium water on the kinetic characteristics of sodium uptake in yolk-sac fry. A possible mechanism for the long-term adaptation of teleosts in acidified natural waters is also proposed.  相似文献   

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