KINETICS OF BLOOD-BRAIN BARRIER TRANSPORT OF PYRUVATE, LACTATE AND GLUCOSE IN SUCKLING, WEANLING AND ADULT RATS

Abstract— The kinetics of the uptake from blood to brain of pyruvate, lactate and glucose have been determined in rats of different ages. The carotid artery single injection technique was used in animals anaesthetized with pentobarbital. The rates of influx for each substrate were determined over a range of concentrations for the different age-groups. Data were analysed in terms of the Michaelis-Menten equation with a component to allow for non-saturable diffusion. Values are given for K _m, V _max and K _d. In suckling rats (15-21 days) the V _max values for both pyruvate and lactate were 2.0 μmol g⁻¹ min⁻¹. In 28-day-old rats the V _max values had fallen to one-half and in adults they were less than one-tenth. K _m, values were higher in the younger animals. The rate of glucose transport in suckling rats was half that of 28-day-old and adults although there was no difference with age in the K _m values.
The results are discussed in relation to the net flux of these substrates in and out of brain during different stages of post-natal development.     

Use of perfused cores for evaluating extracellular enzyme activity in stream-bed sediments

Abstract β-Glucosidase activity was investigated in stream-bed sediments using 4-methylumbelliferyl-β- d -glucopyranoside (MUF-β-Glc) as a model substrate. In a perfused core technique, water containing MUF-β-Glc was perfused up through sediment cores. β-glucosidase activity quantified from the release of fluorescent MUF in water discharge from the cores. At low rates of perfusion, maximum β-glucosidase activity ( V _max) in perfused sediments was similar to that in suspended (unperfused) sediments. Substrate affinity( K m)was higher in the suspended sediments. V _maxand K _m both increased when the perfusion rate was raised, although naturally-low substrate concentrations could mean that variability in perfusion rates has little effect on enzyme activity in the field. V _max was uninfluenced by whether ground or stream water was perfused through the sediments, but K _m was higher in cores perfused with groundwater. Increasing concentrations of glucose in the perfusion water resulted in a progressive inhibition of β-glucosidase activity. Although natural concentrations of glucose were low, the high turnover of enzymatically-released glucose probably means that β-glucosidase activity could be regulated by product concentration.     

Tyrosine Hydroxylase from Bovine Striatum: Catalytic Properties of the Phosphorylated and Nonphosphorylated Forms of the Purified Enzyme

Abstract: The properties of purified tyrosine hydroxylase (TH) from bovine corpus striatum, both native and phosphorylated forms of the enzyme, were studied. TH had a tendency toward greater affinity for tetrahydrobiopterin (BH₄) than for the synthetic cofactor 6-methyltetrahydropterin (6-MPH₄), although the maximal velocity of the TH-catalyzed reaction was greater with 6-MPH4. Phosphorylation increased the affinity of TH for cofactor at pH 6.0, with little change in V _max. At pH 7.0, phosphorylation caused increased activation of TH by increasing V _max as well as reducing the K _m for cofactor. The K _m for dopamine was increased twofold by phosphorylation at pH 6.0, but eightfold at pH 7.0. Phosphorylation was not associated with a change in K _m for tyrosine at any pH or with any cofactor studied, although the K _m for tyrosine of TH was cofactor-dependent and seven to eight times greater with 6-MPH₄ than with BH₄ as cofactor. Heparin and NaCl activated native TH at pH 6.0, but not at pH 7.0. Phosphorylated TH was unaffected by heparin or salt at pH 6.0, but was relatively inhibited at pH 7.0. The data are presented in the context of the physiological environment of TH.     

Deamination of 5-Hydroxytryptamine by Both Forms of Monoamine Oxidase in the Rat Brain

Abstract: K _m and V _max values of monoamine oxidase (MAO) A and B towards 5-hydroxytryptamine were determined for rat brain homogenates after the in vitro inhibition of one of the two forms by the selective inhibitors clorgyline and l -deprenyl. K _m values of 178 and 1170μ m , and V _max values of 0.73 and 0.09 nmol·mg protein⁻¹·min⁻¹ towards 5-hydroxytryptamine were found for MAO-A and -B, respectively. The K ₁ for 5-hydroxytryptamine as a competitive inhibitor of β-phenethylamine oxidation by MAO-B was found to be 1400 μm. The significance of these findings is discussed.     

Isolation and properties of free and immobilized β-galactosidase from the psychrotrophic enterobacterium Buttiauxella agrestis (strain NC4)

A study of the β-galactosidase produced by the psychrotrophic bacterium Buttiauxella agrestis has been carried out. This micro-organism was isolated from raw milk and the enzyme isolated using standard methods. Molecular mass was estimated to be 515 kDa. The isoelectric point was close to 4·45. Optimum pH was 7·25. Maximal activity was observed at 50°C and activation energy was estimated to be 39·1 kJ mol^-1. Lactose enhanced thermal stability. Using α-nitrophenyl-β-D-galactopyranoside as the substrate, the K _m was 11 μmol 1^-1 and V _max was 85 U mg^-1 protein. β-Mercaptoethanol and ethanol were inhibitors; glycerol acted as a complex effector. The enzyme required divalent cations for activity while it was inhibited by EDTA. When the enzyme was immobilized in diethyl aminoethylcellulose the optimum pH of activity was 8. K _m was 47 μmol 1^-1 and V _max was 96 U mg^-1 protein.     

Effects of Phospholipases on the Kinetic Properties of Rat Striatal Membrane-Bound Tyrosine Hydroxylase

Abstract: Rat striatal tyrosine hydroxylase can be isolated in both a soluble and a synaptic membrane-bound form. The membrane-bound enzyme, which exhibits lower K _ms for both tyrosine (7 μ M ) and reduced pterin cofactor (110 μ M ) relative to the soluble enzyme (47 μ M and 940 μ M , respectively), can be released from the membrane fraction with mild detergent, and concomitantly its kinetic properties revert to those of the soluble enzyme. Treatment of membrane-bound tyrosine hydroxylase with C. perfringens phospholipase C increased the K _m of the enzyme for tyrosine to 27 μ M and the V _max by 60% without changing the K _m for cofactor. In contrast, treatment of membrane-bound tyrosine hydroxylase with V. russelli phospholipase A₂ increased the K _m for tyrosine to 48 μ M increased the V _max and increased the K _m for cofactor to 560 μ M . The enzyme remained bound to the membrane fraction following both phospholipase treatments. Addition of phospholipids to treated enzyme could partially reverse the effects of phospholipase A₂ treatment, but not the effects of phospholipase C treatment. The kinetic properties of phospholipase-treated, detergent-solubilized tyrosine hydroxylase were identical to those of the control solubilized enzyme. Tyrosine hydroxylase appears to interact with synaptic membrane components to produce at least two separately determined consequences for the kinetic properties of the enzyme.     

Adenosine Transport into Guinea-pig Synaptosomes

Abstract: Kinetics for transport of adenosine into guinea-pig neocortex synaptosomes were studied by incubating them with [¹⁴C]adenosine for up to 30 s. The apparent K _m value of the high-affinity transport system for adenosine was 21.1 μM and the V _max value was 257.3 pmol/min/mg protein. The transport system was inhibited by both compounds structurally related (compounds 554 and 555) and unrelated (dipyridamole) to adenosine. Because electrically stimulated synaptosomes release up to 1.5% of the adenosine derivative content per min, the physiological significance of adenosine uptake is discussed as a possible mechanism to compensate for the loss of adenine nucleotides from synaptosomes preparations.     

Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans

Three exo-glucanases, two endo-glucanases and two β-glucosidases were separated and purified from the culture medium of Aspergillus nidulans. The optimal assay conditions for all forms of cellulase components ranged from pH 5.0 to 6.0 and 50°C and 65°C for exo-glucanases and endo-glucanases but 35°C and 65°C for β-glucosidases. A close relation of enzyme stability to their optimal pH range was observed. All the cellulase components were stable for 10 min at 40–50°C. Exo-II and Exo-III ( K _m, 38.46 and 37.71 mg/ml) had greater affinity for the substrate than Exo-I ( K _m, 50.00 mg/ml). The K _m values of Endo-I and Endo-II (5.0 and 4.0 mg/ml) and their maximum reaction velocities ( V _max, 12.0 and 10.0 IU/mg protein) were comparable. β-Glucosidases exhibited K _m values of 0.24 and 0.12 mmol and V _max values of 8.00 and 0.67 IU/mg protein. The molecular weights recorded for various enzyme forms were: Exo-I, 29000; Exo-II, 72500; Exo-III, 138000; Endo-I, 25000; Endo-II, 32500; β-Gluco-I, 14000 and β-Gluco-II, 26000. Exo- and endo-glucanases were found to require some metal ions as co-factors for their catalytic activities whereas β-glucosidases did not. Hg²⁺ inhibited the activity of all the cellulase components. The saccharification studies demonstrated a high degree of synergism among all the three cellulase components for hydrolysis of dewaxed cotton.     

Choline Uptake in Cultured Human Y79 Retinoblastoma Cells: Effect of Polyunsaturated Fatty Acid Compositional Modifications

Abstract: Choline uptake in Y79 human retinoblastoma cells occurs through two kinetically distinguishable processes. The high-affinity system shows little sodium or energy dependence, and it does not appear to be linked to acetyl CoA: choline O -acetyltransferase. When the cells are grown in a culture medium containing 10% fetal bovine serum, the high-affinity system has a K' _m= 2.16 ± 0.13 μ m and V' _max= 27.0 ± 2.9 pmol min⁻¹ mg⁻¹, whereas the low-affinity system has K' _m= 20.4 ± 1.3 μ m and V' _max= 402 ± 49 pmol min⁻¹ mg⁻¹. Under these conditions, the polyunsaturated fatty acid content of the cell membranes is relatively low. When the polyunsaturated fatty acid content of the microsomal membrane fraction was increased by supplementing the culture medium with linolenic or docosahexaenoic acids (n-3 polyunsaturated fatty acids) or arachidonic acid (n-6 polyunsaturated fatty acid), the K' ^m of the high-affinity choline transport system was reduced by 40–60%. The V' _max also was reduced by 20–40%. Supplementation with oleic acid, the most prevalent monounsaturated fatty acid, did not affect either kinetic parameter. The results suggest that one functional effect of the high unsaturated fatty acid content of neural cell membranes is to facilitate the capacity of the high-affinity choline uptake system to transport low concentrations of choline. This effect appears to be specific for polyunsaturated fatty acids but not for a single type, for it is produced by members of both the n-3 and n-6 classes of polyunsaturated fatty acids.     

ACETYLCHOLINE METABOLISM AND CHOLINE UPTAKE IN CORTICAL SLICES

Abstract— The uptake of [¹⁴C]choline was studied in cortical slices from rat brain after their incubation in a Krebs-Henseleit medium containing either 4.7 m m -KCl (low K), 25 m m -KCl (high K) or 25 m m -KCl without calcium (Ca free, high K). With 0.84 μ m -[¹⁴C]choline in the medium the uptake per gram of tissue was 0.62 nmol after incubation in low K medium, 1.13 nmol after incubation in high K medium and 0.78 nmol after incubation in a Ca free, high K medium. The differences caused by potassium were greater in fraction P₂ than in fractions P₁ and S₂. With 17 and 50 μ m -[¹⁴C]choline in the medium greater amounts of [¹⁴C]choline were taken up, but the effect of potassium on the uptake almost disappeared. The amount of radioactive material in fraction P₂ followed Michaelis-Menten kinetics with K _m values of 2.1 and 2.3 μ m after incubation in low and high K medium, respectively. Hemicholinium-3 only slightly inhibited choline uptake from a medium with 0.84 μ m -[¹⁴C]choline, but it abolished the extra-uptake induced by high K medium. The radioactivity in the slices consisted mainly of unchanged choline and little ACh was formed after incubation in low K medium, but after incubation in high K medium 50% of the choline taken up was converted into ACh. The hemicholinium-3 sensitive uptake of choline, the conversion of choline into ACh and the synthesis of total ACh, were stimulated about 7–8-fold by potassium. It is concluded that in cortical slices from rat brain all choline used for the synthesis of ACh is supplied by the high-affinity uptake system, of which the activity is geared to the rate of ACh synthesis.     

Diaminopimelic acid uptake by Bacillus megaterium: influence of growth conditions and other amino acids

Diaminopimelic acid (DAP) uptake was studied in Bacillus megaterium. The K _m and V _max were determined for bacteria grown with or without DAP. The uptake of DAP was shown to be constitutive and unaffected by the presence of other amino acids (including cystine). The concentration of DAP and lysine in the amino acid pool was examined and a procedure for pulse-labelling cell walls developed.     

Characterization of Serotonin N-Acetyltransferase in the Lateral Eye of the Green Frog Rana perezi: Protective Action of EGTA

Abstract: The kinetics of seRotonin N -acetyltransferase (NAT) from the lateral eye of Rana perezi have been characterized. NAT from ocular tissue reached maximal activity at a phosphate buffer concentration of 250 m M and a pH of 6.5. Reaction linearity was highly conserved within the homogenate fraction range tested (0.033-0.33). The time course of ocular NAT reaction showed a high linearity at 25 and 35°C. K _m and V_max estimations for acetyl-CoA at a 10 m M tryptamine concentration were 63.3 μ M and 4.42 nmol/h per eye, respectively. Regardless of the acceptor amine (tryptamine or serotonin), the K _m was not affected by the acetyl-CoA concentration (50 or 250 μ M ), whereas the V _max was significantly increased at a 250 μ M acetyl-CoA concentration. Ocular NAT showed a higher affinity for serotonin ( K _m= 20.7 μ M ) than for tryptamine ( K _m= 48-60 μ M ); V _max however, was similar for both substrates. Acetyl-CoA does not protect ocular NAT; in contrast, the use of EGTA (4 m M ) in the assay is essential to protect the enzyme because NAT in ocular crude homogenate shows rapid inactivation. This result suggests that intracellular calcium levels are involved in the NAT inactivation mechanisms in frog ocular tissue.     

Metabolism of nitric oxide in soil and denitrifying bacteria

Abstract Production and consumption of NO was measured under anaerobic conditions in a slightly alkaline and an acidic soil as well as in pure cultures of denitrifying Pseudomonas aeruginosa, P. stutzeri, P. fluorescens, Paracoccus denitrificans, Azospirillum brasilense , and A. lipoferum . Growing bacterial cultures reduced nitrate and intermediately accumulated nitrite, NO, N₂O, but not NO₂. Addition of formaldehyde inhibited NO production and NO consumption. In the presence of acetylene NO was reduced to N₂O. Net NO release rates in denitrifying bacterial suspensions and in soil samples decreased hyperbolically with increasing NO up to mixing ratios of about 5 ppmv NO. This behaviour could be modelled by assuming a constant rate of NO production simultaneously with a NO consumption activity that increased with NO until V _max was reached. The data allowed calculation of the gross rates ( P ) of NO production, of the rate constants ( k ), V _max and K _m of NO consumption, and of the NO compensation mixing ratio ( m _c). In soil, P was larger than V _max resulting in net NO release even at high NO mixing ratios unless P was selectively inhibited by chlorate + chlorite or by aerobic incubation conditions. In bacteria, V _max was somewhat larger than P resulting in net NO uptake at high NO mixing ratios. Both P and V _max were dependent on the supply of electron donor (e.g. glucose). Both in soil (aerobic or anaerobic) and in pure culture, the K _m values of NO consumption were in a similar low range of about 0.5–6.0 nM. Anaerobic soil and denitrifying bacteria exhibited m _c values of 1.6–2.1 ppmv NO and 0.2–4.0 ppmv NO, respectively.     

The Uptake of Carnitine by Slices of Rat Cerebral Cortex

Abstract: The properties of carnitine transport were studied in rat brain slices. A rapid uptake system for carnitine was observed, with tissue-medium gradients of 38 ± 3 for L-[¹⁴CH₃]carnitine and 27 ± 3 for D-[¹⁴CH₃]carnitine after 180 min incubation at 37°C in 0.64 mM substrate. Uptake of L- and D-carnitine showed saturability. The estimated values of K _m for L- and D-carnitine were 2.85 mM and 10.0 mM, respectively; but values of V _max (1 μmol/min/ml in-tracellular fluid) were the same for the two isomers. The transport system showed stereospecificity for L-carnitine. Carnitine uptake was inhibited by structurally related compounds with a four-carbon backbone containing a terminal carboxyl group. L-Carnitine uptake was competitively inhibited by γ-butyrobetaine ( K _i= 3.22 mM), acetylcarnitine ( K _i= 6.36 mM), and γ-aminobutyric acid ( K _i= 0.63 mM). The data suggest that carnitine and γ-aminobutyric acid interact at a common carrier site. Transport was not significantly reduced by choline or lysine. Carnitine uptake was inhibited by an N₂ atmosphere, 2,4-dinitrophenol, carbonylcyanide- N -chlorophenylhydrazone, potassium cyanide, n-ethylmaleimide, and ouabain. Transport was abolished by low temperature (4°C) and absence of glucose from the medium. Carnitine uptake was Na⁺-dependent, but did not require K⁺ or Ca²⁺.     

COMPARATIVE STUDIES ON SYNAPTOSOMES: UPTAKE OF [N-Me-3H]CHOLINE BY SYNAPTOSOMES FROM SQUID OPTIC LOBES

Abstract— The uptake of [ N -Me-³H]choline into synaptosomes from squid optic lobes was studied using a Millipore filtration technique. When incubated in an artificial sea water medium at 26°C, but not at 0°C, the synaptosomes rapidly accumulated choline, most of which could be recovered as unchanged free choline. The accumulated choline was readily released by treatment of the synaptosomes with Triton X-100 or exposing them to hypo-osmotic conditions. The influx of choline increased with increasing concentrations of choline and could be resolved into saturable and non-saturable components. Kinetic analysis revealed the presence of two saturable components one of high affinity ( K _m about 2 μ m ) and one of lower affinity ( K _m 25 μ m ). The rate of choline uptake by these synaptosomes was considerably greater than by mammalian brain synaptosomes. Both high and low affinity systems were Na⁺-requiring and inhibited by hemicholinium no. 3, levorphanol and dextrorphan. NaCN, 2,4-dinitrophenol and ouabain also inhibited choline uptake, the high affinity system being particularly sensitive to these agents. It is suggested that the high affinity system is specific for cholinergic terminals.     

Xylanolytic activity of commercial juice-processing enzyme preparations

Of 22 commercial juice-processing enzyme preparations investigated, Clarex ML was found to exhibit the highest xylanase activity. The xylanase from Clarex ML was most active at 50–60°C and pH 5·0–5·5. The K _m and V _max values of the enzyme with oat-spelt xylan as the substrate were 8·6 mg ml⁻¹ and 42 μmol xylose l⁻¹ min⁻¹, respectively. Xylobiose was the main product of enzymatic hydrolysis of xylan.     

Characterization of a secondary uptake system for l-glutamate in Corynebacterium glutamicum

Abstract A new transport system for the uptake of l-glutamate was characterized in Corynebacterium glutamicum strain Δ glu, in which the previously described binding protein-dependent glutamate uptake system is not present. Kinetic characterization revealed a highly specific secondary transport system, dependent on sodium ions. Glutamate uptake showed Michaelis-Menten kinetics, with a K _m of 0.6 mM and a V _max of 15 nmol min⁻¹ (mg dw)⁻¹. For the co-transported sodium ions, a relatively low K _m of 3.3 mM was determined.     

Enzymes involved in ammonia assimilation in the fungus Acremonium persicinum

Abstract Acremonium persicinum grown in batch culture with ammonium tartrate as the nitrogen source possessed an NADP⁺-dependent glutamate dehydrogenase and a glutamine synthetase. Glutamate synthase was not detected under the culture conditions used. Kinetic studies of the NADP⁺-dependent glutamate dehydrogenase at 25°C and pH 7.6 revealed an apparent K _m of 3.2 × 10⁻⁴ M for 2-oxoglutarate and an apparent K _m of 1.0 × 10⁻⁵ M for ammonium ions, with corresponding apparent V _max values of 0.089 and 0.13 μmol substrate converted/min/mg of protein, respectively. Glutamine synthetase was measured by the γ-glutamyl transferase reaction at 30°C and pH 7.55. This transferase reaction of glutamine synthetase had a higher rate at 30°C than at 25°C or 37°C.     

Effect of Electroconvulsive Shock on the Uptake and Release of Noradrenaline and 5-Hydroxytryptamine in Rat Brain Slices

Abstract: The uptake and release of [³H]noradrenaline and [³H]-5-hydroxytryptamine (5-HT) were studied in cerebral cortex slices from rats 30 min and 24 h after a single electroconvulsive shock (ECS) and 24 h after a series of five shocks given over 10 days. Both the K _m and V _max for 5-HT uptake were lower than controls 24 h after a single ECS, whereas after 5 ECS spread over 10 days both parameters remained depressed, though only the fall in V_max was significant. Noradrenaline uptake was not altered after a single ECS, but the V_max and K _m were elevated following chronic ECS treatment. Neither ECS treatment schedule had any effect on the potassium-stimulated release of either transmitter. It is possible that the changes in monoamine uptake seen following ECS are an adaptive response to alterations in the synaptic cleft concentration of these transmitters.     

Kinetic characteristics of the sodium uptake mechanism during the development of embryos and fry of Atlantic salmon, Salmo salar L., in an improved water quality

The kinetics of active sodium uptake in dechorionated embryos, yolk-sac fry and start-feed fry of Atlantic salmon were compared in two groups reared either in low conductivity, untreated, river water (conductivity ∼ 46 μS cm⁻¹, pH 5.75), or in 'improved' river water buffered with sea water (conductivity ∼2200 μS cm ¹, pH 6.56), the latter treatment often being used in commercial hatcheries to avoid problems associated with periodic acidification.
Maximal transport rate ( V _max) increased during development in both groups but was always significantly higher in embryos and fry maintained in untreated river water. Values for K _m were not seen to vary during development up to 12 weeks after hatching and were not significantly different between groups, or from values reported for adult Atlantic salmon in fresh water.
The results are discussed with respect to the influence of Na⁺ concentrations in the perivitelline fluid of developing eggs and in the external medium surrounding fry on V _max and K _m. The ability of fry reared entirely in buffered river water to maintain sodium balance following transfer to untreated river water is also considered.