Kinetic analysis of blood-brain barrier transport of amino acids.

The Michaelis-Menten kinetics of blood-brain barrier transport of fourteen amino acids was investigated with a tissue-sampling, single-injection technique in the anesthetized rat. Tracer quantities of 14C-labelled amino acids and 3H2O, used as a freely diffusible internal reference, were mixed in 0.2 ml of buffered Ringer's solution and injected rapidly into a common carotid artery. Circulation was terminated by decapitation at 15s following injection. A brain uptake index (Ib) was determined from the ratio of 14C dpm in the brain tissue and the injection mixture divided by the same ratio for the 3H2O reference. Brain clearance of tracer concentration of amino acid was saturable when various concentrations of unlabeled amino acid were added to the injection solution. Double reciprocal plots of the saturation data yielded Km (mM) values that ranged from a low of 0.09 mM for arginine to a high of 0.75 mM for cycloleucine. Transport V values were determined from the relationship P = V/Km where P is the blood-brain barrier permeability constant (ml/g per min): P was calculated from the Ib for each amino acid based on a cerebral blood flow of 0.56 ml/g per min and a fractional extraction of 0.75 for the 3H2O reference 15s following carotid injection. The V values ranged from a low of 6.2 nmol/g per min for lysine to a high of 64 nmol/g per min for l-DOPA. Efflux of the tracer amino acid during the 15-s period after injection was assumed to be slow, since the rate constant of cycloleucine from brain to blood was low, 0.11 min-1.     

Fasting and refeeding modulate neutral amino acid transport activity in the basolateral membrane of the rat exocrine pancreatic epithelium: fasting-induced insulin insensitivity

The effects of fasting and refeeding on amino acid transport in the perfused rat exocrine pancreas were investigated using a rapid dual tracer dilution technique. Unidirectional amino acid influx (15 s) was quantified (relative to the extracellular tracer d-mannitol) over a wide range of perfusate concentrations in pancreata isolated frm fed and 24 h, 48 h, and 72 h fasted and 72 h fasted and refed (24 h) animals. In fed animals transport of phenylalamine (1–24 mM) and l-serine (1–50 mM) was saturable and weighted non-linear regression analyses of the overall transport indicated an apparent K_t=10±3mM and V_max=7.0±1.0 μmol/min per g (n = 7) for phenylalanine and K_t=16±3 mM and V_max=20.6±2.1 μmol/min per g (n = 5) for serine. Fasting animals for 24 h or 48 h did not change the kinetics of either phenylalanine or serine transport. After a 72 h fast the rate of phenylalanine transport (V_max=15.9±2.9 μmol/min per g, (n = 5) was enhanced whereas the transport affinity (K_t=11±3 mM) remained unaltered. l-Serine transport was essentially unaltered. When 72 h fasted animals were refed for 24 h the V_max for the phenylalanine transport was reduced to values observed in fed animals. In parallel experiments refeeding had no significant effect on serine transport. Perfusion of pancreata isolated from 72 h fasted animals with bovine insulin (1 mU/ml or 1 μU/ml) did not stimulate either phenylalanine or serine transport. The fasting-induced stimulation of transport may provide a mechanism by which the extracellular supply of essential amino acids as phenylalanine is increased to meet the demands of continued proteolytic and lipolytic enzyme synthesis.     

Amino acid transport in mammalian oocytes

Uptake of ¹⁴C-labelled amino acids into single oocytes was determined using ³H-labelled choline to correct for extracellular space. Cycloleucine, a non-metabolisable amino acid sharing an entry mechanism with methionine and phenylalanine, was transported in accord with Michaelis-Menten kinetics. At extracellular levels below 8 mM, cycloleucine was concentrated within the oocyte. The proportion of sheep oocytes having a functional amino acid transport system (i.e. cycloleucine flux > 1 nmole cm⁻² h⁻¹) was highest in pre-ovulatory follicles (97%), and lowest in atretic follicles (59%). Amino acid fluxes in functional germinal vesicle oocytes were similar at all stages of development studied. An increase in V_max but not K_m during meiotic maturation resulted in a doubling of amino acid uptake in metaphase II oocytes. These increased fluxes were under gonadotropic regulation and were independent of nuclear maturation. Amino acid uptake by mouse oocytes was approximately half that measured in sheep oocytes.     

Effect of pH on H-2H exchange,H2 production and H2 uptake,catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans

(1) The kinetics of isotope exchange catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans have been studied by measuring H²H, H₂ or ²H₂ produced when the enzyme catalyses the exchange between ²H₂ and H₂O or H₂ and ²H₂O. (2) In the ²H₂-H₂O system the measured rate of H₂ production was always higher than that of H²H. The $\text{H}{}_2\text{H}{}^2\text{H}$ ratio remained constant (about 1.70) in the protein concentration range 0.08–1.32 mg. The very rapid formation of H₂ with respect to H²H is consistent with the hypothesis of a heterolytic cleavage of ²H₂ into a deuteron and an enzyme hydride that can exchange with the solvent. (3) In the H₂-²H₂O system, the exchange rate was much lower than in the ²H₂-H₂O system, indicating a marked isotopic effect of ²H₂O. (4) The H-²H exchange activity, determined from the initial velocity of H²H formation, is optimal at pH 4.5. A second maximum of activity is observed at pH 8.3. The pH value of 4.5 is also the pH optimum for H₂ production while at pH 8.3–8.5 there is a maximum of H₂ oxidation activity. (5) In ordinary H₂O the K_m for hydrogen uptake estimated either from H₂ consumption or from benzyl viologen reduction was 0.06–0.07 μM for both H₂ and ²H₂ indicating a strong affinity of the enzyme for hydrogen at pH 8.3–8.5. Shifting from H₂O to ²H₂O does not affect the K_m of the enzyme for H₂ but lowers the V_max value about 10-fold. The K_m for benzyl viologen and methyl viologen was 0.08 and 2 mM, respectively.     

Purification and characterization of an intracellular N-terminal exopeptidase from Streptococcus durans

An intracellular N-terminal exopeptidase isolated from cell extracts of Streptococcus durans has been purified 470-fold to homogeneity (specific activity of 12.0 μmol/min per mg). In the absence of thiol compounds, the purified aminopeptidase undergoes a slow oxidation with a 70% loss of activity, which can be restored by the addition of 2 mM β-mercaptoethanol. The purified aminopeptidase (M_r 300 000) preferred L-peptide and arylamide substrates with small nonpolar or basic side chains. SDS electrophoresis yielded a single protein band corresponding to a molecular weight of 49 400, suggesting that the native enzyme is a hexameric protein. The enzyme-catalyzed hydrolysis of $\text{L}\text{-}\text{alanyl}\text{-p-}\text{nitroanilide}$ exhibited a bell-shaped pH dependence for log V_max/K_m(pK₁ = 6.35; pK₂ = 8.50) while the log V_max versus pH profile showed only an acid limb (pK = 6.35). Methylene blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino acid analysis indicated that this photooxidative loss of activity corresponded to the modification of one histidine residue per enzyme monomer. N-Ethylmaleimide (100 mM) caused a 78% reduction in enzyme activity. Treatment of the enzyme with 1.0 mM hydrogen peroxide resulted in the oxidation of two cysteine residues per enzyme monomer and caused a 70% decrease in the catalytic activity.     

A role for membrane transport in modulation of intramuscular free glutamine turnover in streptozotocin diabetic rats

We wished to examine the effects of diabetes on muscle glutamine kinetics. Accordingly, female Wistar rats (200 g) were made diabetic by a single injection of streptozotocin (85 mg/kg) and studied 4 days later; control rats received saline. In diabetic rats, glutamine concentration of gastrocnemius muscle was 33% less than in control rats: 2.60 ± 0.06 μmol/g vs. 3.84 ± 0.13 μmol/g (P < 0.001). In gastrocnemius muscle, glutamine synthetase activity (V_max) was unaltered by diabetes (approx. 235 nmol/min per g) but glutaminase V_max increased from 146 ± 29 to 401 ± 94 nmol/min per g; substrate K_m values of neither enzyme were affected by diabetes. Net glutamine efflux (AZ concentration difference × blood flow) from hindlimbs of diabetic rats in vivo was greater than control values (?30.0 ± 3.2 vs. ?1.9 ± 2.6 nmol/min per g (P < 0.001) and hindlimb NH₃ uptake was concomitantly greater (about 27 nmol/min per g). The glutamine transport capacity (V_max) of the Na-dependent System N^m in perfused hindlimb muscle was 29% lower in diabetic rats than in controls (820 ± 50 vs. 1160 ± 80 nmol/min per g (P < 0.01)), but transporter K_m was the same in both groups (9.2 ± 0.5 nM). The difference between inward and net glutamine fluxes indicated that glutamine efflux in perfused hindlimbs was stimulated in diabetes at physiological perfusate glutamine (0.5 mM); ammonia (1 mM in perfusate) had little effect on net glutamine flux in control and diabetic muscles. In Intramuscular Na⁺ was 26% greater in diabetic (13.2 μmol/g) than control muscle, but muscle K⁺ (100 μmol/g) was similar. The accelerated rate of glutamine release from skeletal muscle and the lower muscle free glutamine concentration observed in diabetes may result from a combination of; (i), a diminished Na⁺ electrochemical gradient (i.e., the net driving force for glutamine accrual in muscle falls); (ii), a faster turnover of glutamine in muscle and (iii), an increased V_max/K_m for sarcolemmal glutamine efflux.     

Blood-brain barrier transport ofL-pipecolic acid in various rat brain regions

Blood-brain barrier transport ofL-[l-¹⁴C]pipecolic acid was studied in the rat by single intracarotid injection using³H₂O as a diffusible internal standard. Brain uptake index (BUI) forL-[¹⁴C]pipecolic acid (0.036 mM) was found to be 18.1, 10.5, and 12.6 for the cerebral cortex, brain stem, and cerebellum, respectively which was substantially higher than that reported for its analogL-proline in the whole brain. Influx ofL-pipecolic acid into the brain was concentration dependent and differed significantly between the cerebral cortex and the brain stem, and between the cerebral cortex and the cerebellum, but not between the brain stem and the cerebellum. Kinetic study ofL-pipecolic acid influx revealed a low- and a high-capacity uptake mechanisms. The low-capacity saturable component hasK _m values ranging from 38 to 73 μM, andV _max values ranging from 10 to 13 nmol/g/min for the three brain regions. The nonsaturable component has aK _m of 4 mM, aV _max of 200 nmol/g/min and similar diffusion constant (K _d) (0.03 to 0.06 mlg^?1 min^?1) for all three brain regions. A possible role of the two-component brain uptake mechanism in the regulation of the neuronal function ofL-pipecolic acid was suggested.     

Energization of sulfate transport in yeast

Sulfate uptake by Saccharomyces cerevisiae is stimulated about 12-fold by preincubation of cells with 1% d-glucose or 1% ethanol. The $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ remains unchanged (0.34–0.38 mM), the $\text{J}{}_{\mathrm{<mtext>mar</mtext>}}$ increase from 18–20 to 195–230 and 170–185 nmol/min per g dry wt., respectively, after glucose and ethanol preincubation. The stimulation involves protein synthesis (it is suppressed by cycloheximide), has a half-time of 18 min and requires mitochondrial respiration (no or low effect in respiration-deficient mutants and those lacking ADP-ATP transport in mitochondria, as well as after anaerobic preincubation of the wild-type strain, and in low-phosphate cells). The presence of NH₄⁺ and some amino acids (e.g., leucine, aspartate, cysteine and methionine) depressed the stimulation while that of cationic amino acids (typically arginine and lysine) and of K⁺ increased it by 50–80%. The stimulated (i.e., newly synthesized) transport system was degraded with a half-life of about 10 min.     

Mechanism of action of Clostridium perfringens enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes

Purified enterotoxin from the bacterium Clostridium perfringens rapidly decreased the hormonally induced uptake of α-aminoisobutyric acid in primary cultures of adult rat hepatocytes. At 5 min after toxin addition the decrease in α-aminoisobutyric acid uptake appeared not due to increased passive permeation (estimated with l-glucose) or to increased α-aminoisobutyric acid efflux. When short uptake assay times were employed a depression of α-aminoisobutyric acid influx was observed in toxin-treated hepatocytes. The depression of α-aminoisobutyric acid influx was correlated with a rapid increase in intracellular Na⁺ (estimated using ²²Na⁺) apparently effected by membrane damage. In contrast, the uptake of cycloleucine in the presence of unlabeled α-aminoisobutyric acid (assay for Na⁺-independent amino acid uptake) by hepatocytes treated with toxin for 5 min was decreased to only a small extent or not at all depending upon experimental design. At later times, C. perfringens enterotoxin increased the exodus of l-glucose, $\text{3-O-}\text{methylglucose}$ and α-aminoisobutyric acid from pre-loaded cells indicating that the toxin effects progressive membrane damage. When enterotoxin was removed by repeated washing after 5–20 min the decay of α-aminoisobutyric acid uptake ceased and appeared to undergo recovery towards the hormonally induced control level. The degree of recovery of α-aminoisobutyric acid uptake was inverse to the length of time of exposure to toxin. Adding at 10 min specific rabbit antiserum against C. perfringens enterotoxin without medium change also reversed the effect of toxin on increased intracellular ²²Na⁺, and on the exodus (from preloaded cells) of α-aminoisobutyric acid, L-glucose, and $\text{3-O-}\text{methylglucose}$.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Volume and sound velocity changes associated with coil- transition of poly(S-carboxymethyl L-cysteine) in aqueous solution

The measurements were made for the volume and the sound velocity changes (ΔV and ΔU) on titrating the sodium salt of poly (S-carboxymethyl L -cysteine) with dilute HCl. For the reaction, ? COO^? + H⁺ → ? COOH, ΔV per mole of H⁺ bound was + 12. 7 ml and +11. 4 ml in salt-free and 0. 2 M NaCl solutions, respectively. Corresponding ΔU was about ?13 cm/sec in salt-free polymer solution where 11.5 mM carboxylate ion reacts with equimolar hydrogen ion. ΔV associated with the coil-to-β transition was found to be +2. 35 ml in H₂O and +1. 90 ml in 0. 2 M NaCl per mole of amino acid residue, respectively. These values are larger than those obtained for the coil-to-helix transition of poly (L -glutamic acid). ΔU for the transition was about ?30 cm/sec in salt-free solution of polymer concentration 0.0115 mole/liter. Possible sources of ΔV and ΔU for reaction; coil → β, are (1) the formation of void volume and (2) the changes in the extent of solvation in amide linkage and in side chain.     

SYNTHETIC AMINO ACIDS AND THE NATURE OF l-DOPA TRANSPORT AT THE BLOOD-BRAIN BARRIER

—The blood-brain barrier transport of amino acids has been measured using the carotid injection technique in the rat. The synthetic amino acids, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) and α-(methylamino)isobutyric acid (MeAIB), were model substrates in the Ehrlich cell for the leucine (L) and alanine (A) neutral amino acid transport mechanisms, respectively. The uptake (±)b-[carboxyl-¹⁴C]BCH at the same rate for the five brain regions tested suggested a similarity between regions for the L transport mechanism. At injectant concentrations of 0·1 mm (similar to naturally occurring aromatic neutral amino acids), BCH was mainly taken up by a saturable mediated transport mechanism (K₁, 0·16 mm and V_max, 0·03/μmol/g per min). At higher concentrations, uptake by a nonsaturable or diffusional mechanism could be demonstrated. When BCH was added as a second amino acid to l -[3-¹⁴C]DOPA, the saturable component of l -DOPA transport was significantly inhibited. MeAIB had no measurable effect on the rate of l -DOPA transport. These results suggested that the mediated transport mechanism for l -DOPA at the cerebral capillaries is similar to the l -neutral amino acid transport system.     

Saturation behavior of ascites tumor cell chloride exchange in the presence of gluconate

Steady state Cl^? flux across the Ehrlich mouse ascites cell membrane was studied when gluconate replaced Cl^? in the external medium. Saturation behavior was observed; $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was 23.9 mM Cl^? and V was 758 μmol · g^?1 dry weight · h^?1. The cells lost K⁺, Cl^? and H₂O, consistent with relative impermeability to gluconate, and the Cl^? efflux rate coefficient was elevated. The results indicate that a major portion of Cl^? exchange occurs as a membrane transport process and suggest that the process is sensitive to intracellular Cl^? levels.     

Mechanistic studies of epoxide hydrolase utilizing a continuous spectophotometric assay

A precise continuous photometric assay has been devised and utilized for mechanistic studies of chicken and rat liver microsomal epoxide hydrolase (EH). The assay is based on monitoring the hydration of p-nitrostyrene oxide (PNSO) at 310 nm. Rat liver EH hydrates S-(+)- and R-(?)-PNSO differentially, the K_m and V values for the former being ca. four times those for the latter; in contrast, enantiomeric differences are negligible with chicken liver EH. With rat EH V increases slightly from pH 7 to 8 and then falls rapidly from pH 8 to 9.5; K_m remains constant from pH 7 to 8 and then increases steadily from pH 8 to 9.5. In 86 mol% D₂O the solvent isotope effect on V ($\text{H}{}_2\text{O}\text{D}{}_2\text{O}$) is 1.103 ± 0.015. Both rat and chicken EH show a 3% inverse isotope effect for the hydration of [7-²H]PNSO and a 4% normal isotope effect for the hydration of [8-²H₂]PNSO. These observations are discussed in terms of the possible participation of acid as well as base catalysis in the enzymatic mechanism.     

Bimodal effects of cellular amino acids on Na+-dependent amino acid transport in ehrlich cells

Cells depleted of amino acids show lower rates of glycine or aminoisobutyric acid uptake than do freshly isolated cells. In the amino acid-depleted cells, addition of valinomycin stimulates amino acid influx at least to the level observed in freshly isolated cells. In cells containing high levels of cellular amino acids, valinomycin has little effect on influx of amino acids. It is concluded that the transport of amino acids in freshly isolated cells is elevated compared to depleted cells because the cells are hyperpolarized by the continuous loss of cellular amino acids during the transport assay. During this hyperpolarization by amino acid loss, transport of amino acids is not further stimulated by valinomycin at low external [K⁺] ($\text{10 mM ± 5 mM}$).With the exception of preloading with glycine, cells preloaded with a single amino acid to a concentration greater than 20 mM show reduced rates of glycine and aminoisobutyric acid influx at early times (less than 15 min) compared to amino acid-depleted cells. The reduction of infiux is transient and by 30 min, influx is greater in preloaded than in amino acid-depleted cells.Knowing that increases and decreases in the membrane potential are achieved by using varying external [K⁺] in the presence of valinomycin and propranolol, and using amino acid-depleted cells, it can be shown that an increased membrane potential increases the $\text{V}$ for glycine and aminoisobutyric acid influx. A decrease in the potential difference results in a decreased $\text{V}$. Changes in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ also occur when the membrane potential is varied.     

Fungal proteases and the mammalian kinin system. II. Kinin hydrolysis by brinolase.

Brinolase, a thrombolytic fungal protease capable of forming vasoactive kinins, has been shown to hydrolyze kinins after their formation. Using synthetic bradykinin as a substrate, the kinetics and mechanism of hydrolysis have been elucidated, evidently explaining the apparently low kinin formation $\text{in vivo}$, Bradykinin hydrolysis proceeded rapidly $\text{in vitro}$ with a pH optimum of 7.0–7.5, and a half-life of 5.1 min, using 250 ng/ml bradykinin and 50 μg/ml brinolase. The K_m was 3.2×10^?6 M and the V_max was 4.6 × 10^?8 mol/liter/min, using 5 μg/ml brinolase. Two-dimensional paper fractionation of the brinolase-bradykinin digest revealed the presence of free arginine amongst the five peptide fragment spots.