Choline fluxes in primary nerve cell cultures. Correlation with the endocellular metabolism of choline

The fluxes of choline across the plasma membrane were measured in primary nerve cell cultures from chick embryo cerebral hemispheres containing neurons and supporting cells.The incubation of cells with exogenous concentrations of choline far below the concentrations present in the growth medium (～30–50 μM) and in the range of the high affinity uptake mechanism (about 0.5 μM) profoundly affected the steady state of the endocellular free choline levels. The kinetics of the uptake were dependent upon the endocellular status of the choline pool since after preincubation in the absence of choline two K_ms are observed (K_m1: 0.8 μM; V_max1: 44.8 pmol/mg protein/2 min; K_m2: 14.3 μM, V_max2: 333.3 pmol/mg protein/2 min) while only one mechanism can be found when the endocellular pool of choline was kept in steady state conditions (K_m: 14.3 μM, V_max: 545.5 pmol/mg protein/2 min). The presence of an homoexchange phenomenon was suspected since choline efflux could be increased by increasing the concentrations of choline in the incubation medium.The results suggest that the movement of choline into nerve cells in culture appears to be mediated by a single mechanism which is regulated by the endocellular status of the choline pool.     

Creatine kinase activity of urinary bladder and skeletal muscle from control and streptozotocin-diabetic rats

The urinary bladder depends on intracellular ATP for the support of a number of essential intracellular processes including contraction. The concentration of ATP is maintained constant primarily via the rapid transfer of a phosphate from creatine phosphate (CP) to ADP catalyzed by the enzyme creatine kinase (CK). Since muscular pathologies associated with diabetes are in part related to intracellular alterations in metabolism, we have characterized the CK activity in both skeletal muscle and urinary bladder from control and streptozotocin-diabetic rats.The following is a summary of the results: 1) Bladder tissue from control rats showed linear kinetics with a V_max = 390 nmoles/mg protein/min, and a K_m = 275 µM. 2) Urinary bladder tissue isolated from diabetic rats displayed biphasic kinetics with V_max = 65 and 324 nmoles/mg protein/min, and K_m's = 10 µM and 190 µM respectively. 3) Skeletal muscle isolated from control rats showed linear kinetics with an approximate V_max of 800 nmoles/mg protein/min and a K_m of 280 µM CP. 4) Homogenates of skeletal muscle from diabetic rats showed complex kinetics not separable into distict component forms. 5) The K_m for ADP for both skeletal muscle and bladder was approximately 10 µM.These studies demonstrate that whereas bladders isolated from both control and diabetic rats possess a low-affinity isomer(s) of CK with similar maximum enzymatic activity, there is a high affinity isomer present within the urinary bladder muscle of diabetic rats that is not present in bladder tissue isolated from control rats. Skeletal muscle isolated from both diabetic and control rats exhibited a maximal activity 2 to 3 times higher than that of the bladder.     

KINETICS OF AMINO ACID INFLUX INTO NITELLA FLEXILIS1

The uptake of amino acids by Nitella flexilis has been investigated. Influx of glycine, alanine, and valine appears to be a diffusive process. Influx ranged from 0.14 to 0.06 and 0.04 pmoles/(cm)(sec), respectively. Aspartic acid uptake is an active transport mechanism. The V_max is 2.8 pmoles/(cm)(sec); the transport constant (Michaelis constant) K_m, 7.8 × 10^?3 M. The uptake of arginine is apparently due to 2 transport systems, one with a V_max and K_m of 3.1 pmoles/(cm)(sec) and 3.2 × 10^?3M, respectively. The second system has a V_max of 1.4 pmoles/(cm)(sec) and a K_m of 2.1 × 10^?4 M. The possibility that the second system is diffusive has been considered.     

Regulation of Sertoli cell cyclic adenosine 3':5' monophosphate phosphodiesterase activity by follicle stimulating hormone and dibutyrl cyclic AMP

Total phosphodiesterase activity was measured in Sertoli cell culture after exposure to isobutyl-methyl-xanthine, dibutyryl cyclic AMP and FSH. After 24 hr of incubation both FSH and dibutyryl cAMP caused a significant increase in total phosphodiesterase activity of Sertoli cell homogenates (control: 66 ± 16 pmoles/min/mg protein; FSH: 291 ± 25 pmoles/min/mg protein; dibutyryl cAMP: 630 ± 70 pmoles/min/mg protein). FSH stimulation was potentiated by isobutyl-methyl-xanthine. Both in the presence and absence of xanthine, the induction of phosphodiesterase was dependent on the FSH concentration, with maximal stimulation achieved with 0.5–1.0 μg FSH/ml. The induction of phosphodiesterase activity by hormone was abolished by cycloheximide treatment. The data suggest that FSH regulates phosphodiesterase activity via changes of cAMP levels in Sertoli cell in culture.     

A COMPARATIVE STUDY ON THE SYNTHESIS OF THE NATRIURETIC HORMONE DOPAMINE IN OK AND LLC-PK1CELLS

TheV_maxvalues (in nmol/mg protein/15 min) for AAAD in OK cells (0.94±0.08) were found to be significantly (P<0.01) lower than those observed in LLC-PK₁cells (4.37±0.08). However, in both cell lines decarboxylation reaction was a saturable process with similarK_mvalues (OK cells=1.1 mm (0.3, 1.8); LLC-PK₁cells=1.8 mm (1.6, 2.1)). Contrariwise to OK cells, decarboxylation ofl -DOPA to dopamine in LLC-PK₁cells followed a linear (7.6±0.1 pmol/mg protein/min) non-saturable kinetics till 120 min of incubation. The formation of dopamine from increasing concentrations ofl -DOPA (10 to 500 μm ) followed a non-linear kinetics in both cell lines; the process ofl -DOPA decarboxylation was saturated at low concentrations ofl -DOPA with an apparentK_mvalue of 11 μm (0.2, 22.6) in OK cells and 27.4 μm (11.1, 43.7) in LLC-PK₁cells. The formation of dopamine in LLC-PK₁cells (V_max=2097±113 pmol/mg protein/6 min) was 13.7-fold that occurred in OK cells (V_max=153±10 pmol/mg protein/6 min). In conclusion, LLC-PK₁cells appear to be endowed with a greater ability to form dopamine from exogenousl -DOPA when compared to OK cells.     

Position specificity of an o-dihydroxycoumarin glucosyltransferase from tobacco cell suspension culture

A partially purified UDP-glucose: o-dihydroxycoumarin glucosyltransferase from tobacco cell culture required an intact coumarin ring system with o-dihydroxy groups for highest activity. The enzyme exhibited strict position specificity towards the 7-OH group of both esculetin and daphnetin with the formation of cichoriin and daphnin, respectively. The apparent K_m values were 111 and 95 μM, while the V_max for esculetin was 26.4 pkat/mg protein.     

Metabolism of (5E)-6a-carboprostaglandin I2 by rhesus monkey lung 15-OH prostaglandin dehydrogenase

(5E)-6a-Carbaprostaglandin I₂ (carbacyclin) was oxidized to (5E)-15-dehydro-6a-carbaprostaglandin I₂ (15-dehydrocarbacyclin) by partially purified rhesus monkey lung prostaglandin dehydrogenase (PGDH). The (5E)-15-dehydro-6a-carbaprostaglandin I₂ was isolated by preparative thin-layer chromatography and identified by gas chromatography-mass spectrometry. A Lineweaver-Burke plot gave an apparent K_m value of 2.9 μM and a V_max of 35.7 nmoles carbacyclin oxidized/mg protein/min. These values are similar to previously reported K_m and K_max values for PGI₂ and PGE₁.     

Melanosomal Tyrosine Transport in Normal and Pink-eyed Dilution Murine Melanocytes

Tyrosine is the endogenous substrate for melanin production within melanosomes, but the method of tyrosine transport into the melanosome has not been investigated. In the mouse, melanogenesis is disrupted by mutations in the p gene resulting in the pink-eyed dilution phenotype; it has been suggested that the p gene codes for a tyrosine transport protein. We determined that normal (melan-a) melanosome-rich granular fractions take up 10 μm [³H]tyrosine at 21.1 ± 6.1 (SEM, standard error of the mean) pmol/min/mg protein (N=7) compared with 21.3 ± 5.8 SEM pmol/min/mg protein (N=5) for pink-eyed dilution, whose plasma membrane tyrosine transport was also normal (K_m 89 μM; V_max 302 pmol/min/mg cell protein). We also demonstrated that pink-eyed dilution melanosomes are immature by virtue of their low density, high hexosaminidase activity, and lack of pigment. These data indicate that a tyrosine transport system exists in the melanosomal membrane and that the p gene does not encode a tyrosine transporter of critical importance.     

Adenosine transport by cultured glial cells from chick embryo brain

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [³H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with K_m = 370 μM and V_max = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na⁺ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (K_m = 12 μM; V_max = 0.34 nmol/ min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N⁶-benzyladenosine. In decreasing order of potency, N⁶-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.     

Ibuprofen Inhibits Arylamine N-Acetyltransferase Activity in the Bacteria Klebsiella pneumoniae

Ibuprofen, one of the nonsteroidal anti-inflammatory drugs, inhibited arylamine N-acetyltransferase activity of Klebsiella pneumoniae both in vitro and in vivo. The NAT activities of Klebsiella pneumoniae were inhibited by ibuprofen in a dose-dependent manner both in vitro and in vivo. In vitro, the NAT activity was 0.675 ± 0.028 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.506 ± 0.002 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In vivo, the NAT activity was 0.279 ± 0.016 nmol/min/10¹⁰ colony forming units (CFU) for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.228 ± 0.008 nmol/min/10¹⁰ CFU for the acetylation of 2-aminofluorene. The inhibition of NAT activity by ibuprofen was shown to persist for at least 4 h. For in vitro examination, the values of apparent K _m and V _max were 1.08 ± 0.05 mM and 9.17 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the reaction mixtures, the values of apparent K _m and V _max were 1.19 ± 0.01 mM and 6.67 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. For in vivo examination, the values of apparent K _m and V _max were 1.24 ± 0.48 mM and 4.18 ± 1.06 nmol/min/10 × 10¹⁰ CFU, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the culture, the values of apparent K _m and V _max were 0.95 ± 0.29 mM and 2.77 ± 0.37 nmol/min/mg protein, respectively, for 2-aminofluorene, respectively. This report is the first finding of ibuprofen inhibition of arylamine N-acetyltransferase activity in a strain of Klebsiella pneumoniae. Received: 28 January 1997 / Accepted: 12 February 1997     

Adenine nucleotide metabolism of blood platelets. VIII. Transport of adenine into human platelets

Adenine uptake into human blood platelets is a carrier-mediated process with a K_m of 159±21 nM and a V of 100±10 pmoles/min per 10⁹ platelets (in citrated platelet-rich plasma). The Q₁₀ was 2.53±0.22. A pH optimum was found at 7.5. Washing of the platelets increased the K_m to 453±33 nM and V to 397±38 pmoles/min per 10⁹ platelets. The change in shape induced in platelets by ADP was accompanied by an increase in V (2 times) and K_m (1.5 times).Guanine (K_i 50 μM), hypoxanthine (K_i 390 μM), adenine-N′-oxide (K_i 40 μM), adenosine (K_i 100 μM), RA 233 (K_i 75 μM) and papaverine (K_i 15 μM) acted as competitive inhibitors. Adenosine at low concentrations, and prostaglandin E₁ gave inhibition at only high adenine levels. A similar inhibition was obtained with 2-deoxy-d-glucose. Sulfhydryl-group inhibitors, pyrimidines and ouabain had no effect.     

Effect of indomethacin on cyclic AMP phosphodiesterase activity in myometrium from pregnant rhesus monkeys

Our results indicate that indomethacin inhibits cyclic AMP phosphodiesterase in the myometrium of the pregnant rhesus monkey under in vitro as well as in vivo conditions. Kinetic data on extracts of myometrium from pregnant rhesus monkeys indicated two cyclic AMP phosphodiesterase activities. The apparent K_m value for the high affinity enzyme averaged 3.9 μM and for the low affinity enzyme 23 μM; the V_max values averaged 0.56 and 1.4 nmoles cyclic AMP hydrolized per mg protein min^?1 respectively. When indomethacin was added to the myometrial extracts, the activity of the high K_m phosphodiesterase was competitively inhibited, with an average K_i of 200 μM; the low K_m enzyme was noncompetitively inhibited with an average K_i of 110 μM. Experiments on myometrial slices demonstrated that 10 μM indomethacin potentiated the effect of PGE₁ and epinephrine on cyclic AMP levels, presumably by inhibiting the phosphodiesterase activity. The uterine relaxing effect of indomethacin is generally attributed to the inhibition of prostaglandin synthetase activity. However, treatment of pregnant rhesus monkeys with therapeutic doses of indomethacin resulted in a significant inhibition of myometrial cyclic AMP phosphodiesterase activity in association with uterine relaxation and prolongation of gestation.     

1,4-Methylhistamine is a specific substrate for type B monoamine oxidase

1,4-Methylhistamine was characterized as substrate for monoamine oxidase (MAO) in rat liver mitochondria. The $\text{K}$_m and $\text{V}$_max values were 38.8 μM and 6.33 nmoles/mg protein/60 min, respectively. The inhibition experiments with clorgyline and deprenyl, the selective inhibitors for type A and type B MAO, showed that 1,4-methylhistamine was specific for type B MAO.     

Rat Liver Catechol-O-Methyltransferase Kinetics and Assay Methodology

Abstract

In mammals, catechol-O-methyltransferase (COMT) is distributed throughout various organs, the highest activities being found in the liver and kidney. However, comparisons of the kinetic parameters are difficult to perform, since the experimental procedures in the enzyme assay vary quite considerably. The present work was aimed at studying the optimal liver COMT assay conditions for determining the kinetics of the enzyme. The COMT assay was performed with liver homogenates from 60 days old male Wistar rats with adrenaline (AD) as the substrate. Time course experiments using 100 μM S-adenosyl-L-methionine (SAMe) and 300 μM AD showed linearity of O-methylation reaction upto 10min. Using 100μM SAMe, V_max (nmol mg protein' h^?1) and K_m (μM) values progressively decreased respectively from 22.1 and 104.8 at 5mindown to 5.8 and 24.62 at 60 min incubation periods. This decrease was not due to end-product inhibition. Using 2500 μM AD, K_m values (μM) for the methyl donor SAMe increased progressively from 174 at 5 min upto 1192.5 at 60 min; upto 30 min of incubation F_max values did not change. When a 5 min incubation period and 500 μM SAMe were used, V_max and K_m values for liver COMT were 63.4 nmol mg protein^?1h^?1 and 261.1 μM, respectively. It is concluded that an incubation period of 5 min and a SAMe concentration of 500 μM provide optimal conditions for the liver homogenate COMT assay.     

Transport of thymidine during the cell cycle in mitotically synchronized CHO cells

The kinetics of total uptake of thymidine into the cell were determined for cells which had been mitotically synchronized, plated into scintillation vials and pulsed with five concentrations of [³H]-thymidine at various times during the cell cycle. From Lineweaver-Burk plots of these rates, V_max and K_m values were determined for the transport of thymidine. The V_max values ranged from a low of 2.0 pmoles/ min/10⁶ cells in mid-G1 to a high of 99.7 in mid-S before a decline in late S and G2. K_m values displayed only a 5-fold range in values.     

Kinetic properties, and location of nonspecific phosphomonoesterases in subcellular fractions of fasciola hepatica

Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The K_m for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a V_max of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a V_max of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10^?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis.     

Characterization and inhibition of invertases in sugar cane juice

(NH₄)₂SO₄ fractionation followed by Sephadex G-200 chromatography of sugar cane juice gave an acid invertase with MW of 380 000 and 23.5% carbohydrate and a neutral invertase with MW of 66 000 and 22% carbohydrate. For acid invertase, K_m is 2.8 mM and V_max is 2.7 μmol sucrose hydrolysed/hr/mg protein. For neutral invertase, K_m and V_max are 0.32 mM and 2.8 μmol hydrolysed/hr/mg protein, respectively. Inhibition of both invertases by either lauryl sulfate or metasilicate is not competitive.     

Transport and metabolic effects of α-aminoisobutyric acid in saccharomyces cerevisiae

α-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a K_m for α-aminoisobutyric acid of 270 μM, a V_max of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1–4.3. α-Aminoisobutyric acid is also transported by a minor system(s) with a V_max of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The α-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. α-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH₄⁺. During nitrogen starvation α-aminoisobutric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that α-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because α-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.     

Role of periplasmic trehalase in uptake of trehalose by the thermophilic bacterium Rhodothermus marinus

Trehalose uptake at 65°C in Rhodothermus marinus was characterized. The profile of trehalose uptake as a function of concentration showed two distinct types of saturation kinetics, and the analysis of the data was complicated by the activity of a periplasmic trehalase. The kinetic parameters of this enzyme determined in whole cells were as follows: K_m = 156 ± 11 μM and V_max = 21.2 ± 0.4 nmol/min/mg of total protein. Therefore, trehalose could be acted upon by this periplasmic activity, yielding glucose that subsequently entered the cell via the glucose uptake system, which was also characterized. To distinguish the several contributions in this intricate system, a mathematical model was developed that took into account the experimental kinetic parameters for trehalase, trehalose transport, glucose transport, competition data with trehalose, glucose, and palatinose, and measurements of glucose diffusion out of the periplasm. It was concluded that R. marinus has distinct transport systems for trehalose and glucose; moreover, the experimental data fit perfectly with a model considering a high-affinity, low-capacity transport system for trehalose (K_m = 0.11 ± 0.03 μM and V_max = 0.39 ± 0.02 nmol/min/mg of protein) and a glucose transporter with moderate affinity and capacity (K_m = 46 ± 3 μM and V_max = 48 ± 1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose-poor environments (trehalose concentrations up to 6 μM); at higher concentrations trehalose is assimilated primarily via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased 60% in response to osmotic stress. The nature of the trehalose transporter and the physiological relevance of these findings are discussed.     

In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants <Emphasis Type="Italic">CYP2C19*23</Emphasis> and <Emphasis Type="Italic">CYP2C19*24</Emphasis>

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4′-hydroxylation activity of CYP2C19*23 (V _max 111.5 ± 16.0 pmol/min/mg, K _m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V _max 101.6 + 12.4 pmol/min/mg, K _m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K _m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V _max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CL_int = V _max/K _m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CL_int value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K _m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V _max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V _max/K _m, the CL_int value of CYP2C19 WT was 785 folds of CYP2C19*23. K _m and V _max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5′-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.