Glucuronidation and sulfation of 7-hydroxycoumarin in liver matrices from human, dog, monkey, rat, and mouse

Summary Uridine 5′-diphospho-N-acetylgalactosamine glycosyltransferases (UGTs) and sulfotransferases (SULTs) are 2 phase II enzymes that are actively involved in detoxification processes as well as in drug metabolism. Compared with cytochrome P450 enzymes, the role of UGTs and SULTs in drug metabolism has received little attention. Liver microsomes, S9 fractions, and cryopreserved hepatocytes from human, dog, cynomolgus monkey, mouse, and rat were used as matrices in the study. Single compound, 7-hydroxycoumarin (7-HC), along with necessary cofactors was dosed into the matrices and incubated at 37° C; formation of two metabolites, 7-HC-glucuronide and 7-HC-sulfate, was determined with liquid chromatography with tandem mass spectrometry. Within the same species, the UGTs activities in microsomes and S9 fractions were comparable. In addition, UGTs activities in cryopreserved hepatocytes were lower than in the other matrices. Also, the SULTs activities were much higher in S9 fractions than in cryopreserved hepatocytes and microsomes. Species differences on UGTs and SULTs activities were also observed. The results indicated that S9 fractions, microsomes, and cryopreserved hepatocytes might be useful for UGTs metabolism study, whereas S9 fractions appear to be the most appropriate matrix for both UGTs and SULTs metabolism. Species differences with respect to phase II metabolism also need to be taken into consideration when selecting an in vitro system to evaluate various aspects of drug metabolism.     

In vitro and in vivo conjugation of dietary hydroxycinnamic acids by UDP-glucuronosyltransferases and sulfotransferases in humans

Hydroxycinnamic acids are a class of phenolic antioxidants found widely in dietary plants. Their biotransformation in the human organism primarily involves Phase II conjugation reactions. In this study, activities of UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) towards major dietary hydroxycinnamic acids (caffeic, dihydrocaffeic, dihydroferulic, ferulic and isoferulic acids) were investigated. Conjugate formation was evaluated using human liver and intestinal S9 homogenates, and in vitro characterization was carried out using recombinant human UGTs and SULTs. Analysis of the kinetics of hydroxycinnamic acid conjugation in human S9 homogenates revealed that intrinsic clearance (V_max/K_m) is much greater for sulfation than for glucuronidation. Assessment of activity using a panel of recombinant human SULTs showed that SULT1A1 is most active in the sulfation of caffeic, dihydrocaffeic and isoferulic acids, while SULT1E1 is most active in the sulfation of ferulic and dihydroferulic acids. Only isoferulic acid was significantly glucuronidated by human liver S9 homogenates, explained by the high activity of liver-specific UGT1A9. Studies on the kinetics of active SULTs and UGTs demonstrated a markedly lower K_m for SULTs. To further corroborate our findings, we carried out an intervention study in healthy humans to determine the hydroxycinnamic acid conjugates in urine after consumption of hydroxycinnamate-rich coffee (200 ml). Analysis showed that sulfates are the main conjugates in urine, with the exception of isoferulic acid, which is mainly glucuronidated. These data suggest that sulfates are the predominant hydroxycinnamic acid conjugates in humans, and that SULT mediated sulfation is a major factor determining the bioavailability of hydroxycinnamic acids in vivo.     

Kinetics of phosphate absorption by mycorrhizal and non-mycorrhizal Scots pine seedlings

Kinetics of net phosphate (P_i) uptake was measured on intact ectomycorrhizal and non‐mycorrhizal Pinus sylvestris seedlings using a semihydroponic cultivation method. The depletion of P_i in a nutrient solution was assessed over a 160–0.2 μM P_i gradient. Growth of the pine seedlings was P limited and measurements were performed 7 and 9 weeks after inoculation. Three ectomycorrhizal fungi were studied: Paxillus involutus, Suillus bovinus and Thelephoraterrestris. P_i uptake was extremely fast in plants colonised by P. involutus. The P_i concentration dropped below 0.2 μM within 4–5 h. In plants colonised with S. bovinus this occurred in 5–6 h and in plants associated with T. terrestris 8 h were needed to run through the whole concentration range. Non‐mycorrhizal plants of similar size and nutrient status decreased P_i to a concentration between 1 and 2 μM in 18 h. Data were curve fitted to a two‐phase Michaelis‐Menten equation. The apparent kinetic constants, K_m and V_max, for the high affinity P_i uptake system of the pine roots could be estimated accurately. V_max of this system was up to 7 times higher in pines associated with P. involutus than in non‐mycorrhizal seedlings. The intact extraradical mycelium greatly increased the absorption surface area of the roots (V_max). Non‐mycorrhizal plants had a K_m between 7.8 and 16.4 μM P_i. Plants mycorrhizal with P. involutus had K_m values between 2.4 and 7.2, plants colonised with S. bovinus had a K_m between 5.1 and 12.3, and seedlings associated with T. terrestris had a K_m from 4.6 to 10.1 μM P_i. All 3 ectomycorrhizal fungi had a strong impact on the P_i absorption capacity of the pine seedlings. The results also demonstrated that there is substantial heterogeneity in kinetic parameters among the different mycorrhizal root systems.     

Activation and inhibition of mitochondrial adenosine triphosphatase by various anions and other agents

The activating or inhibiting actions of a variety of anion species and of oligomycin, aurovertin and Dio-9 on the ATPase of a sonic particle preparation of rat liver mitochondria have been characterized by measurements of the relevantV _max,K _i andK _m values.The normalV _max was increased by a factor near 7 by the anions: dichromate, chromate, pyrophosphate, orthophosphate, orthoarsenate and sulphate. The fully activating concentration varied from about 2 mM for dichromate to 150 mM for sulphate. The increase inV _max was accompanied by a time-dependent decrease in (K _i)ADP, but there was no change in (K _m)ATP. The increase inV _max by the activating anions was abolished by aurovertin; but in presence of oligomycin, the lowV _max was increased by the activating anions by the same factor as theV _max in absence of oligomycin.Certain anions, notably azide, decreasedV _max, but did not affect (K _i)ADP or (K _m)ATP. The decrease inV _max by azide and oligomycin were approximately additive. Even at high concentration, Dio-9 was without detectable effect on the ATPase, but it had a gramicidinlike effect on the intact mitochondria.The specificity of the ATPase for ATP relative to GTP was found to be attributable to the high value of (V _max)ATP compared with (V _max)GTP. The values of (K _m)ATP and (K _m)GTP were virtually the same.Some rationalization of these and other supporting observations is attempted in terms of present knowledge of the constitution of the ATPase complex.     

Albumin Stimulates the Activity of the Human UDP-Glucuronosyltransferases 1A7, 1A8, 1A10, 2A1 and 2B15, but the Effects Are Enzyme and Substrate Dependent

Human UDP-glucuronosyltransferases (UGTs) are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA) significantly enhances in vitro activities of UGTs, a limiting factor in in vitro–in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2–4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction’s K _m, increasing its V _max, or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions’ K _m are concerned. In the cases of V _max values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V _max increases. Additionally, the BSA effects may be UGT subfamily dependent since K _m decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V _max increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs.     

In vitro fenoprofenyl–coenzyme a thioester formation: Interspecies variations

In vitro coenzyme A thioester formation from (?)-(R)-fenoprofen (FPF) and palmitic acid has been studied using liver microsomes from rat, guinea pig, sheep, and dog. In every species with both palmitic acid or (?)-(R)-fenoprofen, the Lineweaver–Burk plot was linear in the substrate concentration range used and as a consequence agrees with the involvement of only one isoenzyme (or different isoenzymes of similar K_m values). The V_max values for the thioesterification of (?)-(R)-fenoprofen present large species variations from 2.1 ± 1.0 with sheep liver microsomes to 60.6 ± 11 nmol/min/mg with dog liver microsomes. These values statistically significantly correlate (r = 0.94) to the V_max values observed when palmitic acid was used as a substrate. Furthermore palmitic acid inhibited (?)-(R)-fenoprofen–CoA formation in the same extent in all animal species. The stereoselectivity of the thioesterification was also species dependent. © 1995 Wiley-Liss, Inc.     

Purification and characterization of benzoyl-CoA ligase from a syntrophic,benzoate-degrading,anaerobic mixed culture

Summary The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optimum was 37–40° C, the optimum pH around 8.0 and optimal activity required 50–100 mM TRIS-HCI buffer, pH 8.0 and 3–7 mM MgCl₂; MgCl₂ in excess of 10 mM was inhibitory. The activation energy for benzoate was 11.3 kcal/mol. Although growth occured only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3- and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The K _m values for benzoate, 2-, 3- and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 mM, the V _max values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a K _m of 0.17 mM and a V _max of 1.05 U/mg and for ATP a K _m of 0.16 mM and a V _max of 1.08 U/mg was determined. Benzoate activation was inhibited by more than 6 mM ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (K _i) for pyrophosphate was 5.7 mM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found. Correspondence to: J. Winter     

Stereoselective sulfate conjugation of isoproterenol in humans: Comparison of hepatic,intestinal, and platelet activity

The stereochemistry of sulfate conjugation of isoproterenol (ISO) was examined with human liver, intestine, and platelets as the phenolsulfotransferase (PST) enzyme source and PAP³⁵S as the cosubstrate. With the hepatic cytosol, two distinct sulfation reactions were identified, a high affinity reaction (K_m 5 to 50 μM) and a low affinity reaction (K_m 360 to 2,900 μM). The efficiency of sulfation (V_max/K_m) for both reactions was 5-fold higher for (+)- than for (?)-ISO. When the hepatic PSTs were resolved by ionexchange chromatography, it could be shown that the high affinity reaction was catalyzed by the monoamine (M) form and the low affinity reaction by the phenol (P) form of PST. Only the high affinity (M form) sulfation was detected in the jejunal cytosol with a V_max/K_m value 6.1-fold higher for (+)- than for (?)-ISO. Finally the platelet, as a potentially useful model tissue, also demonstrated only the high affinity M form reaction with a V_max/K_m value 5.7-fold higher for (+)- than for (?)-ISO. In summary, this study has shown that sulfation of ISO by PSTs in various human tissues is stereoselective and favors the inactive (+)-enantiomer over the active (?)-enantiomer by about 5-fold, a finding which should be considered in the therapeutic use of chiral drugs cleared by sulfate conjugation. © 1993 Wiley-Liss, Inc.     

Kinetics of nitrate uptake by different species from nutrient-rich and nutrient-poor habitats as affected by the nutrient supply

The species Urtica dioica L., Plantago major ssp. major L., Plantago lanceolata L., Hypochaeris radicata L. ssp. radicata and Hypochaeris radicata ssp. ericetorum Van Soest were grown under high and low nutrient conditions (1/4 Hoagland and 2% of 1/4 Hoagland further called the 100% and 2% treatment, containing 3.75 mM NO^-₃ and 0.075 mM NO^-₃, respectively). After a certain period half of the plants were transferred from low to high or high to low nutrients, yielding the 100%/2% and the 2%/100% treatments. The kinetics of nitrate uptake in the range of system I of the five species grown under the different nutrient conditions were measured during a three week experimental period. The nitrate uptake of all the species showed the characteristic features of Michaelis-Menten kinetics. Under low nutrient conditions the apparent V_max of U. dioica expressed per g dry root was lower than under high nutrient conditions. For H. radicata ssp. radicata and for H. radicata ssp. ericetorum the reverse was found. The V_max values of P. major ssp. major were almost the same for the two treatments. The apparent V_max in young plants of P. lanceolata was higher in the 100% treatment than in 2%; whereas the reverse was found in mature plants. The results are explained in relation to the relative growth rate, the shoot to root ratio and the natural environment of the species. The apparent K_m values were not influenced by the different treatments. Differences in K_m between the species, if any, were very small. It is suggested that the V_max is a more important parameter for the distribution of plant species in the field than the K_m. The rate of nitrogen accumulation was calculated from growth data and the contents of nitrate and reduced nitrogen. It is concluded that the V_max of system I for nitrate uptake in most cases was sufficient to explain the observed growth rates.     

Trypanosoma lewisi: alterations in membrane function in the rat.

DEAE-cellulose-purified Trypanosoma lewisi from 4-day (dividing trypanosomes) and 7-day (non-dividing trypanosomes) infections in rats were compared for initial uptake of glucose, leucine, and potassium. Glucose entered the parasitic cells by mediated (saturable) processes, whereas leucine and K⁺ entered by mediated processes and diffusion. Glucose entry was significantly elevated in 4-day cells (V_max 4.00 ± 1.02 nmoles/ 1 × 10⁸ cells/min) with respect to 7-day cells (V_max 1.83 ± 0.62 nmoles 1 × 10⁸ cells/min). Likewise, the affinity of the glucose carrier was significantly greater in 4-day cells (K_m = 0.30 ± 0.02 mM) than in 7-day cells (K_m = 0.59 ± 0.11 mM). When leucine and K⁺ transport were compared in 4- and 7-day populations, significant elevations in the rate of entry (V_max) of both substrates were observed for 4-day cells; K_m values for leucine and K⁺ were not altered by the stage of infection. For leucine, the V_max and K_m for 4-day cells were 2.40 ± 0.50 nmoles/1 × 10⁸ cells/30 sec and 78 ± 7 μM, respectively; corresponding values in 7-day cells were 1.06 ± 0.02 nmoles/1 × 10⁸ cells/30 sec and 66 ± 11 μM. For K⁺, the V_max and K_m for 4-day cells were 15.97 ± 0.38 nmoles/1 × 10⁸ cells/min and 1.2 mM, respectively; corresponding values in 7-day cells were 4.76 ± 1.82 nmoles/1 × 10⁸ cells/min and 1.05 mM. The observed increase in the rate of K⁺ entry into 4-day cells was attributable to enhanced influx; no significant difference in the rate of K⁺ efflux was noted when 4- and 7-day cells were compared ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of K⁺ leak for 4- and 7-day cells were 68.1 ± 9.3 and 67.9 ± 15.2 min, respectively). Potassium influx was ouabain insensitive. Membrane function in 7-day cells was not uniformly inhibited. No significant difference in the activity of the membrane-bound enzyme, 5′-nucleotidase, was observed when 4- and 7-day cells were compared.     

Temperature sensitivity of soil enzyme kinetics under N‐fertilization in two temperate forests

Soil microbes produce extracellular enzymes that degrade carbon (C)‐containing polymers in soil organic matter. Because extracellular enzyme activities may be sensitive to both increased nitrogen (N) and temperature change, we measured the effect of long‐term N addition and short‐term temperature variation on enzyme kinetics in soils from hardwood forests at Bear Brook, Maine, and Fernow Forest, West Virginia. We determined the V_max and K_m parameters for five hydrolytic enzymes: α‐glucosidase, β‐glucosidase, β‐xylosidase, cellobiohydrolase, and N‐acetyl‐glucosaminidase. Temperature sensitivities of V_max and K_m were assessed within soil samples subjected to a range of temperatures. We hypothesized that (1) N additions would cause microbial C limitation, leading to higher enzyme V_max values and lower K_m values; and (2) both V_max and K_m would increase at higher temperatures. Finally, we tested whether or not temperature sensitivity of enzyme kinetics is mediated by N addition. Nitrogen addition significantly or marginally significantly increased V_max values for all enzymes, particularly at Fernow. Nitrogen fertilization led to significantly lower K_m values for all enzymes at Bear Brook, but variable K_m responses at Fernow Forest. Both V_max and K_m were temperature sensitive, with Q₁₀ values ranging from 1.64–2.27 for enzyme V_max and 1.04–1.93 for enzyme K_m. No enzyme showed a significant interaction between N and temperature sensitivity for V_max, and only β‐xylosidase showed a significant interaction between N and temperature sensitivity for K_m. Our study is the first to experimentally demonstrate a positive relationship between K_m and temperature for soil enzymes. Higher temperature sensitivities for V_max relative to K_m imply that substrate degradation will increase with temperature. In addition, the V_max and K_m responses to N indicate greater substrate degradation under N addition. Our results suggest that increasing temperatures and N availability in forests of the northeastern US will lead to increased hydrolytic enzyme activity, despite the positive temperature sensitivity of K_m.     

Temperature sensitivities of extracellular enzyme Vmax and Km across thermal environments

The magnitude and direction of carbon cycle feedbacks under climate warming remain uncertain due to insufficient knowledge about the temperature sensitivities of soil microbial processes. Enzymatic rates could increase at higher temperatures, but this response could change over time if soil microbes adapt to warming. We used the Arrhenius relationship, biochemical transition state theory, and thermal physiology theory to predict the responses of extracellular enzyme V_max and K_m to temperature. Based on these concepts, we hypothesized that V_max and K_m would correlate positively with each other and show positive temperature sensitivities. For enzymes from warmer environments, we expected to find lower V_max, K_m, and K_m temperature sensitivity but higher V_max temperature sensitivity. We tested these hypotheses with isolates of the filamentous fungus Neurospora discreta collected from around the globe and with decomposing leaf litter from a warming experiment in Alaskan boreal forest. For Neurospora extracellular enzymes, V_max Q₁₀ ranged from 1.48 to 2.25, and K_m Q₁₀ ranged from 0.71 to 2.80. In agreement with theory, V_max and K_m were positively correlated for some enzymes, and V_max declined under experimental warming in Alaskan litter. However, the temperature sensitivities of V_max and K_m did not vary as expected with warming. We also found no relationship between temperature sensitivity of V_max or K_m and mean annual temperature of the isolation site for Neurospora strains. Declining V_max in the Alaskan warming treatment implies a short‐term negative feedback to climate change, but the Neurospora results suggest that climate‐driven changes in plant inputs and soil properties are important controls on enzyme kinetics in the long term. Our empirical data on enzyme V_max, K_m, and temperature sensitivities should be useful for parameterizing existing biogeochemical models, but they reveal a need to develop new theory on thermal adaptation mechanisms.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Circadian Variation in the Uptake of Tryptophan by Cortical Synaptosomes of the Rat Brain

The kinetics of the high affinity uptake system for L-tryptophan (L-Try)have been measured over 24 hr in cortical synaptosome preparations of rat brain. Both the K_m and V_max, of the uptake process showed a statistically significant 24 hr variation. The highest K_m value, 6.71 ± 10^-5 M, was measured at the beginning of the light phase and the lowest value, 4.23 ± 10^-5 M, 6 hr into the dark phase. V_max was highest at the end of the dark phase (10.43 nmol/mg/5 min) and lowest (4.80 nmol/mg/5 min) 3 hr into the dark phase. In contrast, there was no variation over 24 hr in the V_max/K_m ratio. These results suggest that the high affinity uptake process serves to ensure a constant rate of L-tryptophan entry into the neuron in the face of circadian or ultradian variations in extracellular concentration of tryptophan.     

Kinetics of Transport and Phosphorylation of 2-Fluoro-2-Deoxy-d-Glucose in Rat Brain

Abstract: The kinetics of transport across the blood-brain barrier and metabolism in brain (hemisphere) of [¹⁴C]2-fluoro-2-deoxy-d -glucose (FDG) were compared to that of [³H]2-deoxy-d -glucose (DG) and d -glucose in the pentobarbital-anesthetized adult rat. Saturation kinetics of transport were measured with the brain uptake index (BUI) method. The BUI for FDG was 54.3 ± 5.6. Nonlinear regression analysis gave a K_m of 6.9 ± 1.1 mM and a V_max of 1.70 ± 0.32 μmol/min/g. The K₁ for glucose inhibition of FDG transport was 10.7 ± 4.4 mM. The kinetic constants of influx (k₁) and efflux (K₂) for FDG were calculated from the K_m, V_max, and glucose concentrations of the hemisphere and plasma (2.3 ± 0.2 μmol/g and 9.9 ± 0.4 mM, respectively). The transport coefficient (k_{1 FDG}/k₁glucose) was 1.67 ± 0.07 and the phosphorylation constant was 0.55 ± 0.16. The predicted lumped constant for FDG was 0.89, whereas the measured hexose utilization index for FDG was 0.85 ± 0.16. Conclusion: The value for the lumped constant can be predicted on the basis of the known kinetic constants of FDG and glucose transport and metabolism, as well as brain and plasma glucose levels. Knowledge of the lumped constant is crucial in interpreting data obtained from ¹⁸FDG analysis of regional glucose utilization in human brain in pathological states. We propose that the lumped constant will rise to a maximum equal to the transport coefficient for FDG under conditions of transport limitation (hypoglycemia) or elevated glycolysis (ischemia, seizures), and will fall to a minimum equal to the phosphorylation coefficient during phosphorylation limitation (extreme hyperglycemia).     

Glucose utilization by 6pgd genotypes in Lolium perenne

To gain a deeper understanding of the mechanisms underlying associations between allozyme genotypes and rates of respiration in Lolium perenne, V_max K_m and rates of glucose flux through glycolysis and the pentose phosphate pathway were estimated for the three genotypes of the 6pgd locus. K_m V_max and V_max/K_m differed significantly among genotypes. Values of K_m for the 11, 12, and 22 genotypes were 0.29, 0.25, 0.13, while the values of V_max/K_m for die 11, 12, and 22 genotypes were 3.79, 3.85, 6.70. Flux through the pentose shunt did not differ among genotypes at 20 °C, but at 35 °C the rates of flux for the 11, 12, and 22 genotypes were 0.15, 0.25 and 0.42, respectively. Thus, the 6PGD allozyme genotypes differ markedly in both enzyme kinetic characteristics and in flux through a metabolic pathway. These associations reveal potentially causal relationships between allozyme genotypes and rates of respiration.     

Hydroxycinnamic Acid Transferases in the Biosynthesis of Acylated Betacyanins: Purification and Characterization from Cell Cultures of Chenopodium rubrum and Occurrence in Some Other Members of the Caryophyllales*

An acyltransferase from cell cultures of Chenopodium rubrum was purified 515-fold with a 2.5 % recovery. This enzyme catalyzes the transfer of hydroxycinnamic acids from 1–0-hydroxycinnamoyl-/β-glucose to the C-2 hydroxy group of glucuronic acid of amaranthin (betanidin 5-O-glucuronosylglucose). The invivo products formed are celosianin I (4-coumaroylama-ranthin) and celosianin II (feruloylamaranthin). The enzyme can be classified as l-0-hydroxycinnamoyl-β-glucose: amaranthin O-hydroxycinnamoyl-transferase (EC 2.3.1.-). Its molecular weight was determined by gel filtration column chromatography to be ca. 69.5 kDa. Maximal rate of product formation was found to be at pH 5.6. The isoelectric point of the enzyme was at pH 4.7. The reaction temperature maximum was at 37 °C and the apparent energy of activation was calculated to be 44.5kJ mor^?1. The enzyme showed a V_max of 910pkat (mg protein)^?1 with amaranthin as acceptor and feruloylglucose as acyl donor. The ratios of V_max/K_m values for sinapoyl-, feruloyl, caffeoyl- and 4-coumaroylglucoses were found to be 100:56:56:40. Donor competition experiments support the conclusion that one single enzyme is responsible for the ester formation with the different hydroxycinnamic acids. From the possible acceptors tested, only amaranthin (15S configuration) and isoamaranthin (15R) were esterified with K_m values of 280 and 800 μM, respectively. Catalytic effectivity (V_mix/K_m) was found at a relative ratio 15S:15R of 100:42. Betanin (betanidin 5-O-glucoside) and gomphrenin I (betanidin 6-O-glucoside) were not accepted. Some other acylated betacyanin-containing members of four families of the Caryophyllales were investigated and showed the same type of hydroxycinnamoyltransferase activity with 1-O-hydroxycinnamoylglucose as acyl donor, but with different acceptor molecule specificities.     

Cocaine and butyrylcholinesterase (BChE): Determination of enzymatic parameters

In humans, the plasma enzyme, butyrylcholinesterase (E.C. 3.1.1.8), metabolizes cocaine to the water-soluble, pharmacologically inactive compounds, ecgonine methylester and benzoic acid. Homogenous enzyme was purified from human plasma and used to determine the enzyme kinetic parameters of K_m and V_max with cocaine as the substrate. The K_M (11.9 μM) indicates that cocaine is tightly bound to the four active sites of the native tetramer. The V_max (1.17 μM/min) is 50-fold greater than cocaine catalytic antibodies. Administration of purified human butyrylcholmesterase to a cocaine-intoxicated patient would be expected to shift the metabolism to the inactive metabolites and reduce the toxicity.     

The Effect of Photosynthetic Enhancement on Photorespiration in Sinapis alba

High external concentrations of potassium were found to promote light-induced growth of cucumber cotyledons similarly to the effects previously observed for the growth induced by cytokinins. At 40 mM KCl, the response to white light was 3.6 times greater than in the absence of KCl. The promotive effect of calcium on the growth induced by cytokinin was not observed for light. In 40 mM KCl and 10 mM CaCl₂, the responses to light and cytokinin were similar and additive. Both near-red and far-red light induced growth at low intensities. The response to white light at low intensities was sharply increased with higher intensity up to 24 μE m^-2 s^-1 and only slightly increased above that level. Abscisic acid was found to inhibit strongly the responses to light and cytokinin. The inhibition was greater in the presence of KCl than in its absence and thus abscisic acid appeared to inhibit primarily by its interference with potassium uptake. Kinetic analysis found the light response promoted by potassium to be inhibited competitively by abscisic acid, with the light response having a K_m of 34 mM KCl and a V_max of 115 mg/cotyledon × 4 days. The inhibition of the cytokinin response by abscisic acid was noncompetitive in relation to potassium, having a K_m of 1 mM KCl and a V_max of 50 mg/cotyledon × 4 days. It is suggested that cytokinin, light and abscisic acid have primary properties affecting membrane permeability and that their interaction with potassium is an explanation of many similarities between light and cytokinin responses and their inhibition by abscisic acid.