Crown ether activates cholesterol oxidase in low water media

Kinetic studies of cholesterol oxidase-catalysed oxidation of cholesterol in water/2-propanol mixtures showed a decrease of V _max/K _m values on the increase of concentration of the organic co-solvent. Addition of 18-crown-6 to the reaction medium results in an increase of V _max up to 16 times, and V _max/K _m up to 8.4 times, enhancing the activity of cholesterol oxidase in 2-propanol/water (88:12 v/v) to 3.5 times compared to the level observed in 46% 2-propanol.     

Comparisons Between the Kinetic Behaviour of the Muscle Carnitine Palmitoyltransferase I of a Higher and Lower Vertebrate

The V_max of rat muscle mitochondrial CPT I toward the coenzyme A derivatives of 16:0, 16:1n-7, 18:1n-9, and 22:6n-3 were far lower than those recorded previously for this enzyme in rat liver at the same temperature (37°C). However, the V_max of 7.0 nmol · min⁻¹ · mg mitochondrial protein⁻¹ for linoleoyl CoA (18:2n-6), which was the greatest recorded for the five acyl CoAs examined in muscle, was similar to that in liver. These comparisons presumably reflect a difference in the essential fatty acid requirements of these two rat tissues. Although the V_max values for CPT I in the musculature of a lower vertebrate (larval lamprey) at 20°C were similar to those exhibited toward the coenzyme A derivatives of 16:0, 16:1n-7, 18:1n-9, and 22:6n-3 by the CPT I of rat musculature at 37°C, the corresponding V_max toward 18:2n-6 (3.2 nmol · min⁻¹ · mg mitochondrial protein⁻¹) was lower. The latter relatively low activity may spare from oxidation this essential fatty acid, which is in low abundance in the diet of larval lampreys. Although the V_max values toward the four nonessential fatty acids in larval lamprey muscle were similar to those in rat muscle, the corresponding K_0.5 values were lower, thus indicating that the musculature of larval lampreys has a high capacity for energy generation through β-oxidation.     

Stabilities and rates in the laccase/TEMPO-catalyzed oxidation of alcohols

The influence of alcohol, 4-acetylamino,2,2,6,6′-tetramethylpiperidinyloxy (4-acetylamino-TEMPO) and laccase (from Trametes versicolor, TvL) concentration in the aerobic oxidation of furfuryl alcohol was investigated. Studies show that the K_m for 4-acetylamino-TEMPO is around 6.3 mM (V_max=0.18 mM min^?1) using 6.6 U mL^?1 of laccase and a furfuryl alcohol concentration of 140 mM. Under these optimized conditions, the reaction rate is still dependent on the concentration of enzyme in solution. Laccase can be reused, with a residual activity of around 25%. An important conclusion is that laccase is not stable in the presence of oxoammonium salts, presumably due to degradation via oxidation of essential amino acid residues or the glycosyl moieties on the periphery of the enzyme.     

Esterification of 2-methylalkanoic acids Catalysed by Lipase from Candida rugosa: Enantioselectivity as a Function of water Activity and Alcohol Chain Length

Esterifications catalysed by immobilised lipase from Candida rugosa (CRL) in cyclohexane at constant water activity (a_w = 0.76) were studied using 2-methyl substituted octa-, nona- or decanoic acids and n-alcohols of varying chain length as substrates. The importance of controlling the water activity and choosing the right alcohol for obtaining maximum enantioselectivity is demonstrated. The immobilised lipase was easily recovered without loss of activity and enantioselectivity.     

The uptake of zinc by erythrocytes under near-physiological conditions

The in vitro uptake of zinc by erythrocytes was measured under near-physiological conditions, using⁶⁵Zn as a radioactive tracer. Because of the presence of serum albumin—a strong zinc ligand—a low concentration of medium free zinc was maintained. Under these conditions a high-affinity carrier for zinc transport over the cell membrane was identified. With human erythrocytes, a Michaelis constant (K _m ) of 0.2 nM with respect to free medium zinc was measured and aV _max of 4.5 nmoles Zn transported per h/g dry wt. TheK _m for medium Zn increases when the size of the internal erythrocytic Zn pool is augmented, whereasV _max remains virtually unchanged. A model to explain this phenomenon is proposed. It is suggested that this phenomenon could underlie observations, confirmed here, that the in vitro uptake of Zn by animal erythrocytes depends on the Zn status of the animal.     

Evidence for the Sequential Formation of Two Complexes Between an Uptake Inhibitor, GBR 12783 [1-[2-(Diphenylmethoxy)ethyl]-4-(3-Phenyl-2-Propenyl)piperazine], and the Neuronal Transporter of Dopamine

Abstract : Incubation of a crude synaptosomal fraction from rat striatum with GBR 12783 at 37°C produced an inhibition of the specific uptake of [³H]dopamine that increased with time. The inhibition increased when GBR 12783 was present during preincubation and incubation (IC₅₀ = 1.85 ± 0.1 nM) instead of incubation alone (IC₅₀ = 25 ± 3.5 nM). Time-course studies of uptake inhibition demonstrated that a first collision transporter-inhibitor complex (TI) was formed immediately after addition of GBR 12783 so that the initial uptake velocity (V_o) decreased for increasing concentrations of inhibitor (K_i≥ 20 nM). TI slowly isomerized to a more stable complex TI^* (K^*_i≤ 5 nM) with a value of t_1/2 = 20-270 s. Fits of data to model 2 in which the steady-state uptake (V_S) is set to zero were generally preferred, suggesting that formation of TI^* could tend to irreversibility, as a consequence of a very low reverse isomerization. As expected, k, V_o, and V_S tended to steady-state values in an asymptotic manner for high concentrations of GBR 12783. GBR 12783 at 2.5 nM produced a mixed inhibition of the uptake, with an increase in K_M and a decrease in V_max ; these effects were improved for 10 nM GBR 12783 and at 20°C. These results are discussed in relation to previous data concerning [³H]GBR 12783 binding. The present work gives the first experimental demonstration that dopamine uptake blockers can act according to a two-step mechanism of inhibition ; this is of great interest, because these inhibitors can oppose the effects of cocaine or amphetamine on the transporter according to a reaction that is partly nondependent on the concentration of the abused agent.     

Δ53β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate-binding co-operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Δ⁵3β hydroxysteroid dehydrogenase activity transforms biologically inactive Δ⁵3β hydroxy steroids into the active Δ⁴3-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for Δ⁵3β HSD we have analysed the initial velocity rates (V_o) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20α hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 μm) of the dioestrous and proestrous rat ovary. The results are mean ± S.E.M. The relationship between DHEA concentration (0 to 50 μM) and Δ⁵3β HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/V_o versus 1/S plot, the x intercept being positive. Using a 1/V_o versus 1/S² plot the V_max was determined to be 1·0 ± 0·08 μmol min^?1 mg^?1 CL (n = 6). The Hill constant was 2·7 ± 0·02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 ± 1 μmoles (n = 6). In the corpora lutea cells of the proestrous ovary, the V_max for DHEA transformation was unchanged (0·95 ± 0·04 μmol min^?1 mg^?1, n = 3) whilst the S_0·5 was significantly increased to 27 ± 0·1 (p < 0·01, n = 3). The Hill constant remained positive being 2·9 ± 0·2 (n = 3). NAD⁺ binding to 3β HSD in regressing corpora lutea of the proestrous ovary has been demonstrated previously to be hyperbolic and fit the classical Michaelis-Menten model.¹ Extending the analysis of NAD⁺ binding to the regressing corpus luteum of the dioestrous rat ovary revealed similar kinetic characteristics to that seen with the proestrous enzyme, the apparent V_max and K_m being 0·84 ± 0·04 μmol min^?1 mg^?1 CL (n = 3) and 27 ± 7 μmol 1^?1 (n = 3) respectively. The Hill constant was 1·1 ± 0·03 (n = 3), indicating no co-operativity of co-factor binding.     

Production and initial characterisation of the xylan-degrading system of Phanerochaete chrysosporium

We report the optimum conditions for the degradation of oat spelt arabinoxylan and a preliminary characterisation of the inducible xylan-degrading system of the lignin-degrading white-rot fungus Phanerochaete chrysosporium. Xylanase activity was optimal at pH 5.0 and 50°C; see attached sheet the maximum reaction velocity (V_max) of the system was 3.86 units (U) mg^–1 protein with arabinoxylan as substrate and the substrate concentration giving half V_max (S_0.5) was 0.52 mg ml^–1. At concentrations of arabinoxylan greater than 15 mg ml^–1 excess substrate inhibition was observed. Xylose at 0.9 mm inhibited activity to the extent of 50%. Xylanase activity increased as a function of the dilution of the enzyme preparation prior to assay. It was resolved into four peaks by using a DEAE-Biogel column; the material in these peaks differed with respect to xylan solubilisation and the formation of reducing sugars. Electrofocusing gels allowed visualisation of several bands of activity corresponding to each peak. The arabinoxylan degradation system of P. chrysosporium is therefore composed of multiple components. Correspondence to: P. Broda     

Improved production of ethyl-(R)-2-hydroxy-4-phenylbutyrate with pretreated Saccharomyces cerevisiae in water/organic solvent two-liquid phase systems

Baker's yeast pretreated with α-phenacyl chloride was employed to improve the enantioselectivity of the asymmetric reduction of ethyl-2-oxo-4-phenylbutyrate (EOPB) to ethyl-(R)-2-hydroxy-4-phenylbutyrate ((R)-EHPB) and maintain a high activity of the yeast. A water/organic solvent two-liquid phase system was also introduced to overcome the strong substrate and product inhibition of the enzyme; the highest catalytic activity and enantioselectivity were obtained in a water/benzene two-liquid phase system. When the reduction was catalyzed with pretreated yeast (300 mg mL^?1 buffer) in the water/benzene two-liquid phase system (V_aq/V_ben=20:40), 41.9% molar conversion of EOPB and 87.5% e.e. of (R)-EHPB were obtained in 48 h, using pH 8.0 phosphate buffer with 1.5% (v/v) of ethanol added as a co-substrate at 30°C, even with an initial EOPB concentration of 400 mM and a final EHPB concentration as high as 167.7 mM.     

The response of photosynthetic model parameters to temperature and nitrogen concentration in Pinus radiata D. Don

Responses of photosynthesis (A) to intercellular CO₂ concentration (c_i) in 2-year-old Pinus radiata D. Don seedlings were measured at a range of temperatures in order to parametrize a biophysical model of leaf photosynthesis. Increasing leaf temperature from 8 to 30°C caused a 4-fold increase in V_cmax, the maximum rate of carboxylation (10.7–43.3 μol m^?2 s^?1 and a 3-fold increase in J_max, the maximum electron transport rate (20.5–60.2 μmol m ^?2 s^?1). The temperature optimum for J_max was lower than that for V_cmax, causing a decline in the ratio J_max:V_cmax from 2.0 to 1.4 as leaf temperature increased from 8 to 30°C. To determine the response of photosynthesis to leaf nitrogen concentration, additional measurements were made on seedlings grown under four nitrogen treatments. Foliar N concentrations varied between 0.36 and 1.27 mol kg^?1, and there were linear relationships between N concentration and both V_cmax and J_max. Measurements made throughout the crown of a plantation forest tree, where foliar N concentrations varied from 0.83 mol kg^?1 near the base to 1.54 mol kg^?1 near the leader, yielded similar relationships. These results will be useful in scaling carbon assimilation models from leaves to canopies.     

The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3 beta-hydroxy-delta 5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary

Summary A technique which allows for the continuous microdensitometric monitoring (at 3.3 s intervals) of an enzyme reaction has been applied to a study of 3-hydroxy-⁵-steroid dehydrogenase activity in regressing corpora lutea cells of unfixed tissue sections (5µm thick) of the pro-oestrous rat ovary. Initial reaction rates for NAD⁺ reduction by the activity of the enzyme were maintained for less than 3 min at all the concentrations (0-500µmol/l) of co-factor employed. The relationship between NAD⁺ concentration and enzyme activity under initial velocity rate conditions was hyperbolic and maximum enzyme activity was seen when the NAD⁺ concentration was greater than 250µmol/l. The apparent Michaelis—Menten constant (K _m) was 29µmol/l and V_max 0.63µmol NAD⁺ reduced/min/cm³ corpora lutea.     

Potassium transport in Chlamydomonas reinhardtii: isolation and characterization of transport-deficient mutant strains

Putative potassium-transport-deficient mutant strains of Chlamydomonas reinhardtii Dang. were induced by ultra-violet mutagenesis and were identified by their dependence on abnormally high concentrations of potassium for growth. Potassium transport studies employing ⁸⁶Rb as a tracer were carried out with wild-type cells and with three independently isolated KDP (potassium-dependent phenotype) clones. Wildtype cells exhibit two transport activities. Transport activity A was expressed when cells were grown in medium supplemented with 10 mM KCl. The transporter with type-A activity does not discriminate between either Rb⁺ or K⁺ as a substrate and has a K_m for Rb⁺ equal to 1 mM and a V_max equal to 31 nmol Rb⁺ h^-1 10^-6 cells. Transport activity B was expressed when cells were starved of potassium for 24 h. The transporter with type-B activity prefers K⁺ to Rb⁺ as a substrate; it has a K_m for Rb⁺ equal to 2.5 mM and a V_max equal to 210 nmol Rb⁺ h^-1 10^-6 cells. All three mutant clones exhibit transport activity comparable to type-A when grown in 10 mM KCl. When starved of potassium for 24 h, two KDP clones demonstrate no transport activity and the third clone continues to exhibit only type-A activity.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DES diethylstilbesterol - KDP potassium-dependent phenotype     

Regulation of the structure and catalytic properties of plasma membrane H<Superscript>+</Superscript>-ATPase involved in adaptation of two reed ecotypes to their different habitats

The properties and kinetics of ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activities of plasma membrane H⁺-ATPase from the two reed ecot ypes, swamp reed (SR) and dune reed (DR), were investigated. The pH optimum of the plasma membrane H⁺-ATPase in both reed ecotypes was similar but the sensitivity of the enzyme to the reaction medium pH seemed to be higher in DR than that in SR. Compared to SR, the DR exhibited a higher V_max value for ATP hydrolysis whereas the K_m value was almost similar in both reed ecotypes. The PNPP hydrolysis of the plasma membrane H⁺-ATPase was also studied in both reed ecotypes at increasing PNPP concentrations. K_m and V_max for PNPP hydrolysis showed great differences in the two reed ecotypes and in DR the K_m and V_max values were 2- and 10-fold, respectively, higher than those in SR. The ATP hydrolysis activity of the plasma membrane was markedly inhibited by hydroxylamine in both reed ecotypes, and the percentage inhibition of ATP hydrolysis rate seemed higher in DR than that in SR. In addition, the structure or property of the C-terminal end of the plasma membrane H⁺-ATPase were also different in the two reed ecotypes. These data suggest that different isoforms of the plasma membrane H⁺-ATPase might be developed and involved in the adaptation of the plant to the long-term drought-prone habitat.This research was supported by Natural Science Foundation of China (No. 30270238 & No. 30470274) and the National Key Basic Research Special Funds of China (G1999011705).     

Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii

The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg⁻¹. The enzyme required Mg²⁺ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH optimum of 6.4), and the apparent K _m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V _max of 243 U mg⁻¹) and 1.47 mM (apparent V _max of 197 U mg⁻¹), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent cations including Na⁺ and K⁺. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K _m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V _max of 2.9 U mg⁻¹) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared to ATP and pyrophosphate-dependent phosphofructokinases. Received: August 14, 1998 / Accepted: December 2, 1998     

A Facile Method for Preparation of Polymerizable, Optically Active Ketoprofen Prodrug by Irreversible Lipase-catalysed Resolution

Summary An irreversible resolution of ketoprofen prodrug was developed by lipase-catalysed hydrolysis using corresponding vinyl ester as activated substrate in organic medium. The product obtained, (S)-ketoprofen vinyl ester would be used as a potential prodrug and a significant monomer for polymeric drug. Lipozyme^? immobilized from Mucor miehei showed the highest selectivity and activity after enzyme screening. The effect of solvent, water amount in the reaction medium and reaction temperature on the activity and enantioselectivity of Lipozyme^? was studied. Polymerizable, optically active ketoprofen prodrug could be obtained with excellent enantioselectivity (ee >99%, E ~ 400) in a mixture of dioxane/water (97.5/2.5, v/v) at 25 °C.     

Extracellular phosphatase activity in sediments of the Breitenbach,a Central European mountain stream

Activity of extracellular phosphatases (phosphomonoesterases) was measured in sandy streambed sediments of the Breitenbach, a small unpolluted upland stream in Central Germany. Fluorigenic 4-methylumbelliferyl phosphate served as a model substrate. Experiments were conducted using sediment cores in a laboratory simulation of diffuse groundwater discharge through the stream bed, a natural process occurring in the Breitenbach as well as many other streams.Streambed sediments contained high levels of particulate phosphorus, but concentrations of dissolved phosphorus in the interstitial water were 3 to 4 orders of magnitude lower. These interstitial concentrations were similar to those in the stream and groundwater. Extracellular phosphatase activity was high in the streambed sediments. These enzymes probably contribute significantly to the flux of phosphorus in sediment by hydrolyzing phosphomonoesters, making free phosphate available to the sediment microorganisms.Factors influencing the kinetic parameters V _max (maximum activity) and apparent K _m (enzyme affinity) of phosphatase were discharge rates of water through the sediment, water quality (ground- or stream water), and substrate (phosphomonoesters) as well as dissolved ortho-phosphate concentrations. Enzymes are supposed to be effective at limiting substrate concentrations, where, in this study, changes in discharge rates had little influence on rates of hydrolysis. Higher V _max and lower K _m values were found during percolation of groundwater through the sediment cores, compared with stream water. This indicates that rates of hydrolysis were higher with groundwater, both at substrate limitation and at substrate saturation. This was probably a consequence of the lower levels of dissolved ortho-phosphate in the groundwater.     

Properties of the phosphate and phosphite transport systems of Phytophthora palmivora

Germlings of Phytophthora palmivora possess at least two systems for the uptake of inorganic phosphate (P_i). The first is synthesized on germination in medium containing 50 M P_i and has a K_m of approx. 30 M (V_max=7–9 nmol P_i/h·10⁶ cells). The second is synthesized under conditions of P_i-deprivation and has a higher affinity for P_i (K_m=1–2 M), but a lower V_max (0.5–2 nmol P_i/h·10⁶ cells). The fungicide phosphite likewise enters the germlings via two different transport systems, the synthesis of which also depends on the concentration of P_i in the medium. The K_m of the lower affinity system is 3 mM (V_max=20 nmol phosphite/h·10⁶ cells) and that of the higher affinity system is 0.6 mM (V_max=12 nmol/h·10⁶ cells). P_i and phosphite are competitive inhibitors for each other's transport in both systems. However, whereas mM concentrations of phosphite are necessary to inhibit P_i transport, only M concentrations of P_i are required to inhibit phosphite transport. A third system of uptake for P_i also exists, since when phosphate-deprived cells are presented with mM concentrations of P_i, they transport the anion at a very high rate (around 100 nmol/h·10⁶ cells). High rates of transport of phosphite are also observed when these cells are presented with mM concentrations of this anion.     

Characterization of the oxaloacetate decarboxylase and pyruvate kinase-like activities of Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinases

Two members of the ATP-dependent class of phosphoenolpyruvate carboxykinases (PEPCKs) (Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens) have been comparatively studied with regard to their oxaloacetate (OAA) decarboxylase and pyruvate kinase-like activities. The pyruvate kinase-like activities were dependent on the presence of Mn²⁺; at the same concentrations Mg²⁺ was not effective. These activities were synergistically activated by a combination of both metal ions. V _max for these activities in A. succiniciproducens and S. cerevisiae PEPCKs was 0.13% and 1.2% that of the principal reaction, respectively. The OAA decarboxylase activity was nucleotide independent and, with decreasing order of effectiveness, these activities were supported by Mn²⁺ and Mg²⁺. AMP is an activator of these reactions. V _max for the OAA decarboxylase activities in A. succiniciproducens and S. cerevisiae PEPCKs was 4% and 0.2% that of the PEP-forming reaction, respectively.     

Steady-state kinetics of the photosystem I reaction in chloroplasts of Dunaliella which contain variable concentrations of plastocyanin

The endogenous plastocyanin (PC) concentrations of Dunaliella cultures were varied from 0.3 to 3.1 molecules per pigment 700 (P700) by decreasing the Cu⁺ supply of the nutrient. With these cultures the amount of PC which is sufficient for maximum photosynthesis in intact cells was determined to be about 1 to 1.5 PC/P700. Chloroplasts were also prepared from these cells and were employed in enzyme kinetic measurements of the PSI reaction from ascorbate reduced diaminodurene (DAD) to methylviologen/O₂. The k _m value for DAD in this reaction was 106 M. A decrease of the endogenous PC concentration caused no change of the k _m value but affected the V _max in the DAD-dependent reaction. A similar interference of the PC concentration on the maximum reaction rate could also be observed when the light intensity was varied.     

EFFECT OF THE SINKING RATE OF TWO DIATOMS (THALASSIOSIRA SPP.) ON UPTAKE FROM LOW CONCENTRATIONS OF PHOSPHATE1

The effect of the sinking rate, or rate of medium flow (φ) on the rate of phosphate incorporation (V) by the planktonic diatoms Thalassiosira fluviatilis Hust. and T. pseudonana Hasle & Heimdal in batch and chemostat cultures was determined by passing medium at defined flow rates (0.5–25.0 mm·min^?1) over algae on membrane filters. At concentrations from 1 to 100 μg phosphorus·l^?1 V, increases with increasing velocity of flow, approaching a maximum value (V_m) as described by the empirical relationship: where K_φ is the sinking rate value when V = 1/2 V_m+ V_o and V_o is the uptake at 0 rate of flow. By comparing uptake at controlled flow with uptake in a vigorously stirred medium, the phosphate concentration in the cell boundary layer can be determined. The sinking rate that reduces the phosphate concentration in the boundary layer to half of nominal concentration in the medium is much lower for the larger T. fluviatilis than for T. pseudonana. For both diatoms, it is inversely related to the nominal concentration.