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.     

Effect of cadmium on survival,osmoregulation and gill structure of the Sunda prawn,Macrobrachium sintangense (de Man), at different salinities

The prawn Macrobrachium sintangense is likely to be subjected to occasional exposure to combined metal and saline stressors in its natural environment. This research evaluated the acute toxicity (96?h LC₅₀) of cadmium (Cd) on the prawn M. sintangense, with respect to the osmoregulatory capacity (OC) of prawns and to document histological changes in the gills after exposure to sublethal Cd concentrations at different salinities. The 96?h LC₅₀ of Cd to M. sintangense decreased with increasing salinity. The 96?h LC₅₀ values were 89.12 (72.53–109.50), 681.26 (554.20–837.46) and 825.37 (676.99–1006.27) μg CdL^?1 at 0, 10 and 20 ppt, respectively. The OC of prawns exposed to 30?μg?CdL^?1 at 0 ppt and to 300?μg?CdL^?1 at10 ppt decreased significantly compared with that of control prawns exposed to 0 and 10 ppt respectively. Swelling, hyperplasia and necrosis of gill lamellae resulting in the loss of marginal canals were observed in the gills of prawns exposed to 30?μg?CdL^?1 at 0 ppt and to 300?μg?CdL^?1 at 10 ppt for 7?days.     

Dimeric chlorite dismutase from the nitrogen‐fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non‐azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite ‘dismutase’, Cld). Beside the water‐splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen–oxygen bond. All cyanobacterial Clds have a truncated N‐terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [k_cat 1144 ± 23.8 s^?1, K_M 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 10⁶ M^?1 s^?1]. The resting ferric high‐spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of ?126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low‐spin complex with k_on = (1.6 ± 0.1) × 10⁵ M^?1 s^?1 and k_off = 1.4 ± 2.9 s^?1 (K_D ～ 8.6 μM). Both, thermal and chemical unfolding follows a non‐two‐state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure–function relationships of Clds. We ask for the physiological substrate and putative function of these O₂‐producing proteins in (nitrogen‐fixing) cyanobacteria.     

Uptake kinetics of nitrate,ammonia and phosphate by the dinoflagellate Heterocapsa circularisquama isolated from Hiroshima Bay,Japan

Heterocapsa circularisquama is a harmful dinoflagellate whose first bloom in Hiroshima Bay, Japan, appeared in 1992. As suggested by the authors’ group, in the Seto Inland Sea including Hiroshima Bay, oligotrophication particularly the reduction of phosphate starting 1980 is severe. The bloom caused serious damage to the bay's extensive oyster culture. In the present study, the uptake kinetics of nitrate, ammonia, and phosphate by this species were experimentally investigated. The maximum uptake rate (ρ_max) and the half‐saturation constant (Ks) were 0.41 pmol cell^?1 h^?1 and 4.45 μM, respectively, for nitrate, 2.02 pmol cell^?1 h^?1 and 11.1 μM for ammonium, and 0.079 pmol cell^?1 h^?1 and 1.79 μM for phosphate. The maximum specific uptake rates (V_max) for nitrate, ammonia, and phosphate were estimated to be 8.95, 44.1, and 21.3 day^?1, respectively. A comparison of V_max/Ks, which is also an index of affinity to nutrients, between this species and others suggested that H. circularisquama can utilize nitrate and ammonia efficiently, but not phosphate. Considering both reports describing that H. circularisquama has the ability to utilize dissolved organic phosphorus (DOP) and the DOP concentration is higher than phosphate in Hiroshima Bay, it was concluded that H. circularisquama became dominant due to the phosphate reduction measure.     

SPECIFICITY AND FUNCTION OF GLYCERALDEHYDE‐3‐PHOSPHATE DEHYDROGENASE IN A FRESHWATER DIATOM,ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE)1

The plastidic glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) catalyzes the only reductive step in the Calvin cycle and exists as different forms of which GapC1 enzyme is present in chromalveolates, such as diatoms. Biochemical studies on diatoms are still fragmentary, and, thus, in this report, GAPDH from the freshwater diatom Asterionella formosa Hassall has been purified and kinetically characterized. It is a homotetrameric enzyme with a molecular mass of ~150 ± 15 kDa. The enzyme showed Michaelis–Menten kinetics with respect to both cofactors, NADPH and NADH, with a 16‐fold greater catalytic constant for NADPH. The K_m for NADPH was 140 μM, the lowest affinity reported, while the catalytic constant, 815 s^?1, is the highest reported. The K_m for NADH was 93 μM, and the catalytic constant was 50 s^?1, both are similar to reported values for other types of GAPDH. The GapC1 enzyme, like the Chlamydomonas reinhardtii A₄ GAPDH, exhibits a cooperative behavior toward the substrate, 1,3‐bisphosphoglyceric acid (BPGA), with both cofactors. Mass spectrometry analysis showed that when GapC1 enzyme was purified without reducing agents, it copurified with a small protein with a mass of 8.2 kDa. This protein was recognized by antibodies against CP12. When associated with this protein, GAPDH displayed a lag that disappeared upon incubation with reducing agent in the presence of either BPGA or NADPH as a consequence of dissociation of the GAPDH/CP12 complex. Thus, as in other species of algae and higher plants, regulation of GapC1 enzyme in A. formosa may occur through association‐dissociation processes linked to dark‐light transitions.     

Urease inhibitors from Hypericum oblongifolium WALL.

The bioassay-guided fractionation of H. oblongifolium has led to the isolation of potent urease inhibitors 1–3. The structures were elucidated by NMR and mass spectroscopic techniques. Compound 2 showed a potent enzyme inhibition activity (IC₅₀ 20.96?±?0.93), which is comparatively higher than that for the standard thiourea (IC₅₀ 21.01?±?0.51 μM). Compounds 1 and 3 also showed a significant activity, with IC₅₀ 37.95?±?1.93 and 138.43?±?1.23 μM, respectively. The sub crude fractions (F1, F2, F3, and F4) were tested in vitro for their urease inhibition activity. Fractions F2 and F4 showed significant activity with IC₅₀ 140.37?±?1.93 and 167.43?±?3.03 μM, respectively.     

Purification, characterization and inhibition of sterol C24-methyltransferase from Candida albicans

Solubilized sterol C24-methyltransferase (24-SMT) was purified to homogeneity from a cell extract of the yeast Candida albicans (Ca) by anion exchange chromatography, gel permeation chromatography and fast performance liquid chromatography using a Mono Q column. The purified enzyme has an apparent molecular mass of 178 kDa on gel permeation chromatography and 43 kDa on SDS/PAGE, indicating that it is composed of four identical subunits. The substrate requirement of the native enzyme has an optimal specificity for zymosterol with associated kinetic constants of K_m 50 μM and k_cat of 0.01 s⁻¹. The product of the enzyme incubated with zymosterol was fecosterol. Inhibition of the catalyst was observed with substrate analogs designed as transition state analogs (25-azalanosterol, K_i = 54 nM and 24 (R,S),25-epiminolanosterol, K_i = 11 nM) or as mechanism-based inactivators (26,27-dehydrozymosterol, K_i 9 μM) and k_inact = 0.03 min⁻¹) of the C24-methylation reaction. Product analogs ergosterol and fecosterol, but neither cholesterol nor sitosterol, inhibited activity affording K_i values of 20 and 72 μM, respectively. Ammonium and thia analogs of the intermediates of the sterol C24-methyl reaction sequence were effective growth inhibitors exhibiting IC₅₀ values that ranged from 3 to 20 μM.     

Growth kinetics of top and bottom cultures of Saccharomyces spp. in a chemostat using sugarbeet molasses

Growth kinetics were evaluated for three yeast strains of the genus Saccharomyces. Two topfloating strains, SF 115 and SF 116 and one flocculant yeast SF 104 were analyzed in pure and mixed cultures in 1-liter continuous fermentation experiments in a chemostat. Growth was monitored for 72 h at 30°C in a medium containing sugarbeet molasses and 1.0 g/liter each of NH₄H₂PO₄ and urea. SF 115 and SF 116 were found to have lower μ_max values of 0.290 and 0.296 h^?1, respectively, than SF 104, which had a μ_max of 0.364 h^?1. The two top-floating yeasts (SF 115 and SF 116) demonstrated greater affinity for the substrate and utilized substrates at a greater rate. They have K₈ values of 4.03 × 10^?3 M and 3.798 × 10^?3 M, respectively, compared to 9.06 × 10^?3 M for SF 104. A mixed culture of SF 116 and SF and SF 104 was found to have a μ_max of 0.426 h^?1 with a K_s of 6.924 × 10^?3 M. SF 115 grown in mixed culture with SF 104 exhibited a μ_max of 0.473 h^?1 with a K_s of 7.975 × 10^?3 M. In both cases, the SF 104 was the dominant microbe in mixed culture systems.     

Association of β-amyloid peptide fragments with neuronal nitric oxide synthase: Implications in the etiology of Alzheimers disease

Neuronal nitric oxide synthase (nNOS) was purified on DEAE-Sepharose anion-exchange in a 38% yield, with 3-fold recovery and specific activity of 5 µmol.min^?1.mg^?1. The enzyme was a heterogeneous dimer of molecular mass 225?kDa having a temperature and pH optima of 40°C and 6.5, K_m and V_max of 2.6 μM and 996 nmol.min^?1.ml^?1, respectively and was relatively stable at the optimum conditions (t_½?=?3?h). β-Amyloid peptide fragments Aβ_17–28 was the better inhibitor for nNOS (K_i?=?0.81 µM). After extended incubation of nNOS (96?h) with each of the peptide fragments, Congo Red, turbidity and thioflavin-T assays detected the presence of soluble and insoluble fibrils that had formed at a rate of 5?nM.min^?1. A hydrophobic fragment Aβ_17–21 [Leu₁₇ – Val₁₈ – Phe₁₉ – Phe₂₀ – Ala₂₁] and glycine zipper motifs within the peptide fragment Aβ_17–35 were critical in binding and in fibrillogenesis confirming that nNOS was amyloidogenic catalyst.     

SSF production,purification and characterization of chitin deacetylase from Aspergillus flavus

The production of an extracellular chitin deacetylase (CDA) produced by Aspergillus flavus under solid-substrate fermentation (SSF) using wheat bran as substrate was optimized using statistical methods. The CDA production in SSF increased 1.79-fold in comparison to the unoptimized basal level medium. It was purified to a final purity of 3.94-fold by ammonium sulphate precipitation, ion-exchange chromatography, and gel-permeation chromatography (GPC) consecutively and further characterized. The molecular mass of the enzyme was estimated to be about 28?kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and GPC analysis. The optimum pH and temperature of the purified enzyme were pH 8.0 and 50?°C, respectively. Additionally, the effect of some cations and other chemical compounds on the CDA activity was studied. A marginal increase in enzyme activity was observed with metal ions mainly Mn²⁺ and Zn²⁺. No inhibition of the enzyme was observed by the end product, that is, acetate up to 70?mM concentration. The K_m and k_cat values of the enzyme were determined to be 9.45?mg mL^?1 and 26.72?s^?1 respectively, using colloidal chitin as substrate. Among various substrates tested, glycol chitin and colloidal chitin were deacetylated.     

Purification and Characterization of Aspartic Protease Derived from Sf9 Insect Cells

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS–PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K _m of 0.85 μM. The k _cat and k _cat?K _m values were 13 s^?1 and 15 s^?1 μM^?1 respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K _i, of 25 pM.     

The Inhibition of Clostridium chauvoei (Jakari strain) Neuraminidase Activity by Methanolic Extracts of the Stem Barks of Tamarindus indicus and Combretum fragrans

The inhibition of neuraminidase from Clostridium chauvoei (jakari strain) with partially purified methanolic extracts of some plants used in Ethnopharmacological practice was evaluated. Extracts of two medicinal plants, Tamarindus indicus and Combretum fragrans at 100–1000 μg/ml, both significantly reduced the activity of the enzyme in a dose-dependent fashion (P < 0.001).

The estimated IC₅₀ values for Tamarindus indicus and Combretum fragrans were 100 and 150 μ/ml respectively. Initial velocity studies conducted, using fetuin as substrate revealed a non-competitive inhibition with the V_max significantly altered from 500 μmole min^?1 mg^?1 to 240μmole min^?1 mg^?1 and 340 μmole min^?1 mg^?1 in the presence of Tamarindus indicus and Combretum fragrans respectively. The K_M remained unchanged at 0.42 mM. The computed Index of physiological efficiency was reduced from 1.19 min^?1 to 0.57 min^?1 and 0.75 min^?1 with Tamarindus indicus and Combretum fragrans as inhibitors respectively.     

Purification and partial characterization of a gibberellin 2β-hydroxylase fromPhaseolus vulgaris

A gibberellin 2β-hydroxylase has been purified from mature seeds ofPhaseolus vulgaris. The enzyme is of molecular weight 36,000 and has the characteristics of a dioxygenase; the cofactors areα-ketoglu-tarate, Fe²⁺ and ascorbate, and activity is stimulated by catalase. The V_max of the enzyme is 6.86 nmole h^?1 mg^?1, and the Km values for [1,2-³H₂]GA₁ andα-ketoglutarate are 0.085 μM and 21 μM, respectively. The purified enzyme preparation catalyzes hydroxylation of GA₁, GA₄, GA₉, and GA₂₀ but exhibits a marked preference for the 3-hydroxylated gibberellins as substrate.     

Production of precursors for anti-Alzheimer drugs: Asymmetric bioreduction in a packed-bed bioreactor using immobilized D. carota cells

(S)-1-Phenylethanol derivatives, which are the precursors of many pharmacological products, have also been used as anti-Alzheimer drugs. Bioreduction experiments were performed in a batch and packed-bed bioreactor. Then, the kinetics constants were determined by examining the reaction kinetics in the batch system with free and immobilized carrot cells. Also, the effective diffusion coefficient (D_e) of acetophenone in calcium alginate-immobilized carrot cells was investigated. Kinetics constants for free cells, which are intrinsic values, are reaction rate V_max?=?0.052?mmol?L^?1?min^?1, and constants of the Michaelis–Menten K_M?=?2.31?mmol?L^?1. Kinetics constants for immobilized cells, which are considered apparent values, are V_{max, app}?=?0.0407?mmol?L^?1 min^?1, K_{M, app}?=?3.0472?mmol?L^?1 for 2?mm bead diameter, and V_{max, app}?=?0.0453?mmol?L^?1 min^?1, K_{M, app}?=?4.9383?mmol?L^?1 for 3?mm bead diameter. Average value of effective diffusion coefficient of acetophenone in immobilized beads was determined as 1.97?×?10^?6?cm²?s^?1. Using immobilized carrot cells in an up-flow packed-bed reactor, continuous production of (S)-1-phenylethanol through asymmetric bioreduction of acetophenone was performed. The effects of the residence time and concentrations of substrate were investigated at pH 7.6 and 33°C. Enantiomerically pure (S)-1-phenylethanol (ee?>?99%) was produced with 75% conversion at 4-hr residence time.     

β-xylosidase from Selenomonas ruminantium: Immobilization,stabilization, and application for xylooligosaccharide hydrolysis

The tetrameric β-xylosidase from Selenomonas ruminantium is very stable in alkaline pH allowing it to easily immobilize by multipoint covalent attachments on highly activated glyoxyl agarose gels. Initial immobilization resulted only in slight stabilization in relation to the free enzyme, since involvement of all subunits was not achieved. Coating the catalyst with aldehyde-dextran or polyethylenimine, fully stabilized the quaternary structure of the enzyme rendering much more stabilization to the biocatalyst. The catalyst coated with polyethylenimine of molecular weight 1300 is the most stable one exhibiting an interesting half-life of more than 10 days at pH 5.0 and 50?°C, being, therefore, 240-fold more stable than free enzyme. Optimum activity was observed in the pH range 4.0–6.0 and at 55?°C. The catalyst retained its side activity against p-nitrophenyl α-l-arabinofuranoside and it was inhibited by xylose and glucose. Kinetic parameters with p-nitrophenyl β-d-xylopyranoside as substrate were V_max 0.20?μmol.min^?1?mg?prot.^?1, K_m 0.45?mM, K_cat 0.82?s^?1, and K_cat/K_m 1.82?s^?1?mM^?1. Xylose release was observed from the hydrolysis of xylooligosaccharides with a decrease in the rate of xylose release by increasing substrate chain-length. Due to the high thermostability and the complete stability after five reuse cycles, the applicability of this biocatalyst in biotechnological processes, such as for the degradation of lignocellulosic biomass, is highly increased.     

Competitive substrate inhibition of amyloglucosidase from Rhizopus sp. by vanillin and curcumin

Kinetic studies of two glucosylation reactions catalyzed by an amyloglucosidase from Rhizopus sp. leading to the synthesis of vanillin-α/β-D-glucoside from D-glucose and vanillin and curcumin-bis-α-D-glucoside from D-glucose and curcumin were investigated in detail. Initial reaction rates were determined from kinetic runs involving different concentrations of D-glucose and vanillin (5?mM to 0.1?M) or D-glucose and curcumin (5?mM to 0.1?M). Graphical double reciprocal plots showed that the kinetics of the two enzyme catalyzed reactions exhibited Ping-Pong Bi-Bi mechanism where competitive substrate inhibition by vanillin/curcumin led to dead-end amyloglucosidase–vanillin/curcumin complexes at higher concentrations of vanillin/curcumin. An attempt to obtain the best fit of this kinetic model through computer simulation yielded in good approximation, the values of four important kinetic parameters, vanillin-α/β-D-glucoside: k_cat=35.0±3.2 10^?5M?h^?1·mg, K_i=10.5±1.1?mM, K_mD-glucose=60.0±6.2?mM, K_mvanillin=50.0±4.8?mM; curcumin-bis-α-D-glucoside: k_cat=6.07±0.58 10^?5M?h^?1·mg, K_i=3.0±0.28?mM, K_mD-glucose=10.0±0.9?mM, K_mcurcumin=4.6±0.5?mM.     

The inhibitory kinetics and mechanism of glycolic acid on lipase

Abstract

Obesity is prone to cause a variety of chronic metabolic diseases, and it has aroused people’s attention that the rapid increase in the global population of obese people in the past years. As a kind of weight-loss drug acting in the intestine, lipase inhibitor does not enter the bloodstream without producing central nervous side effects. Because they do not affect the metabolism system, lipase inhibitors and obesity have become one of the hot spots in recent years. Glycolic acid is a new substrate analog inhibitor with the value of the semi-inhibitory concentration of lipase is estimated to be 17.29?±?0.14?mM. Using the plots of Lineweaver-Burk, the inhibition mechanism of lipase by glycolic acid was reversible and the inhibition type belongs to competitive inhibition with a K_I value of 19.61?±?0.26?mM. The inhibitory kinetics assay showed that the microscopic velocity constant k₊₀ of inhibition kinetics is 1.79?×?10^?3?mM^?1s^?1, and k_?0 is 0.73?×?10^?3 s^?1. The results of UV full-wavelength scanning on product cumulative, fluorescence quenching and molecular simulation also indicated that glycolic acid and substrate competitive with lipase by binding to Lys137. Thereby glycolic acid inhibiting the oxidation-catalyzed reaction and reducing the product of the enzyme and substrate. This adds a new direction for the search for lipase inhibitors and provides new ideas about the development of anti-obesity drugs.

Communicated by Ramaswamy H. Sarma     

Enzymatic studies on the interaction of myosin and heavy meromyosin with 1,N 6 -ethenoadenosine triphosphate ( ATP), a fluorescent analog of ATP

The fluorescent analog of adenosine triphosphate (ATP)¹ 1,N⁶-ethenoadenosine triphosphate, (εATP), has been utilized as a substitute for ATP in the myosin and heavy meromyosin ATPase systems. For myosin, the analog εATP replaced ATP with a somewhat larger K_m (2.6 × 10^?4 mole ?^?1 for εATP as opposed to 8.8 × 10^?5 mole ?^?1 for ATP), indicating that the apparent affinity of the enzyme for εATP is less than for ATP. Perhaps of more interest, further comparison yielded a V_max for εATP about two and one half times the value for ATP (20 μmole PO₄ sec^?1 g protein^?1 as opposed to 8.1 μmole sec^?1 g protein^?1). Results for the HMM-εATPase system were similar, yielding a K_m value of 1.47 × 10^?4 mole ?^?1 and a V_max of 54.2 μmole PO₄ sec^?1 g protein^?1, as opposed to corresponding K_m and V_max values of 1.23 × 10^?4 mole ?^?1 and 20.4 μmole PO₄ sec^?1 g protein^?1, respectively for the HMM-ATP interaction. The pH dependence of εATPase for both systems was comparable to ATP, suggesting a similarity in the mechanism of hydrolysis of the two nucleotides. Activation of εATPase by Ca²⁺ in the presence of 0.5 M KCl was comparable to ATPase for both systems, but inhibition by Mg²⁺ seemed to be more effective for εATPase. These results indicate that εATP is an excellent substitute for ATP in the myosin and heavy meromyosin systems and because of its insertion into the active site of these muscle proteins, it promises to be a very useful probe for conformation studies at this level.     

Substrate/Inhibitor Properties of Human Deoxycytidine Kinase (dCK) and Thymidine Kinases (Tk1 And Tk2) Towards the Sugar Moiety of Nucleosides,Including O′-Alkyl Analogues

Abstract

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono-and di-O′-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower K_i values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3′-hexanoylamino-2′,3′-dideoxythymidine, with a K_i of ～600 μM for TK1 and ～0.1 μM for TK2. 3′-OMe-dC was a superior inhibitor of dCK to its 5′-O-methyl congener, consistent with possible participation of the oxygen of the (3′)-OH or (3′)-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly α-dT was a good substrate of both TK1 and TK2, with K_i values of 120 and 30 μM for TK1 and TK2, respectively; and a 3′-branched α-L-deoxycytidine analogue proved to be as good a substrate as its α-D-counterpart. Several 5 ′-substituted analogues of dC were     

Tricyclohexyltin hydroxide effects on cationic and substrate activation kinetics of beta-adrenergic–stimulated cardiac Ca2+-ATPase

Previous studies from this laboratory have indicated that tricyclohexyltin hydroxide (Plictran) is a potent inhibitor of both basal- and isoproterenol-stimulated cardiac sarcoplasmic reticulum (SR) Ca²⁺-ATPase, with an estimated IC-50 of 2.5 × 10^?8M. The present studies were initiated to evaluate the mechanism of inhibition of Ca²⁺-ATPase by Plictran. Data on substrate and cationic activation kinetics of Ca²⁺-ATPase indicated alteration of V_max and K_m by Plictran (1 and 5×10^?8M), suggesting a mixed type of inhibition. The beta-adrenergic agonist isoproterenol increased V_max of both ATP- and Ca²⁺-dependent enzyme activities. However, the K_m of enzyme was decreased only for Ca²⁺ Plictran inhibited isoproterenol-stimulated Ca²⁺-ATPase activity by altering both and V_max and K_m of ATP as well as Ca²⁺-dependent enzyme activities, suggesting that after binding to a single independent site, Plictran inhibits enzyme catalysis by decreasing the affinity of enzyme for ATP as well as for Ca²⁺ Preincubation of enzyme with 15 μM cAMP or the addition of 2mM ATP to the reaction mixture resulted in slight activation of Plictran-inhibited enzyme. Pretreatment of SR with 5 × 10^?7M propranolol and 5 × 10^?8M Plictran resulted in inhibition of basal activity in addition to the loss of stimulated activity. Preincubation of heart SR preparation with 5 × 10^?5M coenzyme A in combination with 5 × 10^?8M Plictran partly restored the beta-adrenergic stimulation. These results suggest that some critical sites common to both basal- and beta-adrenergic-stimulated Ca²⁺-ATPase are sensitive to binding by Plictran, and the resultant conformational change may lead to inhibition of beta-adrenergic stimulation.