Calorimetric Glucose Determination by Glucose Oxidase —Methylene Blue

The reaction between glucose and methylene blue, catalyzed by glucose oxidase (GOD)was analysed calorimetrically. The amount of heat produced under saturating methylene blue concentrations ( > 10^?2 mol/1)was measured with glucose concentration and time as parameters (kinetic procedure) Kinetic constants (pseudo one substrate kinetics) were derived from the experimental data: K_M(glucose)= 1.18 × 10^?3 mol/l and V_max = 0.085 J/mg GOD min (3.89 · 10^?6 mol/mg GOD min) Comparison of caloric with optical measurements gave an enthalpy of reaction of 22.52 kJ/mol. Considering the observed substrate inhibition, glucose determinations are possible up to glucose concentrations of 0.1 mol/l.     

Peptides isolated from human liver with specific inhibitory effects on reassociation/reactivation of in vitro dissociated lactic dehydrogenase (LDH-M4 and −H4) isozymes

Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375–1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1 1.27) isoenzymes (LDH-M₄ = muscle type; LDH-H₄ = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M₄ or LDH-H₄. From the amino acid analyses, gel-filtration and PAGE + SDS, molecular weight of 1800 for the M₄ and ≈2700 for the H₄ inhibitor were calculated. An apparent K_i of ≈3 × 10^?5 mM for the H₄ and ≈7 × 10^?5 mM for the H₄ inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M₄-specific) and 2.4 (LDH-H₄-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides reacts with monomers, dimers or a transition state during the tetramerisation process. k₁ for the dimerisation step of M₄ = 2.0 × 10⁵ M^?1 · s^?1 and of H₄ = 8.2 × 10⁴ M^?1 · s^?1; k₂ for the tetramerisation step of M₄ = 2.8 × 10⁵ M^?1 · s^?1 and of H₄ = 1.2 × 10⁵ · M^?1 · s^?1, were calculated, the second step still being the faster one.     

Characterization and Regulatory Properties of Glucose-6-Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The K_m value for glucose-6-phosphate is 1.6 × 10^?4 and 6.3 × 10^?4M at low (1.0–6.0 × 10^?4M) and high (6.0–30.0 × 10^?4M) concentrations of the substrate, respectively. The K_m value for NADP⁺ is 1.4 × 10^?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg²⁺, and Mn²⁺. In the presence of NADPH, the plot of activity vs. NADP⁺ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg²⁺ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.     

Cooperative binding of calcium to glycerinated skeletal muscle fibers

The binding of ⁴⁵Ca²⁺ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg²⁺ (no ATP) binding was half-maximal at 1.4 · 10^?6M Ca²⁺ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 10⁶ M^?1) and the Hill coefficient was 1.0. In the absence of Mg²⁺, binding was half-maximal at 3 · 10^?7 M Ca²⁺ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 10⁷ and 2 · 10⁶ M^?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg²⁺ binding to about half of the total Ca²⁺ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites.     

Immobilization of enzymes based on hydrophobic interaction. II. Preparation and properties of an amyloglucosidase adsorbate

Amyloglucosidase from Aspergillus niger (α-1,4 and 1,6 glucan glucohydrolase, EC 3.2.1.3) was immobilized through adsorption onto a hexyl–Sepharose, containing 0.51 mol hexyl-group per mole of galactose. The adsorption limit of the carrier with respect to this enzyme was about 17 mg per gram wet conjugate. The retention of activity upon immobilization was high, varying from essentially full activity at low enzyme content down to 68% at the adsorption limit. The immobilized preparation, as well as the soluble enzyme, showed apparent zero order kinetics within 60% of the substrate's conversion limit. Product inhibition of the soluble enzyme showed a k₁ of 5 · 10^?2M. In the presence of 3M NaCl, adsorbates were formed more rapidly and with a higher yield of immobilized protein, but with lower specific activity. Conjugates resulting from adsorption of amyloglucosidase in identical concentrations, but at different salt contents, showed comparable activities and operational stabilities. Continuous operation for three months reduced conjugate activity to 40%. The thermal stability of the adsorbate was inferior to that of the soluble enzyme, but was noticeably enhanced in the presence of substrate.     

Characterization of the uptake of sucrose and glucose by isolated seed coat halves of developing pea seeds. Evidence that a sugar facilitator with diffusional kinetics is involved in seed coat unloading

Uptake of ¹⁴C-labelled sucrose and glucose by isolated seed coat halves of pea (Pisum sativum L. cv. Marzia) seeds was measured in the concentration range <0.1 μM to 100 mM. The initial influx of sucrose was strictly proportional to the external concentration, with a coefficient of proportionality (k) of 6.2 μmol·(g FW)^?1·min^?1·M^?1. Sucrose influx was not affected by 10 μM carbonylcyanide m-chlorophenylhydrazone (CCCP), but it was inhibited by 40% in the presence of 2.5 mM p-chloromercuribenzenesulfonic acid (PCMBS). Influx with diffusional kinetics was also observed for glucose (k = 4.8 μmol·(g FW)^?1·min ^?1·M ^?1) and mannitol (k = 5.1 μmol·(g FW)^?1·min^?1·M^?1). For glucose an additional saturable system was found (K_m = 0.26 mM, V _max = 4.2 nmol·(g FW)^?1·min^?1), which appeared to be completely inhibited by CCCP and partly by PCMBS. In contrast to the diffusional pathway, uptake by this saturable system was slightly pH-dependent, with an optimum at pH 5.5. The influx of sucrose appears to be by the same pathway as the efflux of endogenous sucrose, which was inhibited by 36% in the presence of 2.5 mM PCMBS (De Jong A, Wolswinkel P, 1995, Physiol Plant 94: 78–86). It is argued that passive transport may be the only mechanism for sucrose transport through the plasma membrane of seed coat parenchyma cells. The estimated permeability coefficient of the plasma membrane for sucrose (P = 3.5·10^?7 cm·s^?1) is more than 1 × 10⁶-fold higher than that reported for artificial lipid membranes. This relatively high permeability is hypothesized to result from pore-forming proteins that allow the diffusion of sucrose. Furthermore, it is shown that a sucrose gradient across the plasma membrane of the seed coat parenchyma of only 22 mM will suffice to result in the net efflux of sucrose which is required to feed the embryo.     

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.     

Binding of the Bovine and Porcine Pancreatic Secretory Trypsin Inhibitor (Kazal) To Human Leukocyte Elastase: A Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTT – K_a = 6.3 × 10⁴M^?1, δ5G° = -26.9kJ/mol, δH° = +11.7kJ/mol, and δS° = +1.3 × 10² entropy units; porcine PSTI –K_a = 7.0 × 10³M^?1,δG° = -21.5kJ/mol, δH° = +13.0kJ/mol, and δS° = +1.2 × 10² entropy units (values of K_a δG° and δS° were obtained at 21.0°C; values of δH° were temperature independent over the range (between 5.0°C and 45.0°C) explored). On increasing the pH from 4.5 to 9.5, values of K_a for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from ?7.0, in the free enzyme, to ?5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

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.     

Properties of water-insoluble matrix-bound lactate dehydrogenase.

Lactate dehydrogenase enzyme was immobilized by binding to a cyanogen bromideactivated Sepharose 4B-200 in 0.1 m phosphate buffer, pH 8.5. The immobilized enzyme was found to have lower K_m values for its substrates. K_m values for pyruvate and lactate were 8 × 10 ^?5m and 4 × 10^?3m, respectively, an order of magnitude less than the value for the native (free) enzyme. Chicken heart (H₄) lactate dehydrogenase was found to lose nearly all its substrate inhibition characteristics as a result of immobilization. The covalently bound muscle-type subunits of lactate dehydrogenase showed more favorable interaction with the muscle type than with the heart type subunits. An increase in thermal and acid stability of the dogfish muscle (M₄) lactate dehydrogenase as well as a decrease in the percentage of inhibition of enzyme activity by rabbit antisera and in the complement fixation was observed as a result of immobilization. The changes in the properties of the enzyme as a result of immobilization may be attributable to hindrance produced by the insoluble matrix as well as conformational changes in the enzyme molecules.     

Kinetic behavior of penicillin acylase immobilized on acrylic carrier

The usefulness of Lilly's kinetic equation to describe penicillin G hydrolysis performed by immobilized penicillin acylase onto the acrylic carrier has been shown. Based on the experimental results characteristic kinetic constants have been estimated. The effect of noncompetitive inhibition of 6-amino penicillanic acid has not been found. Five components of reaction resistance have been defined. These components were also estimated for the reaction of the native enzyme as well as the Boehringer preparation.List of Symbols C _E g/m³ enzyme concentration - C _P,C _Q mol/m³ product concentrations - C _S mol/m³ substrate concentration - C _SO mol/m³ initial substrate concentration - K _A mol/m³ constant which defines the affinity of a substrate to the enzyme - K _iS mol/m³ substrate inhibitory constant - K _iP mol/m³ PhAA inhibitory constant - K _iQ mol/m³ 6-APA inhibitory constant - k ₃ mol/g/min constant rate of dissociation of the active complex - R(1) concentrational component of reaction resistance - R(2) resistance component derived from substrate affinity - R(3) resistance component due to the inhibition of the enzyme by substrate - R(4) resistance component due to the inhibition of the enzyme by PhAA - R(5) resistance component due to inhibition of the enzyme by 6-APA - r = dC_s/dt mol/m³ min rate of reaction - t min reaction time - (i) relative resistance of reaction     

Kinetics of chloride transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

The kinetics of the light-driven Cl^? uptake pump of Synechococcus R-2 (PCC 7942) were investigated. The kinetics of Cl^? uptake were measured in BG-11 medium (pH_o, 7·5; [K⁺]_o, 0·35 mol m^?3; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 mol m^?3) or modified media based on the above. Net³⁶Cl^? fluxes (?Cl^?_o,i) followed Michaelis-Menten kinetics and were stimulated by Na⁺ [18 mol m^?3 Na⁺ BG-11 ?Cl^?_max= 3·29±0·60 (49) nmol m^?2 s^?1 versus Na⁺-free BG-11 ?Cl^?_max= 1·02±0·13 (54) nmol m^?2 s^?1] but the Km was not significantly different in the presence or absence of Na⁺ at pH_o 10; the Km was lower, but not affected by the presence or absence of Na⁺ [Km = 22·3±3·54 (20) mmol m^?3]. Na⁺ is a non-competitive activator of net ?Cl^?_o,i. High [K⁺]_o (18 mol m^?3) did not stimulate net ?Cl^?_o,i or change the Km in Na⁺-free medium. High [K⁺]_o (18 mol m^?3) added to Na⁺ BG-11 medium decreased net ?Cl^?_o,i [18 mol m^?3K⁺ BG-11; ?Cl^?_max= 2·50±0·32 (20) nmol m^?2 s^?1 versus BG-11 medium; ?Cl^?_max= 3·35±0·56 (20) nmol m^?2 s^?1] but did not affect the Km 55·8±8·100 (40) mmol m^?3]. Na⁺-stimulation of net ?Cl^?_o,i followed Michaelis-Menten kinetics up to 2–5 mol m^?3 [Na⁺]_o but higher concentrations were inhibitory. The Km for Na⁺-stimulation of net ?Cl^?_o,i [K_1/2(Na⁺)] was different at 47 mmol m^?3 [Cl^?]_o (K_1/2[Na⁺] = 123±27 (37) mmol m^?3]. Li⁺ was only about one-third as effective as Na⁺ in stimulating Cl^? uptake but the activation constant was similar [K_1/2(Li⁺) = 88±46 (16) mmol m^?3]. Br^? was a competitive inhibitor of Cl^? uptake. The inhibition constant (Ki) was not significantly different in the presence and absence of Na⁺. The overall Ki was 297±23 (45) mmol m^?3. The discrimination ratio of Cl^? over Br^? (δCl^?/δBr^?) was 6·38±0·92 (df = 147). Synechococcus has a single Na⁺-stimulated Cl^? pump because the Km of the Cl^? transporter and its discrimination between Cl^? and Br^? are not significantly different in the presence and absence of Na⁺. The Cl^? pump is probably driven by ATP.     

Binding of Paeonol to Human Serum Albumin: A Hybrid Spectroscopic Approach and Conformational Study

The purpose of this study was to elucidate the binding of paeonol to human serum albumin (HSA) through spectroscopic methods. The fluorescence quenching of HSA by paeonol was a result of the formation of the HSA–paeonol complex with low binding affinity (K = 4.45 × 10³ M^?1 at 298 K). Thermodynamic parameters (ΔG^○ = –2.08 × 10⁴ J·mol^?1, ΔS^○ = 77.9 J·mol^?1·K^?1, ΔH^○ = 2.41 × 10³ J·mol^?1, k_q = 9.67 × 10¹² M^?1·s^?1) revealed that paeonol mainly binds HSA through hydrophobic force following a static quenching mode. The binding distance was estimated to be 1.91 nm by fluorescence resonant energy transfer. The conformation of HSA was changed and aggregates were formed in the presence of paeonol, revealed by synchronous fluorescence, circular dichroism, Fourier transform infrared spectroscopy, three‐dimensional fluorescence spectroscopy, and resonance light scattering results.     

Neutral Aminopeptidase and Dipeptidyl Peptidase IV Activities in Plasma of Monosodium Glutamate Obese and Food‐deprived Rats

Biometric parameters, glycemia and activity levels of plasma neutral aminopeptidase (APN) and dipeptidyl peptidase IV (DPPIV) were measured in monosodium glutamate obese and food‐deprived rats (MSG‐FD), to analyze the involvement of these enzymes in such situations. Plasma APN was distinguished as sensitive (PSA) (K_m = 7.8 × 10^?5 mol/l) and predominantly insensitive (APM) (K_m = 21.6 × 10^?5 mol/l) to puromycin, whereas DPPIV was sensitive (DPPIV‐DS) (K_m = 0.24 × 10^?5 mol/l) and predominantly insensitive (DPPIV‐DI) (K_m = 7.04 × 10^?5 mol/l) to diprotin A. Although unchanged in the MSG and food‐deprived animals, APM activity levels were closely correlated with body mass, Lee index, and mass of retroperitoneal fat pad in the food deprived, but not in the MSG animals. DPPIV‐DI activity levels decreased by 33% and were correlated with body mass, Lee index, and mass of periepididymal fat pad in the food‐deprived MSG rats. These data suggest that APM and DPPIV‐DI are respectively related to the downregulation of somatostatin in food‐deprived rats, and to the recovery of energy balance in MSG obese rats during food deprivation.     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

Kinetics of nucleotide binding to chloroplast coupling factor (CF1)

Studies of the kinetics of association and dissociation of the formycin nucleotides FTP and FDP with CF₁ were carried out using the enhancement of formycin fluorescence. The protein used, derived from lettuce chloroplasts by chloroform induced release, contains only 4 types of subunit and has a molecular weight of 280 000.In the presence of 1.25 mM MgCl₂, 1 mol of ATP or FTP is bound to the latent enzyme, with K_d = 10^?7 or 2 · 10^?7, respectively. The fluorescence emission (λ_max 340 nm) of FTP is enhanced 3-fold upon binding, and polarization of fluorescence is markedly increased. The fluorescence changes have been used to follow FTP binding, which behaves as a bimolecular process with K₁ = 2.4 · 10⁴ M^?1 · s^?1. FTP is displaced by ATP in a process apparently involving unimolecular dissociation of FTP with k_?1 = 3 · 10^?3 s^?1. The ratio of rates is comparable to the equilibrium constant and no additional steps have been observed.The protein has 3 sites for ADP binding. Rates of ADP binding are similar in magnitude to those for FTP. ADP and ATP sites are at least partly competitive with one another.The kinetics of nucleotide binding are strikingly altered upon activation of the protein as an ATPase. The rate of FTP binding increases to at least 10⁶ M^?1 · s^?1. This suggests that activation involves lowering of the kinetic barriers to substrate and product binding-dissociation and has implications for the mechanism of energy transduction in photophosphorylation.     

Inhibition by indomethacin and aspirin of 15-hydroxy-prostaglandin dehydrogenase in vitro

15-Hydroxyprostaglandin dehydrogenase from bovine lung was purified 7.4 times to a specific activity of 1.4 mU/mg of protein. The isoelectric point was estimated to 5.4 and the molecular weight by gelfiltration to 40,000. K_m for prostaglandin E₁ and for NAD⁺ were found to be 3.4 μM and 1.1 × 10^?4M respectively. The enzyme was inhibited by indomethacin and aspirin. The indomethacin inhibition was found to be non-competitive to prostaglandin E₁ having a K_i=1.4 × 10^?4M and a K_i^′=1.6 × 10^?5M.     

Catalyses by polymer complexes. VII. Enhanced esterolytic reactivity of polymer-coenzyme A,glutathione complexes

The association of coenzyme A(CoASH) and glutathione (GSH) with the water-soluble polymers and their esterolytic reactivities were evaluated through the reaction with p-nitrophenyl acetate in the presence of cationic polymer micelles: partially laurylated poly(2-ethyl-1-vinylimidazole) and poly(4-vinylpyridine). The polymer micelles with high lauryl-group content (more than 12 mol%) markedly accelerated the reaction at very low concentrations of the polymer. Other polymers with no or small lauryl-group content only slightly enhanced the association and the reaction rate. From the rate-polymer concentration profiles, the association constants (K) and the rate constants for thiol coenzymes bound to the polymer (k′_a,bound) were determined: for polymers with more than 12 mol % lauryl-group content, K_CoASH = 1110–2270 M^?1, K_GSH = 170–503M^?1, k′_a,bound at pH 8.65 = 142–341M^?1 sec^?1. k′_a,bound were 20–340 times larger than that observed in the absence of the polymer. The logarithm of k′_a,bound was found to be correlated well with the polymer hydrophobicity, indicating that the hydrophobic environment of the polymer activated the bound thiol anions. On the other hand, the polymer hydrophobicity did not correlate with the association constant.     

Stereoselective binding of isradipine to human plasma proteins

Isradipine (PN 200–110) is a highly potent calcium entry blocker with an asymmetrically substituted dihydropyridine ring (methyl- and isopropylester, respectively). The binding of the (+)-(S)-isradipine and (?)-(R)-isradipine to isolated human serum albumin (HSA, 30 μmol/l) and α₁-acid glycoprotein (AAG, 10 μmol/l) has been studied in vitro over a wide range of isradipine concentrations (0.06–20 μmol/l) using high-performance liquid chromatography (HPLC). HPLC experiments revealed that both isradipine enantiomers were bound to one class of high-affinity binding sites on the AAG molecule (n_(S) = 0.83 ± 0.05, K_a(S) = (1.33 ± 0.25) × 10⁶ 1/mol, n_(R) = 0.85 ± 0.07, K_a(R) = (1.17 ± 0.44) × 10⁷ l/mol). The (R)-enantiomer also exhibited an interaction with the secondary low-affinity binding sites (n′K′_{a (R)} = (2.66 ± 0.65) × 10⁴ l/mol). In contrast, the pharmacologically more potent (+)-(S)-enantiomer was more strongly bound to HSA than its optical antipode (n_(S) = 1.07 ± 0.07, K_a(S) = (1.76 ± 0.26) × 10⁵ l/mol, nK_a(R) = (3.62 ± 0.06) × 10⁴ l/mol). In general, the resulting binding characteristics of individual isradipine enantiomers showed stereoselectivity, but this was opposite for the two most important plasma binding proteins. The process of accumulation of isradipine by human platelets in the therapeutically relevant range (10–80 ng/ml) at 37°C was devoid of stereoselectivity. © 1995 Wiley-Liss, Inc.     

Purification and Characterization of two Extracellular β-Glucosidases from Trichoderma viride ITCC 1433

T. viride ITCC 1433 synthesizes a two component system for the hydrolysis of cellobiose and cellooligodextrins. 80% of the total activity are solubilized during growth. The large protein (A), mol. weight 98 000 d, is glycosylated and slightly acidic (pH = 6.1). The smaller protein (B), mol. weight 70 000 d, is unglycosylated and neutral (pH = 7.2). Both proteins form a two-step system where β-glucosidase A is active at low substrate concentrations (K_M = 2.3 × 10^?4 M cellobiose) while β-glucosidase B covers the range of 10-fold higher cellobiose concentrations (K_M = 1.8 × 10^?3 M). The enzymes are fairly stable with a residual activity of 70% at 50°C after 24 h.