Quinonoid dihydropterin reductase-I: Purification and characterization of the bovine brain enzyme

Quinonoid dihydropterin reductase (DHPR, EC 1.6.99.7) from bovine brain was purified 340-fold over the initial extract to an activity of 76 units/mg of protein with an overall recovery of 10 per cent. Analytical polyacrylamide gel electrophoresis, combined with protein staining, indicated that the single band of purified enzyme could have contained no more than 7% contaminants. Furthermore, using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, only one protein band was observed for the SDS-treated enzyme. Using a sucrose density gradient, the sedimentation coefficient and the molecular weight of the native enzyme were determined to be 3.70 and 47,000± 1,400 respectively. Molecular weight using 10% SDS-gel electrophoresis was found to be 22,400± 230, indicating that the brain reductase is composed of two equivalent subunits. Other characteristics of DHPR which were determined were the isoelectric point (pH 5.7), the K_m, for quinonoid 6,7-dimethyl-dihydropterin (18 μM) and the K_m for NADH (26 μM). Purified brain DHPR was used as an antigen to produce specific antibodies to the enzyme. The specificity of the antibody was established according to the following criteria: (1) the antibody was found to be a potent inhibitor of partially purified and homogeneous DHPR; (2) a single precipitin arc formed upon running either partially purified or homogeneous DHPR against the antibody on a double immunodiffusion plate, or by immunoelectrophoresis; (3) the antibody inhibited DHPR activity from rat brain, rat kidney and rat liver ; and (4) neither inhibition of activity, nor formation of precipitin arcs was exhibited by the antibody when reacted with other catecholamine-synthesizing enzymes partially purified from different rat tissues.     

Characterization and solubilization of the specific binding sites for d-α-tocopherol from human erythrocyte membranes

Previous work from our laboratory has demonstrated the presence of specific binding sites for d-α-tocopherol (vitamin E) in intact human erythrocytes [A. E. Kitabchi and J. Wimalasena, Biochim. biophys. Acta684, 300 (1982)]. The binding was time, temperature and cell concentration dependent. To localize the binding sites, red blood cells were further fractionated; greater than 90% of the tocopherol binding sites were localized on membranes. The washed membrane fraction from normal human erythrocytes has specific binding sites for d-α-tocopherol with properties suggestive of protein receptors. Two binding sites with K_a values of 3.31 × 10⁷ M^?1 and 1.51 × 10⁶ M^?1 were demonstrated, and solubilized d-α-tocopherol binding site complexes were resolved to major component with an M_r of 65,000 and a minor component with an M_r of 125,000.     

Binding of 3H-beta-endorphin to rat brain membranes: characterization of opiate properties and interaction with ACTH

The binding of tritiated beta-endorphin (3H-beta-EP) to brain homogenates is described. This has been difficult to achieve due to the lack of availability of 3H-beta-EP and to technical difficulties associated with high non-specific binding of beta-EP. We now report that 3H-beta-EP binding is saturable, stereospecific, has high affinity and is inhibited by sodium. Its dissociation rate is ten-fold longer than that of naloxone. Its regional distribution exhibits interesting differences from naloxone and enkephalin binding. ACTH1-24 appears to displace it more effectively than it displaces 3H-naloxone. The results are discussed in terms of multiple transmitter systems and the multiple opiate receptor hypothesis.     

Quantitative characterization of multiple binding sites for phencyclidine and N-allylnormetazocine in membranes from rat and guinea pig brain.

Quantitative ligand binding studies have been used to characterize binding sites for N-allylnormetazocine ((+)SKF10,047) (SKF), 1-(1-phenylcyclohexyl) piperidine (PCP), N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) and haloperidol in membranes from the brain of rat and guinea pig under conditions which permitted simultaneous analysis of the binding of both PCP and SKF. Using four labelled ligands (SKF, TCP, PCP and haloperidol), each displaced by the corresponding four unlabelled ligands, four classes of binding sites were observed in membranes from the brain of the rat, corresponding to sigma (sigma), two classes of PCP sites (PCP1, PCP2) and dopamine (D2) sites. The sigma site was suppressed by 50 nM haloperidol, while the PCP1 and PCP2 sites were not. These results were confirmed by studies employing a self- and cross-displacement design and dose-response surfaces for SKF and TCP, with and without blockade by haloperidol of the sigma site. Using mathematical modelling, employing the program LIGAND, it was possible to reject simpler models involving a common "PCP/sigma" site or a model involving only two classes of sites (sigma and PCP). Similar methods were used to identify two classes of sigma binding sites and two classes of PCP binding sites, in membranes prepared from the brain of the guinea pig. The relative potencies of 18 ligands for displacement of (+)[3H]SKF10,047 and [3H]TCP were compared: there were significant qualitative and quantitative differences in the "sigma" binding sites in the brain of rat and guinea pig, while the PCP binding sites were very similar in the two species.     

Polypeptide nanogels with hydrophobic moieties in the cross-linked ionic cores: synthesis,characterization and implications for anticancer drug delivery

Abstract

Polymer nanogels have gained considerable attention as a potential platform for drug delivery applications. Here we describe the design and synthesis of novel polypeptide-based nanogels with hydrophobic moieties in the cross-linked ionic cores. Diblock copolymer, poly(ethylene glycol)-b-poly(l-glutamic acid), hydrophobically modified with l-phenylalanine methyl ester moieties was used for controlled template synthesis of nanogels with small size (ca. 70?nm in diameter) and narrow particle size distribution. Steady-state and time-resolved fluorescence studies using coumarin C153 indicated the existence of hydrophobic domains in the ionic cores of the nanogels. Stable doxorubicin-loaded nanogels were prepared at high drug capacity (30 w/w%). We show that nanogels are enzymatically-degradable leading to accelerated drug release under simulated lysosomal acidic pH. Furthermore, we demonstrate that the nanogel-based formulation of doxorubicin is well tolerated and exhibit an improved antitumor activity compared to a free doxorubicin in an ovarian tumor xenograft mouse model. Our results signify the point to a potential of these biodegradable nanogels as attractive carriers for delivery of chemotherapeutics.     

Spray-dried casein-based micelles as a vehicle for solubilization and controlled delivery of flutamide: Formulation,characterization, and in vivo pharmacokinetics

Novel casein (CAS)-based micelles loaded with the poorly soluble anti-cancer drug, flutamide (FLT), were successfully developed in a powdered form via spray-drying technique. Genipin (GNP) was used to crosslink CAS micelles as demonstrated by color variation of the micelles. Drug solubilization was enhanced by incorporation within the hydrophobic micellar core which was confirmed by solubility study and UV spectra. Spherical core–shell micelles were obtained with a particle size below 100 nm and zeta potential around −30 mV. At low drug loading, FLT was totally incorporated within micellar core as revealed by thermal analysis. However, at higher loading, excess non-incorporated drug at micelle surface caused a significant reduction in the surface charge density. Turbidity measurements demonstrated the high physical stability of micelles for 2 weeks dependent on GNP-crosslinking degree. In a dry powdered form, the micelles were stable for 6 months with no significant changes in drug content or particle size. A sustained drug release from CAS micelles up to 5 days was observed. After i.v. administration into rats, CAS micelles exhibited a prolonged plasma circulation of FLT compared to drug solution. Furthermore, a more prolonged drug systemic circulation was observed for GNP-crosslinked micelles. Overall, this study reports the application of spray-dried natural protein-based micelles for i.v. delivery of hydrophobic anti-cancer drugs such as FLT.     

The competition of (−)-[3H]nicotine binding by the enantiomers of nicotine,nornicotine and anatoxin-a in membranes and solubilized preparations of different brain regions of rat

Summary In order to characterize the properties of nicotinic acetylcholine receptor (nAChR) subtypes in the CNS, the enantiomers of nicotine, nornicotine and anatoxin-a were studied for their ability to displace (–)-[³H]nicotine binding to membranes and solubilized preparations of different brain regions of rats. In hippocampal membranes, (–)-[³H]nicotine binding was stereo-selectively displaced from two sites by (+)- and (–)-nicotine, as well as by (+)- and (–)-anatoxin-a. (–)-Nicotine displayed a larger proportion of high affinity binding sites than did (+)-nicotine, while the proportions of high and low affinity binding sites for (+)-anatoxin-a was the same as that for (–)-anatoxin-a. In cerebellar membranes, the (–)-[³H]nicotine binding was stereoselectively displaced from a single binding site by nicotine and anatoxin-a with Ki values that did not correspond with their K_H and K_L values observed in hippocampus. The (–)-[³H]-nicotine binding was displaced from a single site by both (+)- and (–)-nornicotine with similar K_i values in both hippocampal and cerebellar membranes. In Triton X-100 solubilized preparations, the (–)-[³H]nicotine binding was displaced from a single site by all of the drugs tested and the Ki values for each individual drug were similar in the cortex, hippocampus and cerebellum. These results provided further evidence for pharmacological heterogeneity of membrane bound nAChRs and clearly indicated that detergent solubilization changed the binding properties of nAChRs in rat brain.Correspondence to X. Zhang at the above address     

Purification of the multifunctional calmodulin-dependent protein kinases from lung and liver, and the comparison to the brain enzyme

We purified the multifunctional calmodulin-dependent protein kinases (calmodulin-kinase) from rat lung and rabbit liver, and compared the properties of this enzyme with those of the rat brain enzyme. The lung and liver enzymes had molecular weights (Mr's) of 530,000 and 330,000 with main subunits of 52 and 51 kDa, respectively. Although the lung and liver enzymes cross-reacted with antibodies to the brain enzyme, the immunoreactivity of the lung enzyme was weaker. The substrate specificity of the three enzymes showed differences in the relative reaction rate of phosphorylation. The patterns of phosphopeptides of the lung and liver enzymes were similar to each other and only partly common to that of the 60-kDa subunit of the brain enzyme.     

Lymphocyte and brain neurotoxic esterase: dose and time dependence of inhibition in the hen examined with three organophosphorus esters

Certain organic phosphorus esters produce sensorimotor axonopathy in man and other species. There is an excellent correlation between the capacity of an organophosphorus compound to produce axonopathy and its ability to inhibit brain neurotoxic esterase (NTE) in hens. Because NTE is present in peripheral lymphocytes of both hen and man, it has been suggested that the lymphocyte enzyme might be useful both in experimental and clinical situations as an indicator of exposure to organophosphorus compounds producing axonopathy. Diethyl 4-nitrophenyl phosphate (paraoxon), tri-2-cresyl phosphate (TOCP), methyl 2,5-dichloro-4-bromophenyl phenylphosphonothionate (leptophos), and di-n-butyl-2,2-dichlorovinyl phosphate (di-n-butyl dichlorvos, DBDCV) were used to examine the relationship between lymphocyte and brain NTE inhibition in hens. As expected, paraoxon (0.75 mg/kg) did not inhibit NTE in brain or lymphocytes. TOCP (10 to 100 mg/kg), leptophos (25 to 150 mg/kg), and DBDCV (1.0 to 4.0 mg/kg) inhibited both brain and lymphocyte NTE activity in a dose-related manner with good correlation of inhibition between tissues taken 24 hr after exposure (r2 = 0.53 to 0.67; p less than 0.020 to 0.001). However, correlation of inhibition between tissues taken from animals killed 48 hr after exposures was poor (r2 = 0.15 to 0.30; p less than 0.10 to 0.05), with consistently less inhibition of lymphocyte NTE relative to brain NTE. This study indicates that assay of lymphocyte NTE can provide a good monitor of exposure to axonotoxic organophosphorus compounds within 24 hr between exposure and measurement.     

Purification, characterization and crystallization of Jararacussin-I, a fibrinogen-clotting enzyme isolated from the venom of Bothrops jararacussu.

A fibrinogen-clotting enzyme, Jararacussin-I, was purified from the venom of Bothrops jararacussu by a combination of ion exchange chromatography using Resource 15S resin and affinity chromatography using Benzamidine Sepharose 6B resin. Jararacussin-I displays a molecular mass of 28 kDa as estimated by sodium dodecyl sulphate-PAGE and possesses an isoelectric point of 5.0. The coagulant specific activity of the enzyme was determined to be 45.8 NIHU/mg using bovine fibrinogen as the substrate and the esterase specific activity was determined to be 258.7 U/mg. The protease inhibitors, benzamidine and DTT inhibited the esterase specific activity by 72.4 and 69.7%, respectively. The optimal temperature and pH for the degradation of both chains of fibrinogen and esterase specific activity were determined to be 37 degrees C and 7.4-8.0, respectively. The enzyme was inactivated at both 4 and 75 degrees C. Single crystals of Jararacussin-I were obtained and complete three-dimensional X-ray diffraction data was collected at the Brazilian National Synchrotron Source (LNLS) to a resolution of 2.4A.     

Purification and characterization of a thrombin-like enzyme, elegaxobin, from the venom of Trimeresurus elegans (Sakishima-habu).

A thrombin-like enzyme, named elegaxobin, was purified from the venom of Trimeresurus elegans (Sakishima-habu) by gel filtration on Sephadex G-100, and ion-exchange chromatographies on Q-Sepharose Fast Flow and S-Sepharose Fast Flow. By this procedure, about 8.5 mg of purified enzyme was obtained from 1.1 g of the venom. The purified enzyme showed a single protein band in SDS-polyacrylamide electrophoresis under reducing condition and its molecular weight is 30,000. The specific activity of this enzyme toward tosyl-L-arginine methyl ester (TAME) was 490 TAME units/mg of protein. Elegaxobin clotted only rabbit fibrinogen whereas human and bovine fibrinogens were unaffected. In the fibrinogen-fibrin convertion, the enzyme released only fibrinopeptide A from rabbit fibrinogen, whereas fibrinopeptide B was not released. The N-terminal sequences (Val-Ile-Gly-Gly) of this enzyme was identical to typical sequence of serine proteinases.     

Synthesis and characterization of metal oxide based ion exchanger from chicken egg shell biomass for the removal of arsenic from water

A new metal oxided-based high capacity biosorbent for As(III) was designed and synthesized from egg shell biomass and characterized using FTIR, FE-SEM, EDX, XRD, chemical analysis. For this, raw egg shell (RES) powder was first dissolved with HCl and mixed with ZrOCl₂18H₂O solution then precipitated to obtain hydrated double oxide precipitate (HDOP), which was employed for As(III) removal. As(III) biosorption onto HDOP is fast, depends on pH and As(III) concentration. Pseudo second order kinetics and Langmuir isotherm models successfully described the As(III) biosorption mechanism. HDOP provided exchangeable hydroxide/or chloride ligands for improved biosorption of As(III). Maximum biosorption capacity of HDOP for As(III) from Langmuir isotherm modelling was found to be 40 mg g^?1 at optimum pH 10. Chloride and nitrate cause negligible interference whereas sulphate and phosphate significantly reduced As(III) biosorption capacity of HDOP. HDOP completely removed arsenic from contaminated ground water and the remaining concentration was reached below the safe drinking water standard (10 μg L^?1) set by WHO. Moreover, HDOP exhibited effective regeneration and high stability with As(III) removal up to 8 cycles. Thus, HDOP synthesized from egg shell biomass can be used as a low cost, environmentally benign and high capacity biosorbent for the treatment of arsenic polluted water.     

Selectivity of ligand binding to opioid receptors in brain membranes from the rat, monkey and guinea pig

Conditions for the equilibrium binding to opioid receptor of [3H]sufentanil (mu selective), [3H][D-Pen2,D-Pen5]enkephalin (delta selective), and [3H]U69,593 (kappa selective) were established in membranes from rat brain cerebrum, monkey cortex, or guinea pig cerebellum. The selectivity index of various opioid alkaloids and peptides in binding to the mu, delta, or kappa opioid receptors was expressed as the ratio of their EC50 values in displacing two selective radiolabeled ligands: [3H]sufentanil/[3H](D-Pen2,D-Pen5)enkephalin (selectivity: mu/delta), [3H]sufentanil/[3H]U69,593 (selectivity: mu/kappa), or [3H][D-Pen2,D-Pen5]enkephalin/[3H]U69,593 (selectivity: delta/kappa). High resolution in binding selectivity was observed: in rat brain the mu/delta selectivity for Tyr-D-Ala-Gly-(Me)Phe-Gly-ol and sufentanil were 0.02 and 0.03, whereas for [D-Pen2,D-Pen5]enkephalin and ICI 174,864 they were 1,200 and 998. Compared to mu opiates, the specific binding of delta and kappa agonists was less sensitive to sodium. The results describe a routinely applicable methodological approach for the assessment of selective ligand binding to the mu, delta and kappa opioid receptors in rodent and monkey brain membranes.     

Purification and characterization of a thrombin like enzyme, elegaxobin II, with lys-bradykinin releasing activity from the venom of Trimeresurus elegans (Sakishima-Habu).

A thrombin like enzyme, named elegaxobin II, with Lys-bradykinin releasing activity was purified from the venom of Trimeresurus elegans (Sakishima-habu) by gel-filtration on Sephadex G-100, and ion-exchange chromatography on the Q-Sepharose Fast Flow. By this procedure, about 9mg of purified enzyme was obtained from 1.1g of the venom. The purified enzyme showed a single protein band, the molecular weight of which was estimated to be about 35,000Da by sodium dodecyl sulfate-PAGE) under reducing condition, and this enzyme was found to contain a carbohydrate moiety. The specific activity of this enzyme toward tosyl-L-arginine methyl ester (TAME) was 250 TAME units/mg of protein. This enzyme clotted only rabbit fibrinogen, whereas human and bovine fibrinogens were unaffected. In the fibrinogen-fibrin conversion, this enzyme released only fibrinopeptide A from rabbit fibrinogen, whereas it did not release fibrinopeptide B. Furthermore, elegaxobin II released Lys-bradykinin when the enzyme was incubated with bovine plasma. The esterase activity was inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF), suggesting that this enzyme is a serine protease. The N-terminal sequence (Val-Ile-Gly-Gly) of this enzyme was identical to the typical sequence of serine proteinases.     

Purification and partial characterization of a thrombin-like enzyme from the venom of the bushmaster snake,Lachesis muta noctiv aga

A thrombin-like enzyme has been purified from the venom of Lachesis muta noctivaga (41-fold purification and 43% yield). The steps included: gel filtration with Sephadex G-100, hydroxylapatite and DEAE-cellulose chromatography, and finally Sephadex G-150 filtration (twice). The material was homogeneous in polyacrylamide electrophoresis and gel filtration on Sephadex G-150. The enzyme is a glycoprotein of molecular weight 36,300 as determined by gel filtration. the thrombin-like enzyme hydrolyses tosyl-l-arginine methyl ester (Km = 1·45 × 10^?4 M, Vm = 353 μmole/min·mg, Kcat = 212sec^?1) with an optimum pH of 8·30. The enzyme also hydrolyses α-N-benzoyl-dl-arginine p-nitroanilide and is competitively inhibited by benzamidine, β-naphtamidine and phenylguanidine. Clotting and amidase activities are inhibited by diisopropylfluorophosphate. The enzyme molarity was determined by active site titration with p-nitrophenyl-p′-guanidino benzoate (92% pure). Injected in dogs 2 μg of the purified enzyme reduce, in 30 min, the plasma fibrinogen concentration to values less than 15% of the original level. In vitro the activity for human fibrinogen-fibrin conversion was equivalent to 1650 ± 12NIH thrombin units/mg protein enzyme.     

3H]oxotremorine-M binding to membranes prepared from rat brain and heart: evidence for subtypes of muscarinic receptors

[3H]Oxotremorine-M has been used as a ligand to label muscarinic binding sites on membranes prepared from rat fore-brain and heart. In rat brain membranes, two binding sites could be identified: a high affinity low capacity site and a low affinity high capacity site. In contrast, only a high affinity site could be labelled in heart membranes. The potency order of agonists for the high affinity site in brain membranes and heart membranes was the same, suggesting that these binding sites represent the same subtype of muscarinic receptors. However, the affinity of pirenzepine for the brain high affinity site was higher than that for the heart high affinity site suggesting that these sites may represent different receptor populations. The potency order of agonists for the high affinity and low affinity sites in brain membranes were significantly different suggesting that these binding sites represent pharmacologically distinct binding sites. GTP abolished the high affinity sites in heart and brain membranes, but the low affinity site in brain membranes appeared unaffected. These results are consistent with the hypothesis proposing subtypes of muscarinic receptors.     

Paclitaxel-loaded microparticles and implants for the treatment of brain cancer: preparation and physicochemical characterization

The aim of this study was to prepare different types of paclitaxel-loaded, PLGA-based microparticles and lipidic implants, which can directly be injected into the brain tissue. Releasing the drug in a time-controlled manner over several weeks, these systems are intended to optimize the treatment of brain tumors. The latter is particularly difficult because of the blood-brain barrier (BBB), hindering most drugs to reach the target tissue upon systemic administration. Especially paclitaxel (being effective for the treatment of ovarian, breast, lung and other cancers) is not able to cross the BBB to a notable extent since it is a substrate of the efflux transporter P-glycoprotein. Both, biodegradable microparticles as well as small, cylindrical, glycerol tripalmitate-based implants (which can be injected using standard needles) were prepared with different paclitaxel loadings. The effects of several formulation and processing parameters on the resulting drug release kinetics were investigated in phosphate buffer pH 7.4 as well as in a diethylnicotinamide (DENA)/phosphate buffer mixture. Using DSC, SEM, SEC and optical microscopy deeper insight into the underlying drug release mechanisms could be gained. The presence of DENA in the release medium significantly increased the solubility of paclitaxel, accelerated PLGA degradation, increased the mobility of the polymer and drug molecules and fundamentally altered the geometry of the systems, resulting in increased paclitaxel release rates.