Preparation and characterization of a fully active biotinylated probe of cholecystokinin

In this study we describe the synthesis and purification of biotinylated cholecystokinin-8 (Bio-CCK-8) and characterize its use as a probe for the pancreatic cholecystokinin receptor. CCK-8 (0.1 umoles) was reacted with either radiolabeled d-[8,9(-3)H]biotin succinimide ester (0.5 umoles) or N-hydroxysuccinimidyl-biotin in dimethylformamide and triethylamine, and purified by anion exchange chromatography. Concentrations of Bio-CCK-8 and CCK-8 needed for half-maximal inhibition of [125]I-CCK-8 binding to pancreatic membranes were the same (1.0 and 1.3 nM). Bio-CCK-8 retained full biological activity as determined by stimulation of pancreatic protein secretion from rats, and the biotin group bound to CCK-8 retained its high sensitivity for avidin.     

Preparation and biological properties of biotinylated PhTX derivatives.

We report the synthesis of several highly functionalized biotinylated philanthotoxin (PhTX) analogues (7, 8, 10, 13-16) designed on the basis of earlier structure-activity relationship studies. Despite the extensive modifications, the binding to nicotinic acetylcholine receptor (nAChR) is in the low micromolar range according to an inhibition assay using 3H-thienylcyclohexyl-piperidine (TCP). A patch clamp functional assay gave comparable results. Compounds exemplified by 16, which consists of a biotinylated ligand linked to a bifunctional photoaffinity probe (BPP), represent a new type of probe which should find use in photo-crosslinking studies of ligand receptor interactions.     

Hordeum chilense repetitive sequences. Genome characterization using biotinylated probes

Summary A library of random DNA fragment clones of wild barley Hordeum chilense was screened for clones of repeated nucleotide sequences. Five clones were isolated that gave a stronger hybridization signal in colony and dot blot hybridization with total H. chilense DNA in comparison to Triticum aestivum DNA. Clones labelled with biotinylated nucleotides were used as probes to investigate the repeated sequences organization in the H. chilense genome. Tandemly arranged and interspersed sequences have been found, together with homology differences with related sequences present in T. Aestivum, which could allow the differentiation of H. chilense DNA when it is present in wheat. We show that biotin can replace the use of ³²P in preparing repeated sequence probes for Southern and DNA dot blot analyses.     

Preparation, characterization, and application of biotinylated and biotin-PEGylated glucagon-like peptide-1 analogues for enhanced oral delivery

Glucagon-like peptide-1 (GLP-1) (7-36) is a type of incretin hormone with unique antidiabetic potential. The introduction of orally active GLP-1 offers substantial benefits in the treatment of type 2 diabetes over conventional injection-based therapies. Because the intestinal absorption of GLP-1 is restricted by its natural characteristics, we developed a series of GLP-1 analogues via the site-specific conjugation of biotin-NHS and/or of biotin-poly(ethylene glycol)-NHS at Lys 26 and Lys 34 of GLP-1 (7-36), respectively, in order to improve oral delivery. The resultant GLP-1 analogues, Lys 26,34-DiBiotin-GLP-1 (DB-GLP-1) and Lys 26-Biotin-Lys 34-(Biotin-PEG)-GLP-1 (DBP-GLP-1), were prepared and studied in terms of their chemical, structural, and biological properties. DBP-GLP-1 demonstrated superior proteolytic stability against trypsin, intestinal fluid, and the major GLP-1 inactivation enzyme (dipeptidyl peptidase-IV (DPP-IV)) to native GLP-1 or DB-GLP-1 ( p < 0.001). The in vitro insulinotropic effects of DB-GLP-1 and DBP-GLP-1 showed potent biological activity in a dose-dependent manner, which resembled that of native GLP-1 in terms of stimulating insulin secretion in isolated rat islets of Langerhans. Intraperitoneal glucose tolerance tests (IPGTT) after the oral administration of GLP-1 analogues in diabetic db/db mice demonstrated that DB-GLP-1 and DBP-GLP-1 significantly reduced the AUC 0-180 min of glucose for 3 h by 14.9% and 24.5% compared to that of native GLP-1, respectively ( p < 0.01). In particular, DBP-GLP-1 concentration in plasma rapidly increased 30 min after oral administration in rats, presumably due to improved intestinal absorption. These findings revealed that site-specific biotinylated and biotin-PEGylated GLP-1 is absorbed by intestine and that it has biological activity in vivo. Therefore, we propose that this orally active bioconjugated GLP-1 might be considered as a potential oral antidiabetic agent for type 2 diabetes mellitus.     

Iodoglucagon. Preparation and characterization.

Iodinated derivatives of glucagon containing an average of 1 to 5 g-atoms of 127I per mol have been prepared by reacting the hormone with increasing amounts of iodine monochloride. Their iodoamino acid composition has been determined by ion-exchange chromatography and electrophoresis, following hydrolysis by pronase. Iodination of the two tyrosyl residues occurs first and is nearly complete after addition of a 4-fold molar excess of ICl. Iodination of the single histidyl residue is a later event and does not exceed an average of one atom per residue. Hydrolysis of iodoglucagon by trypsin and subsequent separation of the iodotyrosyl peptides shows that iodine is equally distributed between tyrosyl residues 10 and 13. Crude iodoglucagon containing an average of 1 g-atom of iodine per mol has been resolved into several components of differing iodine content and iodoamino acid composition by chromatography on DEAE-cellulose. Monoiodoglucagon isolated by this procedure shows a single band when analyzed by polyacrylamide gel electrophoresis. Iodoglucagons containing an average of 1 to 4 g-atoms of iodine per mol are more potent than native glucagon in their ability to stimulate adenylate cyclase activity and to bind to glucagon receptors of liver cell membranes of the rat. The maximal increase in biological potency occurring upon iodination is about 5-fold with respect to adenylate cyclase activity, and 2-fold with respect to binding to receptors; tetra and triiodinated derivatives show, respectively, the highest potency. Similar effects occur whether inactivation by liver membranes is inhibited or not, indicating an enhancement in the intrinsic affinity of iodoglucagon for the receptors. Iodination beyong 4 g-atoms per mol slightly decreases the affinity of the hormone for adenylate cyclase and for the receptors. Iodination causes a 2-20 fold decrease in the ability of liver plasma membranes and of blood plasma to inactivate glucagon in vitro; these effects correlate with the degree of iodination. With liver microsomal membranes, a decrease in glucagon inactivation occurs only at iodine contents exceeding 4 g-atoms per mol, and lower degrees of iodination result in opposite effects. Monoiodination causes a 4-6-fold increase in the plasma concentration of glucagon within the first 18 min following a single intrvenous injection of the hormone to rats. More extensive iodination results, in addition, in a marked decrease in the rate of dissappearance of glucagon from the blood. The immunological reactivity of glucagon is little affected by monoidination, but strongly depressed by higher degrees of iodination...     

Generation and characterization of biotinylated recombinant Fab antibody fragment against cortisol

This paper describes the in vivo generation method of biotinylated recombinant Fab antibody fragments. The original molecular vectors for Escherichia coli Fab fragments expression were designed. In vivo biotinylated recombinant antibody Fab fragment against cortisol was generated. The kinetic parameters of interaction of these antibody fragments with cortisol-BSA complex were measured via the biolayer interferometry method. An equilibrium dissociation constant of this interaction K_D is 5.48 × 10^–10 M. The interaction is reversible and it could be competitively inhibited by free cortisol addition. These results could be used in generating of immunoassays for quantitative cortisol determinations. The in vivo biotinylation system under review is universal and suitable for expression of any biotinylated Fab fragments in the E. coli system.     

Preparation of fluorescein-labelled and biotinylated derivatives of polysaccharides for lectin-saccharide binding studies

Fluorescein- and biotin-labelled polysaccharides were prepared using ethylene diamine coupled to a polysaccharide either by carbodiimide reaction to carboxyl group or after periodate oxidation of saccharide residue and the derivative was used for labelling. Labelled hyaluronic acid, chondroitin sulfate and dextran sulfate were prepared.     

Preparation of a gradient biotinylated polyethylene surface to bind streptavidin-FITC

A novel biotinylated gradient surface was designed to bind a streptavidin-FITC. The gradient polyethylene (PE) surface was prepared by a corona treatment with a power increase along the PE length, and successively, biotinylated gradient PE surface was prepared by the reaction of polyethylenimine (PEI) and biotin. Surface properties of the gradient PE surface changed according to PE length and chemical modification. The biotinylated gradient PE surface provided a continuous gradient of streptavidin-FITC through binding of avidin-biotin. The luminosity of fluorescence that corresponds to a streptavidin-bound FITC on the biotinylated gradient PE surface increased gradually on the surface along the PE length.     

Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: cloning and characterization of a C4H from a psoralen producing plant-Ruta graveolens-exhibiting low sensitivity to psoralen inactivation

Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) complete cDNA was cloned from the leaves of Ruta graveolens, a psoralen producing plant. The recombinant enzyme (classified CYP73A32) was expressed in Saccharomyces cerevisiae. Mechanism-based inactivation was investigated using various psoralen derivatives. Only psoralen and 8-methoxypsoralen were found to inactivate C4H. The inactivation was dependent on the presence of NADPH, time of pre-incubation, and inhibitor concentration. Inactivation stoichiometry was 0.9 (+/-0.2) for CYP73A1 and 1.1 (+/-0.2) for CYP73A32. SDS-PAGE analysis demonstrated that [3H]psoralen was irreversibly bound to the C4H apoprotein. K(i) and k(inact) for psoralen and 8-methoxypsoralen inactivation on the two C4H revealed a lower sensitivity for CYP73A32 compared to CYP73A1. Inactivation kinetics were also determined for CYP73A10, a C4H from another furocoumarin-producing plant, Petroselinum crispum. This enzyme was found to behave like CYP73A32, with a weak sensitivity to psoralen and 8-MOP inactivation. Cinnamic acid hydroxylation is a key step in the biosynthesis of phenylpropanoid compounds, psoralen derivatives included. Our results suggest a possible evolution of R. graveolens and P. crispum C4H that might tolerate substantial levels of psoralen derivatives in the cytoplasmic compartment without a depletive effect on C4H and the general phenylpropanoid metabolism.     

Cloning and characterization of Scavidin, a fusion protein for the targeted delivery of biotinylated molecules.

We have constructed a novel fusion protein "Scavidin" consisting of the macrophage scavenger receptor class A and avidin. The Scavidin fusion protein is transported to plasma membranes where the avidin portion of the fusion protein binds biotin with high affinity and forms the basis for the targeted delivery of biotinylated molecules. Subcellular fractionation analysis, immunostaining, and electron microscopy demonstrated endosomal localization of the fusion protein. According to pulse-labeling and cross-linking studies Scavidin is found as monomers (55 kDa), dimers, and multimers, of which the 220-kDa form was the most abundant. The biotin binding capacity and active endocytosis of the biotinylated ligands were demonstrated in rat malignant glioma. Local Scavidin gene transfer to target tissues could have general utility as a universal tool to deliver biotinylated molecules at systemic low concentrations for therapeutic and imaging purposes, whereby high local concentration is achieved.     

Preparation and characterization of armadillo submandibular glycoproteins.

The nine-banded armadillo (Dasypus novemcinctus mexicanus Peters) was chosen for this study so that a comparison could be made of the salivary mucus glycoproteins of an ancient mammalian species with those derived from previously studied, more highly evolved species. Two mucus glycoproteins, armadillo submandibular glycoprotein A and armadillo submandibular glycoprotein B, were prepared from the armadillo submandibular gland by a modification of the method of Tettamanti & Pigman (1968) (Arch. Biochem. Biophys. 124, 41-50). The composition of glycoprotein A is the simplest one among the known mucus glycoproteins. Six amino acids constitute 98.5 mol/100mol of the protein of glycoprotein A and 82 mol/100 mol of that of glycoprotein B. These are serine and threonine (which make up 40-50% of the molar amino acid composition), glutamic acid, glycine alanine and valine. Proline is absent from glycoprotein A and comprises only 2.3% of glycoprotein B. For both glycoproteins, the protein content, as determined by the method of Lowry, Rosebrough, Farr & Randall (1951) (J. Biol. Chem 193, 265-275), with bovine serum albumin as standard, was nearly 60% higher than when determined by the sum of the amino acids. The ratios of total mol of amino acid/total mol of carbohydrate are 1:0.63 for glycoprotein A and 1:0.68 for glycoprotein B, N-Acetylneuraminic acid and N-acetylgalactosamine, in a molar ratio of about 0.35:1.00, are the principal carbohydrates present in both glycoproteins. Neutral sugars seem to be absent from glycoprotein A, but galactose and fucose are present in glycoprotein B. The carbohydrate side chains in glycoprotein A are composed of about two-thirds monosaccharide and one-third disaccharide residues, whereas those of glycoprotein B are more complex. For both glycoproteins, essentially all of the N-acetylgalactosamine was attached O-glycosidically to the hydroxyamino acid residues of the protein core. The linkage of N-acetylneuraminic acid glycoprotein A was extremely sensitive to dilute acid and neuraminidase. Glycoprotein B has chemical properties similar to those of glycoprotein A. However, whereas glycoprotein A was susceptible to both Clostridium perfringens and Vibrio cholerae neuraminidases, only the latter enzyme had an effect on glycoprotein B at pH 4.75. Both glycoproteins were homogeneous by cellulose acetate electrophoresis and ultracentrifugal analyses. The apparent mol.wts. of glycoprotein A and glycoprotein B were 7.8 X 10(4) and 3.1 X 10(4) respectively.     

Preparation, purification and characterization of chlorohaemin.

Preparation of chlorohaemin (CAS 16009-13-5) is performed on the basis of the method of Labbe and Nishida (acetone-acetic acid-SrCl2 method) with some significant modifications. Instead of blood as starting material, a fresh preparation of purified human erythrocytes is used. This avoids any contamination with serum and erythrocyte proteins and lipids of the end product. Special care is taken to remove contaminating globin by introducing some specific purification steps during isolation and recrystallisation. The yield is in the range 65-75% of theory, and the purity of the product is better than 99.9% as shown by elemental analysis and specific tests on various compounds as possible contaminants which originate from the starting material such as lipids and proteins and/or from the different steps of preparation and purification during the procedure. The pure chlorohaemin which is the compound of choice as reference substance for the AHD method in haemoglobinometry is characterized by LSI mass spectrometry (m/z = 616, haemin ion), field desorption-mass spectrometry (m/z = 652), by IR-spectroscopy, and by UV/VIS absorption spectroscopy (pyridine haemochrome spectrum, AHD spectrum).     

Preparation and characterization of bovine aortic actin.

A functional vascular smooth-muscle actin from bovine aorta was purified to homogeneity by an original method and was able to polymerize. Aortic actin is composed of two major isoforms and at least two minor ones. This actin was not phosphorylated by either cyclic AMP-dependent protein kinase or C kinase. The physical properties of aortic actin were found to be very similar to those of skeletal-muscle actin, except for amino acid composition (three tryptophan residues instead of four). The aortic actin and skeletal-muscle actin differ in the extent of activation of the Mg-dependent ATPase of skeletal-muscle myosin.     

Cytochemical characterization of pituitary target cells for biotinylated gonadotropin releasing hormone

These studies describe the application of new cytochemical stains that co-localize a biotin-labeled gonadotropin releasing hormone (GnRH) analog and FSH or LH in the same field or cell. Pituitary monolayer cells were stimulated with the [D-Lys6] GnRH analog or the same analog labeled with biotin. Biotinylated [D-Lys6] GnRH exhibited a higher affinity and was 7-10 X more potent than unlabeled [D-Lys6] GnRH. The avidin-biotin peroxidase complex technique (ABC) was applied to localize the biotinylated GnRH on the cells with the use of a dense black peroxidase substrate. Specificity tests showed that the stain could be eliminated by competition with unlabeled [D-Lys6] GnRH. The GnRH stain was followed by immunocytochemical stains for LH beta, FSH beta or 25-39ACTH with a different peroxidase substrate (amber or orange-red). Stain for GnRH was found on the surfaces of 16% of the cells and 60-90% of the GnRH stained cells also stained for one of the gonadotropins. Most (90-100%) of the gonadotropes showed stain for GnRH. Our studies demonstrate that a potent biotinylated GnRH analog binds cells that can be identified specifically as gonadotropes.     

Two-dimensional crystals of streptavidin on biotinylated lipid layers and their interactions with biotinylated macromolecules.

Streptavidin forms two-dimensional crystals when specifically bound to layers of biotinylated lipids at the air/water interface. The three-dimensional structure of streptavidin determined from the crystals by electron crystallography corresponds well with the structure determined by x-ray crystallography. Comparison of the electron and x-ray crystallographic structures reveals the occurrence of free biotin-binding sites on the surface of the two-dimensional crystals facing the aqueous solution. The free biotin-binding sites could be specifically labeled with biotinylated ferritin. The streptavidin/biotinylated lipid system may provide a general approach for the formation of two-dimensional crystals of biotinylated macromolecules.     

Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

Glucosamine oligomers: 1. Preparation and characterization

Hydrolysis of chitosan in hot concentrated HCl led to chito-oligosaccharides [beta-(1----4) linked 2-amino-2-deoxy-D-glucose]. The time dependence of the distribution was studied. A convenient choice of the conditions for steric exclusion chromatography of these hydrolysates allowed the separation of the first 15 oligomers and of fractions up to DP = 40.