Carbohydrate Analysis of the B2 Bradykinin Receptor from Rat Uterus

Abstract: The B₂ bradykinin receptor purified from rat uterus has an apparent molecular mass of 81 kDa. This is higher than the value of 42 kDa estimated from the sequence data of rat and human B₂ receptors. Carbohydrate analysis of the rat B₂ bradykinin receptor indicated that it was a sialoglycoprotein with three N-linked complex oligosaccharide side chains. This was consistent with the sequence, which has three potential glycosylation sites. The receptor did not appear to possess O-linked carbohydrate side chains. Removal of the N-linked carbohydrates with endo-β- N -acetylglucosaminidase yielded a core protein of 42–44 kDa. The presence of these N-linked carbohydrates explains the discrepancy between the molecular size of the purified receptor protein and that estimated from the sequence. The sequence of the rat receptor suggests an isoelectric point of about pH 7.0, but the purified receptor had an isoelectric point of pH 4.5–4.7. Sialic acid residues on the N-linked side chains are likely to be responsible for the acidic nature of the rat receptor. Carbohydrate does not appear to play a role in ligand-receptor interactions, as deglycosylation did not alter the affinity of the B₂ bradykinin receptor for bradykinin or the B₂-selective antagonist HOE-140.     

B1 and B2 Bradykinin Receptors Encoded by Distinct mRNAs

Abstract: Bradykinin receptors have been subdivided into at least two major pharmacological subtypes, B₁ and B₂. The cDNAs encoding functional B₂ receptors have recently been cloned, but no molecular information exists at present on the B₁ receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the B₁ and B₂ types of receptor. We showed previously that the Human fibroblast cell line W138 expresses both B₁ and B₂ receptors. In this report, we describe oocyte expression experiments showing that the B₁ receptor in W138 human fibroblast cells is encoded by a distinct mRNA ∼2 kb shorter than that encoding the B₂ receptor. We have used an antisense approach in conjunction with the oocyte expression system to demonstrate that the two messages differ in sequence at several locations throughout the length of the B₂ sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B₁-type pharmacology may arise from two independent molecular mechanisms.     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

Purification of the D-2 dopamine receptor from bovine striatum

The D-2 dopamine receptor has been purified 21500 fold from bovine striatal membranes. Solubilized receptor preparation was partially purified by affinity chromatography on a haloperidol adsorbent followed by gel filtration on a Sephacryl S-300 column. The fractions eluted from this column which contained the ligand binding activity were further chromatographed on wheat germ agglutinin conjugated to Sepharose. The resulting receptor preparation displays a major polypeptide band of an apparent molecular weight of 92 kDa, and exhibits a specific binding activity of 2490 pmol spiperone per mg protein. This purified receptor preparation can reabsorb specifically to the haloperidol affinity column indicating that the 92 kDa polypeptide represents the ligand binding unit of the D-2 dopamine receptor.     

Isolation of Putative Benzodiazepine Receptors from Rat Brain Membranes by Affinity Chromatography

Abstract: The benzodiazepine receptor from rat brain was solubilised and purified 5200-fold by affinity chromatography. The affinity column contained an immobilized benzodiazepine (delorazepam) and biospecific elution with 6 m m -chlorazepate was achieved. The purified receptor is apparently homogeneous in SDS-polyacrylamide gel electrophoresis. The native protein had a molecular weight of 240,000, and the subunit one of 60,000. The dissociation constant ( K _D) is 8 n m for [³H]diazepam. A correlation exists between the value of affinity obtained for benzodiazepine derivatives and their known pharmacological effectiveness.     

Affinity purification of pancreastatin receptor-Gq/11 protein complex from rat liver membranes

Pancreastatin, a chromogranin A derived peptide, exerts a glycogenolytic effect on the hepatocyte. This effect is initiated by binding to membrane receptors which are coupled to pertussis toxin insensitive G proteins belonging to the Gq/11 family. We have recently solubilized active pancreastatin receptors from rat liver membranes still functionally coupled to G proteins. Here, we have purified pancreastatin receptors by a two-step procedure. First, pancreastatin receptors with their associated Gq/11 regulatory proteins were purified from liver membranes by lectin absorption chromatography on wheat germ agglutinin immobilized on agarose. A biotinylated rat pancreastatin analog was tested for binding to liver membranes before using it for affinity purification. Unlabeled biotinylated rat pancreastatin competed for 125I-labeled [Tyr0]PST binding to solubilized receptors with a Kd = 0.27 nM, comparable to that of native pancreastatin. The biotinylated analog was immobilized on streptavidin-coated Sepharose beads and used to further affinity purify wheat germ agglutinin eluted receptor material. Specific elution at low pH showed that the receptor protein was purified as an 80-kDa protein in association with a G protein of the q/11 family, as demonstrated by specific immunoblot analysis. The specificity of the receptor band was assessed by chemical cross-linking of the purified material followed by SDS-PAGE and autoradiography. In conclusion, we have purified pancreastatin receptor as a glycoprotein of 80 kDa physically associated with a Gq/11 protein.     

Occurrence of Two Types of Ca2+-Dependent Protein Kinases in the Cytosol Fraction of the Brain

Abstract— Two types of Ca²⁺-dependent protein kinases were demonstrated and partially purified from the cytosol fraction of rat brain by DEAE-cellulose, Sephadex G-200, and calmodulin-affinity column chromatography, using endogenous proteins and chicken gizzard myosin light chains as substrates. The molecular weights of the enzymes were 88,000 (peak I) and 120,000 (peak II) on gel filtration. Peak I had no affinity for calmodulin, whereas peak II had a high affinity for it, with a K _a value of 16.7 n m . The K _a values of peaks I and II for Ca²⁺ were 2.4 and 1.6 μ m , respectively.     

Purification and characterization of two glycoproteins from oligodendroglial plasma membranes

Two major glycoproteins of 99 kDa and 77 kDa have been purified from oligodendroglial plasma membranes. These two glycoproteins exhibit intense binding to the lectin, wheat germ agglutinin. The 99-kDa and 77-kDa glycoproteins were purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography and SDS-polyacrylamide pore gradient gel electrophoresis. Re-electrophoresis of excised gel slices containing the two glycoproteins demonstrated their apparent homogeneity. The isoelectric points of the 99-kDa and 77-kDa glycoproteins were 6.15 and 6.00, respectively. Peptide mapping revealed structural differences between the two glycoproteins. Lectin binding studies with radiolabeled succinylated wheat germ agglutinin demonstrated that the binding of the 99-kDa and 77-kDa glycoproteins to wheat germ agglutinin was due to N-acetyl-D-glucosamine residues in the oligosaccharide side-chains.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

The Role of Vitamin B12 Binding In the Uptake of the Vitamin By Euglena Gracilis

The uptake of [⁵⁷Co]B₁₃ (cyanocobalamin) by Euglena gracilis strain Z (ATCC 12716) occurred in 2 distinct phases-an initial rapid phase followed by a slower secondary phase. This secondary phase appeared after the saturation of the binding sites involved in the initial rapid phase and was energy-dependent and completely inhibited by 2,4-dinitrophenot, KCN and sodium azide. the subcellular localization of labeled cyanocobalamin taken up by the cell was mostly contained in the chloroplast fraction. the time course and the saturation kinetics of B₁₂ uptake by purified chloroplast fraction indicated that this fraction and the intact cell had a similar affinity for the vitamin B₁₂. This suggested that the chloroplasts contained the binding sites for vitamin ₁₂ and might regulate the uptake process in the intact cell. the kinetic properties of the overall ₁₂ uptake mechanism suggested that the initial phase represent the binding of vitamin ₁₂ to the available sites on the chloroplast. the secondary phase may represent the de novo synthesis of new binding sites.     

Glycoproteins in the whorls of membrane produced by oligodendroglia in culture

Two glycoproteins of 99 kDa and 77 kDa which exhibit intense binding to wheat germ agglutinin have been purified from the whorls of membrane produced by oligodendroglia in culture. The whorls of membrane were isolated by gradient centrifugation from purified bovine oligodendroglia maintained in culture. The two glycoproteins were solubilized from the membranes using a non-ionic detergent and purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography, and SDS-polyacrylamide pore gradient gel electrophoresis. HPLC peptide mapping of the 99-kDa and 77-kDa glycoproteins revealed structural differences between the two proteins. Peptide mapping suggested that the 99-kDa glycoprotein from the whorls of membrane may be homologous to that from the plasma membranes. The 77-kDa glycoproteins from both sets of membrane may also be structurally related. Lectin binding studies showed that both glycoproteins from the whorls of membrane bound to wheat germ agglutinin, succinylated wheat germ agglutinin, concanavalin A, and lentil lectin, indicating the presence of high mannose and hybrid type oligosaccharide side-chains.     

Purification of an active receptor for acidic and basic fibroblast growth factor from bovine retina.

Acidic and basic fibroblast growth factors (FGFs) influence cell division and differentiation in retina cells. Their effects are thought to be mainly mediated through stimulation of a specific membrane receptor and subsequent generation of an intracellular signal pathway. In this study, we purified a FGF receptor of 130 kDa from bovine neural retina using wheat germ agglutinin affinity chromatography followed by FGF-affinity chromatography. The isolated receptor showed ligand binding activity with dissociation constants of 0.8 nM and 2 nM for aFGF and bFGF, respectively. Furthermore, binding of aFGF and bFGF to purified receptor resulted in self-phosphorylation, demonstrating that the isolated receptor had an unaltered intrinsic kinase activity.     

Purification of opioid receptor in the presence of sodium ions.

Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents CHAPS and digitonin in the presence of protease inhibitors and 1 M NaCl. The solubilized receptor bound mu-opioid agonists and antagonists with affinities similar to those of native membrane receptor. The affinity of solubilized receptor for the agonist PL017 was greatly reduced by GTP gamma S, suggesting the receptor is still associated with G-protein. The solubilized material was passed through an opioid antagonist (10cd) affinity column and a wheat germ agglutinin column, set up in series, to obtain a partially purified receptor preparation. This partially purified material bound mu-agonist with low affinity and the binding affinity was no longer affected by GTP gamma S. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. Binding of opioid antagonist [3H]diprenorphine to the partially or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed saturable and stereospecific binding for opioid ligands, was predominantly of the mu-type, and exhibited as a diffuse band with a medium molecular mass of 62 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The average specific binding activity of the purified receptor was 18.8 +/- 2.3 pmol/micrograms protein, a value close to the theoretical estimation.     

Enhancement of vitamin B6 levels in seeds through metabolic engineering

As a versatile cofactor for many enzymes catalyzing important biochemical reactions, vitamin B₆ is required for all cellular organisms. In contrast to bacteria, fungi and plants, which have the ability to synthesize vitamin B₆ de novo , animals have to take up the vitamin from their diet. Plants are the major source of vitamin B₆ for animals. The recent identification of vitamin B₆ biosynthetic enzymes PDX1 and PDX2 in plants makes it possible to regulate the biosynthesis of this important vitamin. In this study, we generated Arabidopsis plants overexpressing the PDX1 and/or PDX2 gene and used a liquid chromatography/mass spectrometry/mass spectrometry method to determine the levels of different forms of vitamin B₆ in these transgenic plants. It was found that expression of the PDX genes under control of the CaMV 35S promoter caused only a limited increase in pyridoxine contents in dry seeds but not in shoots or roots. When using the Arabidopsis seed-specific 12S promoter to drive the expression of the PDX genes, the levels of vitamin B₆ increased more than twofold in transgenic plants. Our work demonstrates that it is feasible to enhance vitamin B₆ content in seeds by metabolic engineering.     

Isolation and Characterization of a Cytosolic Phospholipase A2 from Bovine Adrenal Medulla

Abstract: We have recently demonstrated that bovine adrenal medulla contains a soluble phospholipase A₂ (PLA₂), which is localized in the cytosol. In the present study, this PLA₂ was purified 1,097-fold using sequential concanavalin A, hydrophobic interaction, anion exchange, gel filtration, and an additional anion exchange chromatography. The enzyme is activated over the range of 20–1,000 µ M Ca²⁺ and has a pH optimum near 8.0. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a molecular mass of 26 kDa and an isoelectric point of 4.6 as revealed by isoelectric focusing. The cytosolic PLA₂ is not inhibited by NaCl, and the enzymatic activity is stimulated at low concentrations of Triton X-100 (0.01%) and deoxycholate (1 m M ) but inhibited at higher concentrations (0.1% and 3 m M , respectively) of these detergents. Furthermore, heat treatment (57°C, 5 min) reduced the enzymatic activity by 80%, whereas glycerol (30%) increased the activity. p -Bromophenacylbromide, a frequently used irreversible inhibitor of type II PLA₂, has little effect until 100 µ M , and 2–10 m M dithiothreitol totally inactivated the enzyme. The purified PLA₂ displays a preference for phosphatidylcholine as a substrate but hydrolyzes phospholipid substrates with arachidonic acid or linoleic acid esterified at the sn -2 position to the same extent. It is concluded that the chromaffin cell cytosolic PLA₂, which was isolated and characterized in this study, represents a type of PLA₂ that has not been formerly reported in chromaffin cells. Additional research on the chromaffin cell cytosolic PLA₂ will help to reveal whether the enzyme is important for exocytosis.     

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (β-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K _m of 6.5 mg ml⁻¹ and a V _max of 1440 U (mg protein)⁻¹.     

Sucrose synthase from wheat leaves. Comparison with the wheat germ enzyme

Sucrose synthase (UDP glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) was partially purified from wheat ( Triticum aestivum L. cv. San Agustin INTA) leaves and its properties compared with the wheat germ enzyme. The leaf enzyme moved faster in polyacrylamide gel electrophoresis, was more sensitive to SH reagents and crossreacted more slowly with antibody prepared towards the germ enzyme. Kinetic constants were of the same order for all substrates. UDP was a strong inhibitor of the synthesis reaction. MgCl₂ stimulated this reaction and partially reversed UDP inhibition. Molecular weight determined by gel filtration was 380 and 370 kdalton for the leaf and germ enzymes respectively. Both enzymes presented forms of higher molecular weight estimated to around 800 and 1000 kdalton. Neither sucrose synthase from leaves nor from germ were affected by fructose 6-P, fructose 1,6—P₂, glucose 1—P, glucose 6—P, fructose 2,6—P₂ and cAMP.     

Novel Neonicotinoid-Agarose Affinity Column for Drosophila and Musca Nicotinic Acetylcholine Receptors

Abstract: Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [³H]IMI with K _D values of 1–2 n M and B _max values of 560–850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an α-bungarotoxin (α-BGT)-agarose affinity column are known to be α-subunit homooligomers. This study uses 1 - [ N - (6 - chloro - 3 - pyridylmethyl) - N - ethyl]amino - 1 - amino-2-nitroethene (which inhibits [³H]IMI binding to Drosophila and Musca head membranes at 2–3 n M ) to develop a neonicotinoid-agarose affinity column. The procedure—introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamide gel electrophoresis—gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the α-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with ¹²⁵I-α-BGT-4-azidosalicylic acid gives a labeled derivative of 66–69 kDa. The yield is 2–5 µg of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.     

Cell wall component and mycotoxin moieties involved in the binding of fumonisin B1 and B2 by lactic acid bacteria

Aims:  The ability of lactic acid bacteria (LAB) to bind fumonisins B₁ and B₂ (FB₁, FB₂) in fermented foods and feeds and in the gastrointestinal tract could contribute to decrease their bioavailability and toxic effects on farm animals and humans. The aim of this work was to identify the bacterial cell wall component(s) and the functional group(s) of FB involved in the LAB–FB interaction.
Methods and Results:  The effect of physicochemical, enzymatic and genetic treatments of bacteria and the removal/inactivation of the functional groups of FB on toxin binding were evaluated. Treatments affecting the bacterial wall polysaccharides, lipids and proteins increased binding, while those degrading peptidoglycan (PG) partially decreased it. In addition, purified PG from Gram-positive bacteria bound FB in a manner analogue to that of intact LAB. For FB, tricarballylic acid (TCA) chains play a significant role in binding as hydrolysed FB had less affinity for LAB.
Conclusions:  Peptidoglycan and TCA are important components of LAB and FB, respectively, involved in the binding interaction.
Significance and Impact of the Study:  Lactic acid bacteria binding efficiency seems related to the peptide moiety structure of the PG. This information can be used to select probiotics with increased FB binding efficiency.     

Cloning, Characterization, and Chromosomal Localization of a Human 5-HT6 Serotonin Receptor

Abstract: We describe the cloning and characterization of a human 5-HT₆ serotonin receptor. The open reading frame is interrupted by two introns in positions corresponding to the third cytoplasmic loop and the third extracellular loop. The human 5-HT₆ cDNA encodes a 440-amino-acid polypeptide whose sequence diverges significantly from that published for the rat 5-HT₆ receptor. Resequencing of the rat cDNA revealed a sequencing error producing a frame shift within the open reading frame. The human 5-HT₆ amino acid sequence is 89% similar to the corrected rat sequence. The recombinant human 5-HT₆ receptor is positively coupled to adenylyl cyclase and has pharmacological properties similar to the rat receptor with high affinity for several typical and atypical antipsychotics, including clozapine. The receptor is expressed in several human brain regions, most prominently in the caudate nucleus. The gene for the receptor maps to the human chromosome region 1p35–p36. This localization overlaps that established for the serotonin 5-HT_1Dα receptor, suggesting that these may be closely linked. Comparison of genomic and cDNA clones for the human 5-HT₆ receptor also reveals an Rsa I restriction fragment length polymorphism within the coding region.