Characterization of plasma triiodophenol binding proteins in vertebrates and tissue distribution of triiodophenol in Rana catesbeiana tadpoles

We investigated the interaction of 2,4,6-triiodophenol (TIP), a potent thyroid hormone disrupting chemical, with serum proteins from rainbow trout (Onchorhynchus mykiss), bullfrog (Rana catesbeiana), chicken (Gallus gallus), pig (Sus scrofa domesticus), and rat (Rattus norvegicus) using a [(125)I]TIP binding assay, gel filtration chromatography, and native polyacrylamide gel electrophoresis. [(125)I]TIP bound non-specifically to proteins in trout serum, specifically but weakly to proteins in bullfrog serum, and specifically and strongly to proteins in chicken, pig, and rat serum samples. Candidate TIP-binding proteins included lipoproteins (220-320kDa) in trout, albumin in bullfrog, albumin and transthyretin (TTR) in chicken and pig, and TTR in rat. TTR in the chicken, pig, and rat serum samples was responsible for the high-affinity, low-capacity binding sites for TIP (dissociation constant 2.2-3.5×10(-10)M). In contrast, a weak interaction of [(125)I]TIP with tadpole serum proteins accelerated [(125)I]TIP cellular uptake in vitro. Intraperitoneal injection of [(125)I]TIP in tadpoles revealed that the radioactivity was predominantly accumulated in the gallbladder and the kidney. The differences in the molecular and binding properties of TIP binding proteins among vertebrates would affect in part the cellular availability, tissue distribution and clearance of TIP.     

Interaction of chlorinated phenols with thyroxine binding sites of human transthyretin, albumin and thyroid binding globulin

Previous results (Brouwer and van den Berg, Toxicol. Appl. Pharmacol., 85 (1986) 301) indicated preferential binding of a hydroxylated metabolite of tetrachlorobiphenyl to transthyretin (TTR) a carrier of thyroxine (T4). In the present study it was investigated whether the T4 binding site of TTR could be occupied specifically by hydroxylated chlorinated aromatic compounds using chlorinated phenol congeners as model compounds in a competition assay with [125I]T4. Chlorinated aromatics such as 2,3-dichlorobenzene and 3,4,3',4'-tetrachlorobiphenyl, and phenols such as 4-hydroxybiphenyl and phenol were inefficient competitors. All chlorinated phenols tested were competitors for the T4 binding site of TTR. The ranking in competition was pentachlorophenol (PCP) greater than trichlorophenols greater than dichlorophenols greater than monochlorophenols. Structures with chlorine in both ortho positions to the hydroxyl group were more efficient competitors. The relative affinity of binding of pentachlorophenol (PCP) to TTR was about twice that of T4. Scatchard analysis showed that PCP mainly decreased the affinity constant K11 while the binding capacity R1 was not altered, indicating a competitive type of inhibition. PCP was also able to compete with T4 sites on albumin with a relative affinity of 0.25. T4 binding to thyroid binding globulin (TBG) was much less affected by interference of PCP (relative affinity 0.001). The results indicate a specific interaction of chlorophenols with the T4 binding site of TTR.     

Insulin-like growth factor (IGF)-I binding to a cell membrane associated IGF binding protein-3 acid-labile subunit complex in human anterior pituitary gland

The binding characteristics of [(125) I]insulin-like growth factor (IGF)-I were studied in human brain and pituitary gland. Competition binding studies with DES(1-3)IGF-I and R(3) -IGF-I, which display high affinity for the IGF-I receptor and low affinity for IGF binding proteins (IGFBPs), were performed to distinguish [(125) I]IGF-I binding to IGF-I receptors and IGFBPs. Specific [(125) I]IGF-I binding in brain regions and the posterior pituitary was completely displaced by DES(1-3)IGF-I and R(3) -IGF-I, indicating binding to IGF-I receptors. In contrast, [(125) I]IGF-I binding in the anterior pituitary was not displaced by DES(1-3)IGF-I and R(3) -IGF-I, suggesting binding to an IGF-binding site that is different from the IGF-I receptor. Binding affinity of IGF-I to this site was about 10-fold lower than for the IGF-I receptor. Using western immunoblotting we were also unable to detect IGF-I receptors in human anterior pituitary. Instead, western immunoblotting and immunoprecipitation experiments showed a 150-kDa IGFBP-3-acid labile subunit (ALS) complex in the anterior pituitary and not in the posterior pituitary and other brain regions. RT-PCR experiments showed the expression of ALS mRNA in human anterior pituitary indicating that the anterior pituitary synthesizes ALS. In the brain regions and posterior pituitary, IGFBP-3 was easily washed away during pre-incubation procedures as used in the [(125) I]IGF-I binding experiments. In contrast, the IGFBP-3 complex in the anterior pituitary could not be removed by these washing procedures. Our results indicate that the human anterior pituitary contains a not previously described tightly cell membrane-bound 150-kDa IGFBP-3-ALS complex that is absent in brain and posterior pituitary.     

Purification of Transthyretin by High Performance Affinity Chromatography from Human Plasma

Abstract

The partial purification of human transthyretin (TTR) by high performance affinity chromatography with the help of other separation techniques is described in the present paper. A new affinity medium was prepared with a monosized (ca. 10μM particle size) macroporous resin as the support and thyroxine (T₄) as the ligand. The purification of TTR was carried out in a few simple steps involving serum precipitation, anion exchange, Thyroxine affinity chromatography and gel filtration. The overall yield was 29% and the refined TTR contained less than 2% impurities as analyzed by RP-HPLC. When TTR was administrated to the culture medium DMEM of liver tumor strain SMMC-7721, a true inhibition of cell growth (ca. 50%) was observed as an actual decrease in cell number over time.     

Isolation and characteristics of dihydropyridine calcium antagonist receptor from rabbit skeletal muscles

Up to 80% of the dihydropyridine receptor is solubilized from transverse tubules of rabbit skeletal muscle by 3-[(3-cholamidopropyl)-dimethylammonium]-2-oxy-1-propane sulfonate (CHAPSO). The DHP receptor is an oligomeric complex made up of two subunits with molecular masses of 160 and 53 kD as shown by DHP-Sepharose affinity chromatography and SDS gel electrophoresis of specifically eluted proteins. The reduction of disulfide bridges of the 160 kD subunit is accompanied by a decrease in its apparent molecular mass up to 125 kD. A method is proposed for preparative isolation of the DHP receptor which is based on ion-exchange chromatography and WGA-Sepharose chromatography. Individual subunits of DHP receptor were isolated by Sepharose 4B gel filtration in SDS; their amino acid composition was determined. Both the 160 and 53 kD subunits are N-glycosylated, and the oligosaccharide portions make up to 7.5% and 6.6%, respectively.     

Isolation and characterization of the major mannose-binding protein in chicken serum

Mannose-binding activity is abundantly present in chicken serum. The major mannose-binding protein has been isolated from chicken serum by affinity chromatography and gel filtration. The protein consists of two subunits of 75 000 and 26 500 daltons. Unlike hepatic lectins or other mannose-binding proteins, this protein does not require calcium for binding mannose-containing glycoconjugates. The chicken serum mannose-binding protein is immunochemically distinct from the chicken hepatic lectin and rabbit serum mannose-binding protein.     

Isolation of a low molecular mass vasoactive intestinal peptide binding protein.

A vasoactive intestinal peptide (VIP) binding protein was purified in active form by detergent solubilization of lung membranes, gel filtration, VIP-Sepharose affinity chromatography, reverse phase high performance liquid chromatography, and anion exchange chromatography. The mass of this protein was estimated at 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 17 kDa by gel filtration. The binding of VIP by this protein was inhibited by Mg2+, covalent cross-linking of [Tyr10-125I]VIP to the protein produced two radioactive bands at 22 and 26 kDa identified by electrophoresis, and the purified protein exhibited saturable and high affinity binding of VIP and the related neuropeptide, rat growth hormone releasing factor.     

The Dopamine Transporter Is Absent in Parkinsonian Putamen and Reduced in the Caudate Nucleus

The neuronal dopamine transporter/uptake site can be covalently labeled with the photoaffinity probe 1-(2-[bis-(4-fluorophenyl) methoxy]ethyl)-4-[2-(4-azido-3-[125I]iodophenyl)ethyl]piperazine [( 125I]FAPP) and visualized following sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Upon photolysis, [125I]FAPP specifically incorporated into a polypeptide of apparent Mr = 62,000 in membranes from both the putamen and the caudate nucleus of control, Alzheimer's, schizophrenia, and Huntington's diseased brain, and following complete deglycosylation, migrated as an Mr approximately 48,000 polypeptide. In parkinsonian postmortem putamen, however, there was no detectable photoincorporation of [125I]FAPP into the ligand binding subunit of the dopamine transporter. [125I]FAPP did specifically label the Mr 62,000 polypeptide of parkinsonian caudate, although with efficiencies of 20-50% of control. The asymmetrical loss of the dopamine transporter in Parkinson's diseased striatum was confirmed in reversible receptor binding experiments using [3H]GBR-12935 (3H-labeled 1-[2-(diphenylmethoxy) ethyl]-4-(3-phenylpropyl)piperazine). In parkinsonian putamen, mazindol competitively inhibited the binding of [3H]GBR-12935 with an estimated affinity (Ki approximately 2,000 nM) 10 times lower than in controls (Ki approximately 30 nM), while the affinity of maxindol for [3H]GBR-12935 binding in the caudate was equal to that seen with controls (Ki approximately 50 nM). The proportion of [3H]GBR-12935 binding sites recognized by mazindol with high affinity in Parkinson's diseased caudate was, however, reduced by 50-80%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaffinity labeling of the alpha 1-adrenergic receptor using an 125I-labeled aryl azide analogue of prazosin

alpha 1-Adrenergic receptor probes, which can be radioiodinated to yield high specific activity radioligands, have been synthesized and characterized. 2-[4-(4-Amino-benzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquin azoline (CP63,155), an arylamine analogue of the selective alpha 1-adrenergic antagonist prazosin, and its iodinated derivative, 2-[4-(4-amino-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP63,789), bind reversibly and with high affinity (KD = 1 nM and 0.6 nM, respectively) to rat hepatic membrane alpha 1-adrenergic receptors. Conversion of [125I]CP63,789 to the aryl azide yields a photolabile derivative, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP65,526), which prior to photolysis binds competitively and with high affinity (KD = 0.3 nM). Binding of [125I]CP63,789 and [125I]CP65,526 (prior to photolysis) is rapid and saturable. Both ligands identify similar alpha 1-adrenergic receptor binding site concentrations as the parent probe, [3H]prazosin. Specific binding by these iodinated ligands is stereoselective and inhibited by a variety of adrenergic agents with a specificity typical of the alpha 1-adrenergic receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [125I]CP65,526-labeled rat hepatic membranes reveal major protein species with molecular weights of 77K, 68K and 59K. Each protein binds adrenergic ligands with stereoselectivity and with a specificity typical of the alpha 1-adrenergic receptor. Inclusion of multiple protease inhibitors during membrane preparation prior to SDS-PAGE does not alter the labeling of these peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Zinc partitions insulin-like growth factors (IGFs) from soluble IGF binding protein (IGFBP)-5 to the cell surface receptors of BC3H-1 muscle cells

Zinc (Zn(2+)) is a multifunctional micronutrient. The list of functions for this micronutrient expanded with the recent discovery that Zn(2+) retains insulin-like growth factors binding proteins (IGFBPs) on the surface of cultured cells, lowers the affinity of cell-associated IGFBPs, and increases the affinity of the cell surface insulin-like growth factor (IGF)-type 1 receptor (IGF-1R). However, currently there is no information concerning the effect of Zn(2+) on soluble IGFBPs. In the current study, the soluble IGFBP-5 secreted by BC(3)H-1 cells is shown to bind approximately 50% more [(125)I]-IGF-II than [(125)I]-IGF-I at pH 7.4. Zn(2+) is shown to depress the binding of both IGF-I and IGF-II to soluble secreted IGFBP-5; [(125)I]-IGF-I binding is affected more so than [(125)I]-IGF-II binding. Zn(2+) acts by lowering the affinity (K(a)) of IGFBP-5 for the IGFs. Scatchard plots are non-linear indicating the presence of high and low affinity binding sites; Zn(2+) affects only binding to the high affinity site. In contrast, Zn(2+) increases the affinity by which either [(125)I]-IGF-I or [(125)I]-R(3)-IGF-I binds to the IGF-1R, but depresses [(125)I]-IGF-II binding to the IGF-type 2 receptor (IGF-2R) on BC(3)H-1 cells. By depressing the association of the IGFs with soluble IGFBPs, Zn(2+) is shown to repartition either [(125)I]-IGF-I or [(125)I]-IGF-II from soluble IGFBP-5 onto cell surface IGF receptors. Zn(2+) was active at physiological doses depressing IGF binding to IGFBP-5 and the IGF-2R at 15-20 microM. Hence, a novel mechanism is further characterized by which the trace micronutrient Zn(2+) could regulate IGF activity.     

Preparation of the N-(4'-hydroxy-[3'-125I]iodophenethyl)-6-(4-O-diethylstilbestryl)hexanam ide for diethylstilbestrol radioimmunoassays.

The synthesis of a radioiodinated diethylstilbestrol (DES) derivative is described. This derivative was prepared by coupling the previously synthesized active ester of 6-(4-O-diethylstilbestryl)hexanoic acid with mono-[125I]iodotyramine in dry tetrahydrofuran (20 to 22 C, 16 hours). The mono-[125I]iodotyramine was prepared using a chloramine-T method and purified by paper electrophoresis. The final product, N-(4'-hydroxy-[3'-125I]iodophenethyl)-6-(4-O- diethylstilbestryl)hexanamide, was separated by thin-layer chromatography (cyclohexane/ethanol/NH4OH 2.5 N/acetone; 40:50:5:20, v/v/v/v); it was stable for 2 months in ethanol at 4 C and had a specific activity higher than 540 Ci/mmol. The [125I]DES amide synthesized was found to retain the immunoreactivity of DES, since it competed with [3H]DES or DES in an in vitro radioimmunoassay system for the binding sites of a rabbit anti-DES antibody; thus, it seems to be capable of replacing the tritiated tracer used so far in DES radioimmunoassays.     

Isolation and characterization of a phosphatidylinositol-glycan-anchor-specific phospholipase D from bovine brain

In recent years an increasing number of proteins has been shown to be membrane-anchored by a covalently attached PtdIns-glycan residue. In mammalian cells little is known about PtdIns-glycan-specific phospholipases which might play a role in the metabolism of PtdIns-glycan-anchored proteins. In order to identify PtdIns-glycan-specific phospholipases, a rapid and sensitive assay for such enzymes was developed using the PtdIns-glycan-anchored amphiphilic membrane form of acetylcholinesterase as substrate. The rate of product formation was monitored by the increase in soluble hydrophilic acetylcholinesterase in the aqueous phase after separation in Triton X-114. With this assay we established the presence of a PtdIns-glycan-specific phospholipase in bovine brain. This enzyme was soluble and could be partially purified by a heat step followed by chromatography on DEAE-cellulose and by gel filtration on Sepharose CL-6B. PtdIns-glycan-specific phospholipase had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 52 nM) and did not degrade either PtdCho or PtdIns. Hydrophobic labeling of the anchor of the substrate with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) caused a marked decrease in the cleavage rate and methylation of the amino group of the glucosamine residue of the anchor decreased the cleavage rate to zero. Using [125I]TID-labeled substrate, diradylglycerol phosphate was identified as the second product showing that the cleavage specificity of PtdIns-glycan-specific phospholipase was that of a phospholipase D. PtdIns-glycan-specific phospholipase D was inhibited by mercurials, omicron-phenanthroline and EGTA. It was stimulated by Ca2+ in micromolar concentrations indicating that PtdIns-glycan-phospholipase D is a Ca2(+)-regulated enzyme.     

Identification of thyroid hormone receptors in rat liver nuclei by photoaffinity labeling with L-thyroxine and triiodo-L-thyronine

Photoaffinity labeling of rat liver nuclear extract with underivatized thyroid hormones was performed after incubation with 1 nM [3',5'-125I]thyroxine ([125I]T4) or [3'-125I]triiodothyronine [( 125I]T3) by irradiation with light above 300 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the covalently photolabeled nuclear extract revealed four distinct hormone binding proteins of molecular masses 96, 56, 45, and 35 kilodaltons (kDa), respectively. Distribution of the hormone among these proteins was similar for T4 and T3. The 56- and 45-kDa proteins were the most prominently labeled. The specificity of the photoattachment of thyroid hormones to these nuclear proteins was verified by the irradiation of eight randomly chosen proteins and two proteins known to have thyroid hormone binding sites, human thyroxine binding globulin and bovine serum albumin. Only the latter two were photolabeled with [125I]T4. Competition studies performed by incubating nuclear extracts with [125I]T4 or [125I]T3 in the presence of increasing amounts of the corresponding unlabeled hormone (10-, 100-, and 1000-fold molar excess) demonstrated that (1) photoattachment of labeled T3 or T4 to the 56- and 45-kDa proteins was inhibited by 67-78% and 73-85%, respectively, after incubation with a 1000-fold molar excess of unlabeled hormone, (2) in the presence of lower molar excesses of the corresponding competitor (10- and 100-fold), photoattachment of labeled T3 or T4 to the 56- and 45-kDa receptors was gradually inhibited to a similar extent on both proteins, and (3) the 35- and 96-kDa proteins, although having thyroid hormone binding sites, display lower binding activities since the inhibition of photoattachment of labeled T3 or T4 by a 1000-fold molar excess of unlabeled hormone did not exceed 30-42% and 26-49%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and functional characterization of high-affinity urotensin II receptors in rat cortical astrocytes

The urotensin II (UII) gene is primarily expressed in the central nervous system, but the functions of UII in the brain remain elusive. Here, we show that cultured rat astrocytes constitutively express the UII receptor (UT). Saturation and competition experiments performed with iodinated rat UII ([(125)I]rUII) revealed the presence of high- and low-affinity binding sites on astrocytes. Human UII (hUII) and the two highly active agonists hUII(4-11) and [3-iodo-Tyr9]hUII(4-11) were also very potent in displacing [(125)I]rUII from its binding sites, whereas the non-cyclic analogue [Ser5,10]hUII(4-11) and somatostatin-14 could only displace [(125)I]rUII binding at micromolar concentrations. Reciprocally, rUII failed to compete with [(125)I-Tyr0,D-Trp8]somatostatin-14 binding on astrocytes. Exposure of cultured astrocytes to rUII stimulated [(3)H]inositol incorporation and increased intracellular Ca(2+) concentration in a dose-dependent manner. The stimulatory effect of rUII on polyphosphoinositide turnover was abolished by the phospholipase C inhibitor U73122, but only reduced by 56% by pertussis toxin. The GTP analogue Gpp(NH)p caused its own biphasic displacement of [(125)I]rUII binding and provoked an affinity shift of the competition curve of rUII. Pertussis toxin shifted the competition curve towards a single lower affinity state. Taken together, these data demonstrate that rat astrocytes express high- and low-affinity UII binding sites coupled to G proteins, the high-affinity receptor exhibiting the same pharmacological and functional characteristics as UT.     

Effect of gastrointestinal proteases on purified human intrinsic factor-vitamin B12 (IF-B12) complex

Intrinsic factor (IF) from human gastric juice was purified and complexed with vitamin B12 (IF-B12 complex) on Sepharose-vitamin B12 affinity matrix. By labeling studies, using [(57)Co] vitamin B12 and (125)I, the specific B12 binding activity of IF was found to be 23 microg B12/mg protein, and the molecular size by gel filtration 60 kDa. Proteolysis of the IF-B12 complex by sequential treatment with pepsin, trypsin, alpha-chymotrypsin and carboxypeptidase A, followed by chromatography of proteolysed complex and IF-B12 showed higher mobility of proteolysed fraction. Gel filtration, however, showed same molecular size for both proteolysed and the IF-B12 complex. On SDS-PAGE, purified IF-B12 appeared as a single band of 60 kDa. The proteolysed complex had higher mobility on SDS-PAGE and did not bind to zirconium phosphate gel. Immunodiffusion with rabbit antisera had positive reaction with IF-B12, but there was no reaction with the proteolysed sample.     

Purification and partial characterization of a plasma inhibitor of tonin

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange of chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/fo) of 1.95 was calculated from the molecular weight and Stokes radius. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat alpha 1-macroglobulin or human alpha 2-macroglobulin.     

Further evidence for the presence of "septide-sensitive" tachykinin binding sites in tissues possessing solely NK(1) tachykinin receptors

Binding experiments performed with [(125)I]-NKA allowed us to demonstrate the presence of "septide-sensitive" specific binding sites on membranes from rat CHO cells transfected with the NK(1) receptor cDNA (CHO-rat-NK1 cells), human astrocytoma U373 MG, or mouse cortical astrocytes, cells which express NK(1) but neither NK(2) nor NK(3) receptors. In all cases, [(125)I]-NKA was specifically bound with high affinity (2 to 5 nM) to a single population of sites. In the three preparations, pharmacological characteristics of [(125)I]-NKA binding sites were notably different from those of classical NK(1) binding sites selectively labelled with [(125)I]-BHSP. Indeed, the endogenous tachykinins NKA, NPK, and NKB and the septide-like compounds such as septide, SP(6-11), ALIE-124, [Apa(9-10)]SP, or [Lys(5)]NKA(4-10) had a much higher affinity for [(125)I]-NKA than [(125)I]-BHSP binding sites. Interestingly, differences were also found in the ratio of B(max) values for [(125)I]-NKA and [(125)I]-BHSP specific bindings from one tissue to another. These latter observations suggest that these two types of NK(1) binding sites are present on distinct NK(1) receptor isoforms (or conformers). Finally, while several tachykinins and tachykinin-related compounds stimulated cAMP formation or increased inositol phosphate accumulation in CHO-rat-NK1 cells, these compounds only increased the accumulation of inositol phosphates in the two other preparations.     

Salt stimulation of serum insulin-like growth factor binding protein activity

Insulin-like growth factors (IGF)-I and -II are bound to carrier or binding proteins in serum. There are at least two classes of binding protein: a high molecular weight complex and a low molecular weight species that is relatively unsaturated. Total binding capacity in serum generally is determined by incubating [125I]IGF with protein that has been stripped of IGF by acid gel filtration. We found that addition of NaCl to the assay increased binding to stripped guinea pig binding protein to about two to four times the level measured in the absence of salt. Stimulation by NaCl was optimal between concentrations of 0.6 and 1.4 M and also was observed when fetal calf or human sera were used as sources of stripped binding protein or when IGF-II was the ligand. Using chloride salts, the order of activity with respect to cations was Na+ greater than K+ greater than Li+. Na2HPO4 at 0.6 M was as stimulatory as 1.2 M NaCl but 0.6 M Na2SO4 was less effective. NH4HCO3 was as effective as NaCl at 0.6 M. Scatchard plots of data from competitive dilution experiments with [125I]IGF-I and unlabeled IGF-I showed that binding was heterogeneous in the absence of 0.6 M NaCl but linear in its presence. NaCl did not stimulate binding when whole serum was used, but after gel filtration of serum on Sephacryl 200 at pH 8, which does not dissociate IGFs from binding protein, binding to individual fractions was stimulated three- to fourfold by NaCl. Fractions stimulated included those containing the large complex or the unsaturated binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-purification of A1 adenosine receptors and guanine nucleotide-binding proteins from bovine brain

A1 adenosine receptors and guanine nucleotide-binding proteins (G proteins) solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate have been co-purified from bovine cerebral cortex. A portion of solubilized receptors which displays high affinity GTP-sensitive agonist binding (40-50%) adheres tightly to agonist affinity columns composed of N6-aminobenzyladenosine-agarose. A1 adenosine receptors and G proteins are rapidly and selectively coeluted from agonist columns by the addition of 8-p-sulfophenyltheophylline, but only in combination with Mg2+-GTP or N-ethylmaleimide, agents which lower the affinity of receptors for agonists. Purified receptors and G protein alpha-subunits can be detected with the potent A1-selective antagonist radioligand, [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentylxanthine (125I-BW-A844U) and [35S]guanosine 5'-3-O-(thio)triphosphate [( 35S]GTP gamma S), respectively. Pretreatment of solubilized receptors with 0.1 mM N-ethylmaleimide or 0.1 mM R-phenylisopropyladenosine abolishes adsorption of receptors and G proteins to affinity columns. Following removal of 8-p-sulfophenyltheophylline and GTP, purified receptors bind agonists (2 sites) and antagonists (1 site) with affinities similar to crude soluble receptors and typical of A1 receptors. Some receptors may be denatured as a result of purification since only 23% of the radioligand binding sites which adhere to the affinity column can be detected in the eluate. The Bmax of purified receptors, 820 +/- 100 pmol/mg protein (n = 3) is 1800-fold higher than crude soluble receptors. The specific activity of [35S]GTP gamma S binding sites in affinity column eluates is 4640 pmol/mg protein. Assuming a 1:1 stoichiometry, this specific activity indicates that receptor-G protein complexes are greater than 50% pure following affinity chromatography. The photoaffinity labeled purified receptor was identified by polyacrylamide gel electrophoresis as a single band with a molecular mass of 35 kDa which when deglycosylated undergoes a characteristic shift in molecular mass to a sharp band at 32 kDa. In addition to the receptor, silver staining revealed polypeptides with molecular masses of 39 and 41 kDa, which are ADP-ribosylated by pertussis toxin, and 36 kDa corresponding to G protein beta-subunits.     

Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties

6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)(-1), was purified 344-fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and KM and Vmax values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G-200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Ki values and inhibition types were estimated by means of Lineweaver-Burk graphs obtained for NADPH and CO2 products.