DEAMIDATED ACTIVE INTERMEDIATES IN THE IRREVERSIBLE ACID DENATURATION OF RIBONUCLEASE-A

A study has been made on the changes in the enzymatic activity of Ribonuclease-A**-(RNase-A) exposed to highly acidic (pH < 1) aqueous environment. Irreversible alterations of activity were observed when the protein was exposed to an acidic medium for a long period (20 to 60 h). Even prior to these changes in activity RNase-A was found to form intermediates which had very nearly the same activity as the native protein. The primary process in the acid denaturation of RNase-A was observed to be deamidation of the protein leading to the formation of active chromatographically distinct derivatives. The initial product of deamidation, a monodeamidated derivative, has been isolated by chromatography on Amberlite XE-64. This initial deamidation reaction proceeded with very high specificity. The subsequent deamidation reaction is comparatively slower, so that nearly 50% of the native protein could be converted to this derivative before any subsequent deamidation took place. This monodeamidated derivative has been designated RNase-Aa₁. The conversion of RNase-A to RNase-Aa₁ was not accompanied by any changes in the primary structure other than the observed deamidation. Apart from the differences in chromatographic and electrophoretic mobilities, RNase-Aa₁ was found to have very nearly the same activity and physico-chemical properties as the native enzyme. Significance of this specific and faster deamidation of RNase-A in this denaturing medium as well as the biological significance of such deamidation reactions of proteins are discussed.     

Characterization of human growth hormone from acromegalic pituitary tumors

Human growth hormone (HGH) was extracted from acromegalic pituitary tumors at pH 10.5 and precipitated with ammonium sulfate at 20–40% saturation. It was purified on a Sephadex G-100 column to yield monomeric HGH. The tumor-HGH was indistinguishable from the authentic one in polyacrylamide gel electrophoresis at pH 8.3 or in the presence of sodium dodecyl sulfate, high-performance liquid chromatography, radioimmunoassay, peptide map, amino acid composition and N-terminal partial amino acid sequence. The tumor-HGH is active in the tibia assay and body weight gain test in hypophysectomized rats with comparable potency to that of the authentic sample.     

HUMAN SOMATOTROPIN:

The amino terminal 54 residue peptide fragment of human somatotropin, [Cys(Cam)⁵³]-HGH-(I-54), has been synthesized by the solid-phase method. The symmetrical anhydride and active ester coupling methods were used exclusively. The synthetic product was purified by gel filtration, isoelectric focusing, and partition chromatography. It was found to be homogeneous by six additional criteria. In complement fixation experiments the synthetic product was immunologically active with antisera to HGH and [Cys(Cam)⁵³]-HGH-(I-134). Antiserum raised against the synthetic product was immunologically active in the homologous assay and with HGH, [Cys(Cam)⁵³]-HGH-(I-134), and [Cys(Cam)⁵³]-HGH-(15–125). The synthetic fragment exhibited 53% of the activity of [Cys(Cam)⁵³]-HGH-(I-134) in the rat tibia assay.     

Studies on the mechanism of aspartic acid cleavage and glutamine deamidation in the acidic degradation of glucagon

In this study, the polypeptide hormone glucagon was used as a model to investigate the mechanisms of aspartic acid cleavage and glutaminyl deamidation in acidic aqueous solutions. Kinetic studies have shown that cleavage at Asp-21 occurred at significantly slower rates than at Asp-9 and Asp-15 while deamidation rates were similar at the three Gln residues. The role of side-chain ionization in the cleavage mechanism was investigated by determining the pK(a) values of the three Asp residues using TOCSY and NOESY NMR methods. The role of proton transfer was investigated using kinetic solvent isotope effect studies (KSIE). The pK(a) values for the sidechains of Asp-9, Asp-15, and Asp-21 were found to be 3.69, 3.72, and 4.05 respectively. No kinetic solvent isotope effect was observed for the cleavage reaction whereas an inverse effect was observed for deamidation. Based on the lack of sequence effects, pH-rate behavior, and KSIE, the deamidation mechanism was proposed to involve direct hydrolysis of the amide side-chain by water. Based on substrate ionization, pH-rate profiles, and KSIE, the proposed mechanism for Asp cleavage involved nucleophilic attack of the ionized side-chain carboxylate on the protonated carbonyl carbon of the peptide bond to give a cyclic anhydride intermediate.     

Isolation and characterization of monodeamidated derivatives of bovine pancreatic Ribonuclease A

The isolation and characterization of the initial intermediates formed during the irreversible acid denaturation of enzyme Ribonuclease A are described. The products obtained when RNase A is maintained in 0.5 M HCl at 30° for periods up to 20 h have been analyzed by ion-exchange chromatography on Amberlite XE-64. Four distinct components were found to elute earlier to RNase A; these have been designated RNase Aa₂, Aa_1c, Aa_1b, and Aa_1a in order of their elution. With the exception of RNase Aa₂, the other components are nearly as active as RNase A. Polyacrylamide gel electrophoresis at near-neutral pH indicated that RNase Aa_1a, Aa_1b, and Aa_1c are monodeamidated derivatives of RNase A; RNase Aa₂ contains, in addition, a small amount of a dideamidated component. RNase Aa₂, which has 75% enzymic activity as compared to RNase A, consists of dideamidated and higher deamidated derivatives of RNase A. Except for differences in the proteolytic susceptibilities at an elevated temperature or acidic pH, the monodeamidated derivatives were found to have very nearly the same enzymic activity and the compact folded structure as the native enzyme. Fingerprint analyses of the tryptic peptides of monodeamidated derivatives have shown that the deamidations are restricted to an amide cluster in the region 67–74 of the polypeptide chain. The initial acid-catalyzed deamidation occurs in and around the 65–72 disulfide loop giving rise to at least three distinct monodeamidated derivatives of RNase A without an appreciable change in the catalytic activity and conformation of the ribonuclease molecule. Significance of this specific deamidation occurring in highly acidic conditions, and the biological implications of the physiological deamidation reactions of proteins are discussed.     

Pathways of chemical degradation of polypeptide antibiotic bacitracin

We described the main pathways of bacitracin (Bc) decomposition, chromatographically set the position of its major degradation products and evaluated microbiological activity of isolated components of Bc and its degradation products. All processes of Bc decomposition under stress and accelerated test conditions were monitored with HPLC, performed mainly on a new type reversed-phase (RP-18e) monolithic silica column (Chromolith) enabling fast separation times and some of them also on conventional HPLC columns. Diode array detection, preparative HPLC and FAB mass spectrometry were used for identification of individual Bc components. We found that the major decomposition mechanism in water solutions of Bc is oxidation, and in alkaline solutions, deamidation. In oxidation process the components B1, B2 and B3 and A are oxidized into their corresponding oxidative products H1, H2, H3 and F respectively by the same mechanism. A detailed study of oxidative degradation products revealed that HPLC separation with an acid mobile phase caused splitting of peaks of components H2, H3 and F into two peaks but the peak of component H1 did not split due to its special structural properties. For the component A we confirmed gradual formation of desamido product through an intermediate. We found oxidative degradation products of Bc to be relatively stable, and desamido degradation products to be rather unstable. The estimation of kinetics of Bc decomposition was presented with a semi-quantitative model. Microbiological activity of individual isolated active components of Bc was established and the negligible antimicrobial activity of the degradation products was confirmed.     

Effect of protamine on the solubility and deamidation of human growth hormone

The effect of protamine on the solubility and deamidation of human growth hormone (hGH) was investigated. Protamine is an extremely basic peptide. It is isolated from sperm cells of salmon where it can build a complex with DNA due to electrostatic interactions. We hypothesize a similar electrostatic effect between negatively charged hGH and positively charged protamine residues. Arising cationic complexes are stabilized by electrostatic repulsion. This stabilizing complexation allows solubilization of hGH down to a pH of 5.4 (pI 5.3) at concentrations of 3.4 mg/ml. The minimal solubilizing molar ratio between hGH and protamine was found to be at least 1:23 (hGH:protamine) by turbidity and dynamic light scattering (DLS) measurements. Complexation was characterized by small-angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC). Electrostatic binding of protamine to hGH was also observed by a reversal in surface charge, as shown by zeta potential measurements. The presence of protamine did not alter the conformational structure of hGH which was determined by circular dichroism (CD) spectroscopy. A pH of 5.4 is known to slow deamidation of hGH and consequently retardation of hGH deamidation could be detected after 3 months storing by reversed-phase high-performance liquid chromatography (RP-HPLC).     

麦白霉素与麦迪霉素及其组分的体内外生物活性分析与比较

本文用高效液相法及微生物检定法分析了麦白霉素和麦迪霉素各组分的含量及其体内外生物活性,结果表明,二者的A_1、A_2、A_6组分的百分含量有一定差异;A_1组分含量,前者为40％,后者为80％,而A_2、A_3组分则相反前者比后者大3～7倍。从检菌的MIC、兔体内血药浓度及模拟胃液中稳定性比较,各组分及其制剂未发现有明显差异,由于生产的剂型不同影响体内吸收及酸中的稳定性。     

The effect of pizotifen, a serotonin antagonist, and of pirenzepine, a muscarinic antagonist, on hormonal responses to metoclopramide in healthy subjects

To examine whether and how muscarinic or serotoninergic properties might contribute to the hormone stimulating action of the dopamine antagonist metoclopramide, the effect of oral single-dose pretreatment with pirenzepine (Gastrozepin) 50 mg, a muscarinic antagonist, pizotifen (Sandomigran) 1 mg, a serotonin antagonist, or placebo on the responses of prolactin, aldosterone, plasma renin activity (PRA), cortisol (hydrocortisone) and human growth hormone (HGH) to the i.v. injection of metoclopramide 10 mg was studied in 6 young healthy volunteers. For comparison, a mere placebo study was added. Whereas aldosterone response to metoclopramide and PRA remained unchanged, prolactin response was decreased by pizotifen from 43 +/- 6.5 to 24 +/- 9 ng/ml, p less than 0.05. Pizotifen decreased base-line cortisol from 10.5 +/- 0.5 to 6.6 +/- 0.6 microgram/dl and prevented the increase in cortisol after metoclopramide in responsive subjects. Both pizotifen and pirenzepine decreased HGH responsiveness to metoclopramide. It is concluded that cortisol and, partly, prolactin stimulation by metoclopramide are due to its serotoninergic properties and that HGH responsiveness to metoclopramide becomes explainable by both the serotoninergic and muscarinic potencies of the drug.     

Isolation and identification of cyclic imide and deamidation products in heat stressed pramlintide injection drug product

This report summarizes the identification of six cyclic imide [Asu] and two deamidation products from a sample of pramlintide final drug product that had been stressed at 40 degrees C for 45 days. The pramlintide degradation products were isolated by cation exchange high-performance liquid chromatography (HPLC) followed by reversed-phase HPLC. The isolated components were characterized by mass spectrometry (MS), tandem MS (MS/MS) and when necessary, by enzymatic (thermolysin) digestion followed by liquid chromatography/mass spectrometry (LC/MS) and sequence analysis. The isolated products were identified as [Asu14]-pramlintide, [Asu21]-pramlintide, [Asu22]-pramlintide, [Asu35]-pramlintide, [1-21]-succinimide-pramlintide, and [1-22]-succinimide-pramlintide. Also identified were [Asp35]-pramlintide, the deamidation product of pramlintide at Asn35, and [Tyr37-OH]-pramlintide, the deamidation product of the pramlintide amidated C-terminal Tyr. Together these data support those presented earlier (C. Hekman et al., Isolation and identification of peptide degradation products of heat stressed pramlintide injection drug product. Pharm Res 1998;15:650-9) indicating that the primary mechanism of degradation for pramlintide in this pH 4.0 formulation is deamidation, with six of the eight possible deamidation sites observed to undergo deamidation. Gln-10 and Asn-31 are the only two residues subject to deamidation for which none is observed. The data indicate that the cyclic imide products account for approximately 20% of the total thermal degradation while the deamidation products account for 64%. The remaining degradation is due to peptide backbone hydrolysis.     

Tetracaine induces apoptosis through a mitochondrion-dependent pathway in human corneal stromal cells in vitro

Purpose: Tetracaine is a local anesthetic widely used in ocular diagnosis and ophthalmic surgery and may lead to some adverse effects and complications at a clinical dose. To assess the cytotoxicity and molecular toxicity mechanisms of tetracaine, we used human corneal stromal (HCS) cells as an in vitro model to study the effects of tetracaine on HCS cells.

Materials and methods: The cytotoxicity of tetracaine on HCS cells was investigated by examining the changes of cell growth, morphology, viability and cell cycle progressing when HCS cells were treated with tetracaine at concentrations from 10?g/L to 0.078125?g/L. To prove the hypothesis that the cytotoxicity of tetracaine on HCS cells was related with apoptosis induction, we further detected multiple changes in HCS cells, including plasma membrane (PM) permeability, phosphatidylserine (PS) orientation, genomic DNA integrality, and cell ultrastrcuture after treated with tetracaine. Furthermore, the pro-apoptotic signalling pathway induced by tetracaine was explored through detecting the activation of various caspases, the changes of mitochondrial transmembrane potential (MTP), the expression level of Bcl-2 family proteins and the amount of mitochondria-released apoptosis regulating proteins in cytoplasm.

Results: Tetracaine at concentrations above 0.15625?g/L had a dose- and time-dependent cytotoxicity to HCS cells, which resulted cell growth inhibition, proliferation retardation, morphological abnormalities and decreased viability. Meanwhile, we found that the HCS cells treated with tetracaine had typical features associated with apoptosis, including an increase in PM permeability, PS externalization, DNA fragmentation and apoptotic body formation. Tetracaine not only resulted in caspase-3, caspase-8 and caspase-9 activation and disruption of MTP but also downregulated Bcl-2 and Bcl-xL and upregulated Bad and Bax, along with the upregulation of cytoplasmic cytochrome c (Cyt. c) and apoptosis-inducing factor (AIF).

Conclusions: These results suggested that tetracaine-induced apoptosis might be triggered through Fas death receptors and mediated by Bcl-2 family proteins in the mitochondria-dependent pathway. Our findings identified the cytotoxicity and molecular mechanisms of tetracaine, which could provide a reference value for the safety of this medication and prospective therapeutic interventions in eye clinic.     

Naloxone does not modify the hemodynamic and neuroendocrine effects of clonidine in normal humans

The relationship between central opioidergic and noradrenergic central control mechanisms of blood pressure was investigated in normal men by evaluating the interference exerted by naloxone, a specific opiate antagonist, on the cardiovascular (blood pressure and heart rate) and neuroendocrine [human growth hormone (HGH) stimulation] effects of clonidine, a centrally acting alpha-adrenergic agonist, according to two different protocols. In series 1, the effects of placebo (normal saline), clonidine (0.15 mg i.v.), and naloxone (0.4 mg i.v.) were compared with that of clonidine plus naloxone (0.15 and 0.4 mg i.v., respectively), in seven normal male subjects. Clonidine decreased blood pressure and heart rate, and increased HGH levels. Naloxone administered alone (0.4 mg i.v.) did not modify blood pressure, heart rate, and HGH levels, while naloxone (0.4 mg) pretreatment left unaltered the hemodynamic and neuroendocrine effects of clonidine. In series 2, in five additional normal males, the effect of increasing doses of naloxone (0.4, 2.0, and 8.0 mg i.v.) on the pharmacodynamic activity of clonidine (0.15 mg i.v.) was further evaluated. Clonidine alone decreased blood pressure and heart rate and increased HGH levels, while naloxone pretreatment, in the whole range of doses studied, did not significantly modify the action of clonidine. These data suggest that a central opioidergic tone does not modulate the effect of central alpha-noradrenergic stimulation in normal humans.     

Effects of various beta-receptor blockers on metabolic and circulatory parameters during physical exertion

For further elucidation of the effect of various beta-adrenoceptor blockers on metabolism during exercise investigations on the influence of 5 beta-adrenoceptor blockers were carried out in healthy subjects. The 5 beta-adrenoreceptor blockers (acebutolol, metoprolol, penbutolol, pindolol, propranolol) were different regarding cardioselectivity and intrinsic activity (ISA). Each substance was given in 4 different dosages corresponding to 10:20:40:80 mg propranolol. Exercise was performed as bicycle ergometer test over 1 hour. In order to find out different effects during long-term application a medium dosage corresponding to 40 mg propranolol was applied for 4 weeks after the acute trials. The following parameters were measured: heart rate, systolic arterial pressure, parameters of carbohydrate metabolism (glucose, lactate) as well as lipid metabolism (free fatty acids (FFA), cholesterol, triglycerides etc.), serum concentration; for three beta-adrenoceptor blockers hormonal concentrations (catecholamines, insulin, human growth hormone (HGH), adrenocorticotrophic hormone (ACTH] were assessed. The different beta-adrenoceptor blockers demonstrated a varying relation between dosage and effect which was mostly pronounced for propranolol. All beta-adrenoceptor blockers caused a nearly complete blockade of energy release from FFA, and serum glucose concentration decreased. These changes were smaller under the effect of a low dosage of the cardioselective beta-adrenoceptor blocker metoprolol. The hormonal concentrations demonstrated a more pronounced increase of epinephrine during beta-blockade in contrast to only small changes by norepinephrine. Whereas there was no effect to be found on ACTH, HGH increased significantly under beta-blockade but independent of cardioselectivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Poly (epsilon-caprolactone) microparticles containing Levobunolol HCl prepared by a multiple emulsion (W/O/W) solvent evaporation technique: effects of some formulation parameters on microparticle characteristics

The aim of this study was to prepare poly (epsilon-caprolactone) (PCL) microparticles of Levobunolol HC1 (L-HC1) for use as an anti-glaucomatous drug to the eye. The double emulsion (W/O/W) solvent evaporation technique was used for encapsulating L-HC1 as a hydrophilic drug. The study examined the impact of different factors including the pH and volume of the external aqueous phase, the concentration of polyvinylalcohol (PVA) and Pluronic F68 (PF68) used as stabilizers and drug/polymer ratios on the characteristics of the microparticles. Scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) were used to identify the physical state of the drug and polymer. The zeta potential of the particles was also identified. Entrapment efficiency was found to be highest with a 0.5% PVA concentration and 100 mL volume of external aqueous phase at pH 12. The high efficiency was due to a reduction in the degree of drug ionization. The microparticles were spherical and appropriately sized for ophthalmic application. Drug release from the microparticles appears to consist of two components, with an initial rapid release followed by a slower stage. Drug release was slower when the microparticle was incorporated into the thermally reversible gel (Pluronic F127) in comparison to drug release from the free drug incorporated into the gel and drug release from the free microparticle.     

The Stability and Degradation Pathway of Recombinant Human Parathyroid Hormone: Deamidation of Asparaginyl Residue and Peptide Bond Cleavage at Aspartyl and Asparaginyl Residues

Purpose. The stability of recombinant human parathyroid hormone (rhPTH) was examined under acidic to alkaline conditions; its degradation pathways were elucidated from resultant products. Methods. Degradation assay was performed in the pH range 2 to 10 at 40, 50 and 60°C. The approximate molecular mass and pI values of the degradation products were estimated by electrophoresis. FAB-MS peptide mapping and amino acid composition analysis were used to determine these structures. The amount of each respective product was determined by HPLC. Results. At pH2, eight degradation products were found: l-30rhPTH, l-74rhPTH, l-71rhPTH, l-56rhPTH, l-45rhPTH, 46-84rhPTH, 31-84rhPTH and Asp76-rhPTH; these were mainly as a consequence of peptide bond cleavage of the amide bond of Asp. At pH9, five products were found: isoAsp16-rhPTH, Aspl6-rhPTH, Asp57-rhPTH, Asp76-rhPTH, 17-84rhPTH; the main degradation pathway was deamidation of Asn via a cyclic imide intermediate. Degradation products resulting from cleavage at Asp were increased in proportion to the extent that pH was lowered below 5. As pH was increased above 5, so were products resulting from deamidation of Asn. Correspondingly, levels of intact rhPTH were at a peak at pH5. Conclusions. Degradation of rhPTH under acidic conditions predominantly occurs by cleavage at Asp, whereas, above pH5, deamidation of Asn is the more prominent. rhPTH is most stable at pH5.     

Poly (ε-caprolactone) microparticles containing Levobunolol HCl prepared by a multiple emulsion (W/O/W) solvent evaporation technique: Effects of some formulation parameters on microparticle characteristics

Abstract

The aim of this study was to prepare poly (ε-caprolactone) (PCL) microparticles of Levobunolol HC1 (L-HC1) for use as an anti-glaucomatous drug to the eye. The double emulsion (W/O/W) solvent evaporation technique was used for encapsulating L-HC1 as a hydrophilic drug. The study examined the impact of different factors including the pH and volume of the external aqueous phase, the concentration of polyvinylalcohol (PVA) and pluronic® F68 (PF68) used as stabilizers and drug/polymer ratios on the characteristics of the microparticles. Scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) were used to identify the physical state of the drug and polymer. The zeta potential of the particles was also identified. Entrapment efficiency was found to be highest with a 0.5% PVA concentration and 100 mL volume of external aqueous phase at pH 12. The high efficiency was due to a reduction in the degree of drug ionization. The microparticles were spherical and appropriately sized for ophthalmic application. Drug release from the microparticles appears to consist of two components, with an initial rapid release followed by a slower stage. Drug release was slower when the microparticle was incorporated into the thermally reversible gel (Pluronic® F127) in comparison to drug release from the free drug incorporated into the gel and drug release from the free microparticle.     

Nasal absorption kinetics of human growth hormone enhanced by acylcarnitines in rats

Pharmacokinetic analysis revealed that acylcarnitines enhanced overall nasal absorption of human growth hormone (HGH) in rats. Acylcarnitines dramatically shortened onset time, shortened absorption-duration time, and efficiently delivered HGH. HGH administered intranasally with acylcarnitines appeared in the blood within 0.28–2.7 min, far more rapidly than the 11.7 min after intranasal administration of HGH without acylcarnitines (CTL) or the 9.8 min after subcutaneous administration (SC). Absorption-duration times of HGH enhanced by acylcarnitines were in the range of 2.1–3.8 h, which are shorter than those of CTL (7.5 h) or SC (9.8 h). Among acylcarnitines at 1%, lauroylcarnitine chloride (LCC) showed the greatest absolute bioavailability (17.4%), which is 0.73 times the absolute bioavailability value for SC (22.1%). LCC enhancement reached a plateau at a concentration of 0.1%. This nasal absorption enhancement would be suitable for pulsatile administration. A single dose toxicity study also suggested that LCC had similar systemic safety to L-carnitine at a high dose of 100 mg/kg in mice. Further, the potential safety of LCC was ten times greater than palmitoylcarnitine chloride.     

A quantitative cell-based high-content screening assay for the epidermal growth factor receptor-specific activation of mitogen-activated protein kinase

The complexity of mitogen-activated protein kinase (MAPK) signaling pathways and their activation by different stimuli makes assaying the activation of particular MAPKs by specific receptors a challenging problem. The multiplexing capability of quantitative high-content screening (HCS) assays enables the simultaneous monitoring and correlation, in the same cell, of an MAPK's specific activation with a particular receptor's post-signaling behavior, such as its internalization. We demonstrate a cell-based HCS assay to quantify the epidermal growth factor (EGF) receptor-specific activation of the MAPK ERK. Activation was quantified by measuring immunofluorescently labeled phosphorylated extracellular signal-regulated protein kinases (ERK) in the nucleus. Specificity of ERK activation by the EGF receptor was simultaneously confirmed in the same cell by quantitatively monitoring fluorescent EGF's internalization and subsequent intracellular degradation. Quantitative analysis of the temporal behavior of these two activities showed that phosphorylated ERK's accumulation in the nucleus peaked at 5 min before falling to basal levels by 30 min. Cellular accumulation of fluorescent EGF was slower, peaking around 30 min, before being degraded. This assay strategy can serve as a paradigm to study other signaling pathways and their activation by specific receptors. The flexibility and multiplexing capability of HCS assays allow the use of additional targets to further qualify the specificity of response by including other MAPKs or receptors, to rule out cross-talk from competing signaling pathways, or to simultaneously monitor toxicity effects of compounds. This automated, non-subjective, easy-to-use assay procedure provides information rich, quantitative results, and demonstrates the potential of the HCS assay approach in deconvolving intracellular signaling pathways.     

PURIFICATION AND RECEPTOR BINDING PROPERTIES OF COMPLEXES BETWEEN LUTROPIN AND MONOVALENT ANTIBODIES AGAINST ITS α SUBUNIT

A complex between bovine lutropin (LH) and monovalent antibodies (Fab fragments) directed against its α subunit, which is common to the glycoprotein hormones, has been purified by gel filtration and chromatography on concanavalin A-Sepharose. The complex is heterogeneous with respect to molecular size; 70–80% of the hormone is complexed with either two or three Fab fragments. The LH-Fab α complexes retain only about 13% receptor binding activity as compared to LH when measured in a radioligand receptor assay in which the radiolabeled ligand is human choriogonadotropin. (Use of the human hormone as labeled ligand permits direct measurement of competition between receptor and the bovine complex because the α portion of the human hormone does not cross react significantly with antibodies directed against bovine α subunits.) Complex formation does not lead to dissociation of the lutropin into its subunits, as shown with a homologous LH-β immunoassay which distinguishes free β subunit from intact LH. Complexing of LH with Fab-α fragments also causes little or no change in the affinity of the hormone's β subunit for anti-LH-β antibodies indicating that significant changes in β subunit conformation did not occur. The data show that at least two well-separated antigenic regions on the α subunit are exposed to the surface in the intact hormone. They are also in agreement with the proposal that the loss of binding activity to receptor is due to steric effects rather than to changes in conformation or dissociation, and that there may be sites on the α subunit which interact directly with the receptor.     

Evidence that Escherichia coli STb enterotoxin binds to lipidic components extracted from the pig jejunal mucosa.

Escherichia coli strains producing the heat-stable enterotoxin STb cause diarrhoea in pigs, but little is known on the receptor binding step initiating the diarrhoeal process. In the present study, pig jejunal mucosa extracts were tested for the presence of binding component(s) for STb. Jejunal epithelial cells and the mucus layer were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated material was transferred to a polyvinylidene difluoride (PVDF) membrane and overlayed with STb. The results indicated that a band migrating with the tracking dye was bound by STb. This band was not stained by Coomassie blue and was thus regarded as non proteinic but rather as a lipidic component. Thus, total lipid extracts were obtained from the epithelial cells and the mucus layer. Compared to SDS-PAGE on 12% gels, a better separation of the low molecular mass components contained in these extracts was obtained using high-density Phastgel. Most of the components were detected following silver staining but not using Coomassie blue. Interestingly, commercially available pure glycolipids could also be visualized, after separation, only following silver staining. In the total lipid extracts, a band migrating in the 2.5-6.5 kDa range was observed. Using a monoclonal antisulfatide antibody, this band was recognized indicating that sulfatide was, in effect, present in the extract. When pure sulfatide was run on the same gels, it showed the same electrophoretic mobility. In addition, a dose dependent binding of STb to sulfatide could be observed. Taken together, these data suggested that sulfatide present on the jejunal mucosa, could represent a natural target binding molecule for STb.