Hormone action at the membrane level VI. Binding of (—)-[3H]dihydroalprenolol and (±)-[3H]isoproterenol to turkey erythrocytes and correlation with adenylate cyclase activity

The binding of (±)-[³H]isoproterenol and (—)-[³H]dihydroalprenolol to intact turkey erythrocytes was studied using a rapid centrifugation technique. The binding of both ligands is rapid, dissociable, stereospecific and inhibited by (—)-propranolol. The total number of isoproterenol binding sites is 2800 sites/ cell. This consists of a low and high affinity site both of which show stereospecific binding. The high affinity isoproterenol site has a K_d of 15.5—19.5 nM and has 600 sites/cell. The low affinity isoproterenol site has a K_d of 195 nM and has 2200 sites/cell. The binding of (—)-[³H]dihydroalprenolol shows one type of site with a K_d of 7.8 nM and has 2500 sites/cell. The agonists epinephrine, norepinephrine, soterenol and p-hydroxyphenylisoproterenol which were tested by competition for binding showed a 6—25-fold greater affinity for the high affinity site determined by (±)-[³H]isoproterenol as compared to the (—)-[³H]dihydroalprenolol binding site. However, the antagonists propranolol, practolol and metrapolol showed similar affinities for the binding sites as determined by competition of binding of either labeled isoproterenol or dihydroalprenolol. These studies indicate that isoproterenol binding can recognize two independent stereospecific β-adrenergic receptors or can recognize two different conformational states of a single receptor. Provisional calculations are made on the turnover number of adenylate cyclase under physiological conditions using intact erythrocytes. The turnover number is 4000 molecules of cyclic AMP/10 min per high affinity receptor.     

Synthesis of (R,S)-isoproterenol,an inhibitor of tau aggregation,as an 11C-labeled PET tracer via reductive alkylation of (R,S)-norepinephrine with [2-11C]acetone

(R,S)-Isoproterenol inhibits the formation of toxic granular tau oligomers associated with neuronal loss and development of cognitive disorders, and is an attractive drug candidate for Alzheimer’s disease. To elucidate its behavior in the brain by positron emission tomography, we synthesize (R,S)-[¹¹C]isoproterenol by reductive alkylation of (R,S)-norepinephrine with [2-¹¹C]acetone, which was in turn synthesized in situ under improved conditions afforded a decay-corrected radiochemical yield of 54%. The reductive alkylation using NaBH(OAc)₃ as reducing agent in the presence of benzoic acid in DMSO/DMF (60:40 v/v) at 100 °C for 10 min gave (R,S)-[¹¹C]isoproterenol in an 87% radio-high performance liquid chromatography (HPLC) analytical yield. HPLC separation using a strong cation exchange column, followed by pharmaceutical formulation in the presence of d/l-tartaric acid, afforded (R,S)-[¹¹C]isoproterenol with a total radioactivity of 2.0 ± 0.2 GBq, a decay-corrected radiochemical yield of 19 ± 2%, chemical and radiochemical purities of 71% and >99%, respectively, and a molar activity of 100 ± 13 GBq/μmol (n = 3). The overall synthesis time from the end of the bombardment to pharmaceutical formulation was 48 min. A preliminary preclinical PET study in a rat demonstrated the potential of the radioligand for the evaluation of the penetration of (R,S)-isoproterenol in human brain.     

The steroid ligands of estrogen binding proteins in Xenopus laevis liver cytosol are primarily metabolites of 17β-estradiol

The interactions in vitro between [³H]estradiol and liver proteins from Xenopus laevis have been examined to determine if the binding reaction meets criteria of steroid-receptors which may function in the induction of vitellogenesis. Estrogenic hormones associated with proteins in serum and liver cytosol from Xenopus laevis. However, the interactions between soluble liver proteins and estrogens apparently do not result from serum contamination of liver as specific binding was distinguishable by ligand affinity and by differential mobility on polyacrylamide gels. Steroid ligands bound by liver proteins during incubation in vitro were examined by solubility and by thin-layer chromatography. Only a small percentage (13%) of the bound radioactive ligand was recovered as the original tritium-labeled steroid, 17β-estradiol. The major ligand was recovered as a water-soluble metabolite of estradiol which was identified tentatively as an estradiol-glucoside. To investigate whether the protein-bound estradiol metabolite(s) merely masks a small amount of authentic estradiol-receptor complexes or if the metabolite could be an intermediate in estrogen function, isolated liver nuclei were incubated with liver cytosol containing ³H-labeled steroid-protein complexes or with serum protein-bound [³H]estradiol. Nuclei preferentially accumulated ³H-labelea steroids from liver cytosol protein-steroid complexes relative to [³H]estradiol from serum proteins. However, analysis of the steroids recovered in the nuclei after incubation with liver cytosol revealed that both 17β-[³H]estradiol and the ³H-labeled water-soluble metabolite were retained in vitro by nuclei.     

Surface interaction of [3H]norepinephrine with cultured chick embryo myocardial cells

Cultured chick embryo cardiac myoblasts specifically bind [³H]nonrepinephrine. The binding is rapid and reversible. Bound [³H]nonrepinephrine, dissociated by 1 M HCl, can be rebound to fresh cells. β-Adrenergic catecholamines were most potent in displacing [³H]nonrepinephrine from the cellular bindign sites. The binding reaction did not show stereospecificity. α-Adrenergic amines were much less potent. Propranolol, but no phentolamine, competed for the sites. Approximately 2.5 · 10⁶ specific binding sites are present per myocardial cell. The sites appear to be present predominantly at the cell surface in that nonrepinephrine linked to agarose beads competes for th sites. Similarly, the sites were degraded by either trypsin or trypsin bound to agarose. Two different binding constants, K = 2 · 10⁶ and 1 · 10⁵, were observed. Proteolytic enzymes decreased binding whereas certain hospholipases led to an increase in specific binding. Divalent cations at concentrations > 1 mM diminished binding as did chelating agents.     

Direct studies of beta-adrenergic receptors intact frog erythrocytes.

(?) [³H]Dihydroalprenolol, a potent competitive β-adrenergic antagonist can be used to directly study β-adrenergic receptors by ligand binding techniques in an intact cell system, the frog erythrocyte. At 37°, binding reached equilibrium within 1 minute. Upon addition of excess unlabeled propranolol, complete dissociation of receptor bound ligand occurred within 1 minute. The characteristics of (?) [³Hdihydroalprenolol binding to β-adrenergic receptors in intact cells were quite similar to those previously demonstrated with isolated membrane fractions. The equilibrium dissociation constant for (?) [³H]dihydroalprenolol was 1.5 nM. Order of potency of agonists and antagonists in competing for the binding sites was appropriate for the β-adrenergic receptor as was the stereospecificity of binding ((?) isomers more potent than (+) isomers). Saturation studies with these intact cells indicated 1700 binding sites/cell in excellent agreement with the number previously estimated from membrane studies. Preincubation of cells with 10^?5M isoproterenol produced a 36% fall in number of β-adrenergic receptors. It is concluded that (?) [³H]dihydroalprenolol can be used to directly study the properties and regulation of β-adrenergic receptors in intact cell as well as broken cell preparations.     

Potentiometric and spectroscopic studies on the solution molecular structures of copper(II)-[S]-2-[N-(2′-hydroxybenzyl)aminomethyl]pyrrolidine complexes, potential non-steroidal anti-inflammatory agents (NSAIDs)

The purpose of this study was to investigate the complexes formed by copper(II) with potential non-steroidal anti-inflammatory agents (NSAIDs) under physiological conditions. A former study suggested that 2-benzylaminomethylpyrrolidine ligands could be good candidates as potential OIL (OH-inactivating ligand) when complexed to copper(II). In order to assess the chemical behavior as OIL, [S]-2-[N-(2′-hydroxybenzyl)aminomethyl]pyrrolidine (OHbamp) was synthesized and bound to copper(II). Physico-chemical properties were determined at 37 °C in 0.15 M NaCl using glass electrode potentiometry, UV-Vis and circular dichroism spectroscopies, before and after copper(II) complexation. [Cu(OHbamp)(H₂O)₃]⁺ was the main complex found at both physiological and inflammatory pH values, showing appreciable stability at pathological pH compared to copper(II) complexes of histidine, the predominant low-molar-mass ligand of copper(II) in blood plasma. However, neutral species such as [Cu(OHbamp)₂(H₂O)₂] and [Cu(OHbamp)(OH)(H₂O)₃] are predominant only above pH 8, preventing a significant amount of drug from diffusing through membranes at inflammatory pH. In conclusion, copper(II)-OHbamp system does not meet all the requirements to be an OIL. Nevertheless, these results allow us to better identify the chemical features needed for a good OIL candidate.     

Isolation of a palmitoyl CoA-protein complex with properties of the ADP/ATP carrier from bovine heart mitochondria

A palmitoyl CoA-protein complex was isolated from bovine heart mitochondria and purified to homogeneity. The elution profile of the [¹⁴C]palmitoyl CoA bound protein from a hydroxyapatite column was identical to that seen when [³H]carboxyatractylate was used as the bound ligand. A sample of the palmitoyl CoA-protein complex from a peak fraction of the column appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. The mobility of the protein bound with palmitoyl CoA was identical to the one bound with carboxyatractylate and the molecular weight was estimated to be 30,000 daltons. Compared to the stable palmitoyl CoA-protein complex, purification of the unliganded carrier from mitochondria at 22°C resulted in a disaggregated protein. These physical characteristics of the palmitoyl CoA-protein complex correspond to those identified for the $\text{ADP}\text{ATP}$ carrier. The results further confirm the specificity of the fatty acyl CoA ligand for the adenine nucleotide translocase and support the concept that it may be a physiological modulator of adenine nucleotide translocation.     

[3H]norepinephrine binding and lipolysis by isolated fat cells

[³H]norepinephrine was shown to bind to specific sites on isolated fat cells. A Scatchard plot of norepinephrine binding showed two apparent K_a of 1.9 · 10⁶ and 1.2 · 10⁵ LM^?. 1.4 · 10^?4 M Norepinephrine covalently-linked to agarose beads reduced [³H]norepinephrine binding by over 50%. Several structurally related drugs were compared as inhibitors of [³H]norepinephrine binding and as stimulators of lipolysis in preparations of similarly prepared cells. Dose-response curves for norepinephrine, epinephrine and isoproterenol showed the affinities for binding inhibition and for stimulation of lipolysis to be in the same range of 6 · 10^?7-2 · 10^?6 M. Dopamine and dopa were potent inhibitors of [³H]norepinephrine binding at 8.5 · 10^?7 M and 2.0 · 10^?6 M respectively, but did not stimulate lipolysis even at 10^?4 M. Propranolol, a β-adrenergic antagonist, had no effect on [³H]norepinephrine binding at 10^?4 M but completely inhibited catecholamine-stimulated lipolysis at 10^?5 M. Phentolamine, an α-adrenergic antagonist, did not inhibit binding or catecholamine-stimulated lipolysis at 10^?4 M. Ephedrine, metaraminol, phenylephrine and normetanephrine were also ineffective both as [³H]norepinephrine binding inhibitors and as stimulators of lipolysis. The results suggested the catechol ring of catecholamines is more important than the ethanolamine side chain as a requirement for binding, while both an intact catechol moiety and ethanolamine function appear necessary for physiological effect.     

Matrix metalloproteinase activities of turkey (Meleagris gallopavo) bile

The bile from turkey (Meleagris gallopavo) gall bladders was found to contain substantial matrix metalloproteinase (MMP) activities using gelatin, collagen, and casein substrate zymography, [³H]labeled collagen degradation assays, and gelatin–agarose affinity purification. Five major bands corresponding to approximate M_w of 64, 60, 46, 40 and 36 kDa showed gelatinolytic activities. On incubation with p-aminophenylmercuric acetate or thimerosal, the densities of both the 64- and 46-kDa bands decreased with increasing intensities of the 60- and 40-kDa bands. Both the 64- and 60-kDa bands showed collagenolytic activities whereas the caseinolytic activities appeared as diffuse bands corresponding to M_w of approximately 60, 40 and 36 kDa. Using [³H]collagen as substrate, the bile enzymes showed both a time and concentration-dependent degradation, which could be inhibited by the MMP inhibitors such as EDTA, phenanthroline, and N-[(2R)-2-(hydroxyamido carbonylmethyl)-4-methylpentanonyl]-l-tryptophan methylamide, but not by serine and cysteine protease inhibitors like trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane, phenylmethylsulfonyl fluoride or leupeptin. Both 60- and the 40-kDa gelatinolytic bands showed affinity adsorption to a gelatin–agarose matrix. The physiological roles of bile MMPs are not clear, but their involvement in the digestive functions of birds are likely.     

Preparation of AzoDNA and its use in affinity chromatography

A limited coupling reaction between 4-diazobenzoic acid ([¹⁴C]carboxyl) and sheared single-stranded DNA was employed to prepare a ligand capable of bonding covalently with aminopentane Sepharose C1-4B. The ligand AzoDNA demonstrated small changes in ultraviolet absorbance spectra yet, unlike the parent DNA, had a distinct fluorescence emission peak at 400 nm when excited at 292 nm in neutral or alkaline solutions. On hydroxyapatite thermal chromatography the AzoDNA eluted as single-stranded DNA, while following catalytic reduction, the associated fluorescence and [¹⁴C]azobenzoate radioactivity were removed in large part from the derivatized DNA. In the coupling reaction, prior derivatization of the ligand DNA was required for covalent bonding to aminopentane Sepharose C1-4B and, at optimal polydeoxynucleotide concentrations, about 75 μg was bound/ml of packed gel. DNA:DNA hybridization reactions were accomplished using AzoDNA aminopentane Sepharose C1-4B gels with 50% of the hybridized polynucleotide strands being eluted at temperatures approximating the T_m values measured optically. The use of the AzoDNA gel was extended to the hybridization of adenovirus 2 and vaccinia complementary RNA. The viral complementary RNAs were specifically bound to matrices containing the homologous AzoDNA and eluted under conditions consistent with destabilization of RNA:DNA hybrids. These applications indicate the potential utility of AzoDNA-extensor arm affinity chromatography for the isolation of specific viral RNA molecules.     

Physiological responses of <Emphasis Type="Italic">Pterocladiella capillacea</Emphasis> (Rhodophyta,Gelidiales) under two light intensities

Macroalgae must be able to survive in conditions of different light intensities with no damage to their physiological performance or vital processes. Irradiance can stimulate the biosynthesis of certain photoprotective compounds of biotechnological interest, such as pigments and proteins. Pterocladiella capillacea is a shade-grown alga, which play a role key in the balance of marine ecosystems. In addition, it is considered one of the best sources of bacteriological agar and agarose with a wide pharmacological potential. In order to evaluate the photosensitivity in P. capillacea under 60 (control) and moderate light intensity of 300 μmol(photon) m^–2 s^–1, photosynthetic performance and chemical composition were assessed. P. capillacea showed photosensitivity without evidence of photodamage. The results indicate the possibility to increase a growth rate and probably infer productivity in long-term cultivation by stimulation at moderate light intensity. Increasing photosynthetic pigment and protein contents were also observed under medium light, an interesting result for functional ingredient approaches.     

Protease inhibitors and human plasmin: interaction in a whole plasma system.

Using trace amounts of [¹²⁵I]-plasminogen and conventional biochemical techniques, the distribution of the labeled zymogen amongst the various protease inhibitors was studied in whole plasma before and after activation with urokinase and streptokinase. A small percentage of the labeled enzyme was bound to α₂-macroglobulin while a majority was complexed to a component in plasma immunologically distinct from the well known human antiplasmins. The inhibitor was identified as α₂-antiplasmin and confirmed the existence of this antiprotease recently described by others. These data also suggest that the other antiplasmins may play a minor, yet important role in the regulation of plasmin activity under different physiological conditions.     

Enzymatic mechanochemistry. I. The interaction of heavy meromyosin with “immobilized adenosine triphosphate”

ATP was covalently bound to an agarose gel. The insolubilized ATP was found to be capable of specifically binding heavy meromyosin. The adsorbed heavy meromyosin could be eluted by ATP in solution. Both binding and elution by ATP of heavy meromyosin were not much effected by Ca²⁺, Mg²⁺ or EDTA.While the water-soluble polyalanine-myosin was also found to be adsorbed, myosin in 0.5 M KCl did not seem to be adsorbed by agarose-ATP.Both Mg²⁺ and Ca²⁺ appear to activate the splitting of bound ATP by heavy meromyosin to practically the same extent.We prepared water-soluble derivatives of ATP in which ATP underwent the same chemical modification required for its coupling to agarose but in which the agarose component was absent. Their splitting by heavy meromyosin was also activated by Mg²⁺ though to a lesser extent but actin did not influence this reaction.Possible relations between our findings and the various stages of the reaction between myosin and ATP, as well as the potential use of columns filled with insolubilized NTPs for the separation and purification of myosin and of its subfragments, are discussed.     

Regulation by isoproterenol of muscarinic acetylcholine receptor numbers and sensitivity in rat submandibular,but not lacrimal,glands

Chronic administration of DL-isoproterenol, a β-adrenergic agonist, to male Sprague-Dawley rats increased submandibular gland weights by 3 to 4-fold. This increase resulted from a combination of hyperplasia and hypertrophy of secretory cells. Possible effects of this drug regimen on submandibular gland muscarinic acetylcholine receptors were examined by analysis of the binding of the cholinergic antagonist, L-quinuclidinyl [³H]benzilate, to receptors in gland homogenates. Parallel investigations of receptors in exorbital lacrimal glands, an organ that is not grossly affected by chronic isoproterenol treatment, were also carried out. [³H]QNB bound to submandibular receptors with a K_d of 37.8±6.3 pM in control rats and 41.0±4.0 pM in isoproterenol-treated animals, a non-significant difference (P > 0.05). In contrast, the maximal binding level (B_max) is isoproterenol-treated rats, 1.52±0.10 fmol/μg DNA, was depressed by approx. 30% (P<0.05) from that of 2.22±0.16 fmol/μg DNA in control animals. In lacrimal glands, both K_d (61.3±5.3 vs. 53.2±4.0 pM) and B_max (1.74±0.24 vs. 1.78±0.17 fmol/μg DNA) were unchanged by isoproterenol treatment. The affinity of glandular muscarinic receptors for cholinergic agonists was also examined by competition experiments using carbachol. This agonist inhibited [³H]QNB binding to receptors in homogenates from both glands in a dose-dependent fashion. Inhibition constant (K_i) for this interaction were similar in control and isoproterenol-treated lacrimal glands; 53.6±5.4 μM and 66.6±7.9 μM, respectively (P>0.05). In submandibular glands, isoproterenol treatment elicited a highly significant (P < 0.01) shift in K_i from 17.3±1.4 μM to 68.3±5.2 μM. These results demonstrate that chronic administration of isoproterenol to rats results in a reduction in receptor numbers and a decrease in their sensitivity to cholinergic agonists in submandibular, but not lacrimal, glands.     

β2-Adrenergic regulation of prostaglandin D2 receptor in rabbit platelets

[³H]Prostaglandin D₂ binding to rabbit platelets was increased by about 150% in the presence of β-adrenoceptor agonist, isoproterenol. The isoproterenol-induced potentiation of the [³H]prostaglandin D₂ binding gave a bell-shaped dose-response relationship (maximum response at 3·10⁻⁸ M) in a stereospecific manner. Similar and moderate potentiation was obtained with terbutaline. On the other hand, β-adrenoceptor antagonists such as alprenolol, propranolol and butoxamine (β₂-specific) had no potentiating effect on [³H]prostaglandin D₂ binding; rather, they abolished the isoproterenol-induced increase of [³H]prostaglandin D₂ binding. The β₁-specific antagonist, metoprolol, did not have any effect. Rabbit platelets were found to possess one [³H]prostaglandin D₂ binding site (K_d = 6·10⁻⁷ M, B_max = 787 fmol/mg protein). In the presence of isoproterenol at 3·10⁻⁸ M, B_max was increased with unaltering K_d value. Isoproterenol did not increase [³H]prostaglandin E₁, [³H]prostaglandin E₂ and [³H]prostaglandin F_2α bindings to platelets. The potential effect of isoproterenol was mimicked by forskolin, theophylline, dibutyryl cyclic AMP, prostaglandin E₁ and prostaglandin I₂, but it was abolished by 2′, 5′-dideoxyadenosine, an inhibitor of adenylate cyclase, indicating that elevated level of cyclic AMP may be available for the induction of the increase of [³H]prostaglandin D₂ binding. Prostaglandin D₂-induced cyclic AMP synthesis and antiaggregation activity were also augmented in the presence of isoproterenol. These results suggest a β₂-adrenoceptor-mediated cyclic AMP-dependent mechanism for the regulation of prostaglandin D₂ receptor binding in rabbit platelets.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

Fate of oxygen species from O2 activation at dimetal cofactors in an oxidase enzyme revealed by 57Fe nuclear resonance X-ray scattering and quantum chemistry

Oxygen (O₂) activation is a central challenge in chemistry and catalyzed at prototypic dimetal cofactors in biological enzymes with diverse functions. Analysis of intermediates is required to elucidate the reaction paths of reductive O₂ cleavage. An oxidase protein from the bacterium Geobacillus kaustophilus, R2lox, was used for aerobic in-vitro reconstitution with only ⁵⁷Fe(II) or Mn(II) plus ⁵⁷Fe(II) ions to yield [FeFe] or [MnFe] cofactors under various oxygen and solvent isotopic conditions including ^16/18O and H/D exchange. ⁵⁷Fe-specific X-ray scattering techniques were employed to collect nuclear forward scattering (NFS) and nuclear resonance vibrational spectroscopy (NRVS) data of the R2lox proteins. NFS revealed Fe/Mn(III)Fe(III) cofactor states and Mössbauer quadrupole splitting energies. Quantum chemical calculations of NRVS spectra assigned molecular structures, vibrational modes, and protonation patterns of the cofactors, featuring a terminal water (H₂O) bound at iron or manganese in site 1 and a metal-bridging hydroxide (μOH⁻) ligand. A procedure for quantitation and correlation of experimental and computational NRVS difference signals due to isotope labeling was developed. This approach revealed that the protons of the ligands as well as the terminal water at the R2lox cofactors exchange with the bulk solvent whereas ¹⁸O from ¹⁸O₂ cleavage is incorporated in the hydroxide bridge. In R2lox, the two water molecules from four-electron O₂ reduction are released in a two-step reaction to the solvent. These results establish combined NRVS and QM/MM for tracking of iron-based oxygen activation in biological and chemical catalysts and clarify the reductive O₂ cleavage route in an enzyme.     

A support for affinity chromatography that covalently binds amino groups via a cleavable connector arm.

The preparation of a new succinimidyl ester agarose derivative (SEPE-Agarose) is described. This agarose derivative can be used for covalently linking proteins and other ligands containing amino groups to agarose via phenyl ester linkages that can later be broken under mild conditions which should not alter other groups which may be present in proteins such as cystinyl residues and glycosyl residues. SEPE-Agarose is prepared by the reaction of bis[4-[2-(N-succinimidoxycarbonyl)ethyl]phenyl]succinate with an aminoethylcarbamylmethyl derivative of agarose. Studies of the covalent binding and release of trypsin and myoglobin to SEPE-Agarose indicate that gels containing 0.1 to 0.6 μmol protein/ml of gel are obtained by reacting protein (0.5–5 mg/ml) with the N-succinimidyl ester groups in SEPE-Agarose. Protein-linked gel is reasonably stable in dilute phosphate buffers (pH ≤ 7.4, ≤ 25 °C). Protein is released from the gel, however, by treatment at 25 °C with solutions containing nucleophiles such as 1 m imidazole-glycine, pH 7.4, for 4 h, or 1 m hydroxylamine, pH 7, for 10 min. Protein is also released from the gel by treatment with 1 m Tris pH 8.2 for 24 h. SEPE-Agarose should prove useful in affinity chromatography and immunoabsorption when it is difficult or impractical to elute material bound to conventional affinity supports.     

Use of Surface Plasmon Resonance and Biolayer Interferometry for the Study of Protein–Protein Interactions on the Example of an Enzyme of a Glycosyl Hydrolase Subtype (EC 3.2.1) and Specific Antibodies to It

Two previously obtained, full-size, fully human antibodies that reversibly bind the active form of an enzyme belonging to the subtype EC 3.2.1, which is used for substitutive enzymatic therapy in lysosomal storage diseases, have been characterized by surface plasmon resonance and biolayer interferometry methods. It was shown under normal physiological conditions that the designed antibodies specifically bound with the antigen (KD ~ 10^–8 M) and rapidly dissociated at neutral pH in 60% ethylene glycol while leaving the enzymatic activity unchanged. Dan ue to their properties, the developed antibodies can be used in industry as affinity ligand in the isolation of therapeutic substances of the enzyme.