Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.     

Physico-Chemical Characterization of Hamster Interferon

The glycoprotein nature of Syrian hamster interferon was tested on several immobilized lectins. The specific retention of small portion (20%) of interferon activity was observed only on concanavalin A-agarose; Component I of the interferon (not retained) has an apparent molecular weight of 23,500 whereas Component II (retained) is larger, 31,500. The apparent hydrophobicity of Syrian hamster interferon was probed by its chromatography on: (a) straight chain hydrocarbons of varied length; (b) aromatic ligands (aminobenzene, benzylamine, β-phenylethylamine, γ-phenyl-propylamine); ligands listed in (a) and (b) were immobilized to cyanogen bromide-activated agarose (isoure linkage); and (c) phenyl-agarose (Phenyl-Sepharose CL-4B), an aromatic ligand immobilized vi 2-hydroxypropyl arm to the agarose (ether linkage).

For a hydrophobic interaction to occur under physiological solvent conditions, the hydrocarbon arm (isourea 1inkage) must be C₉ to C₁₀ carbon atoms long, whereas the aromatic ligand (isourea linkage) must be removed from the agarose matrix by a molecular arm of C₃ carbon atoms. There is no significant binding of hamster interferon to phenyl-agarose (ether linkage) near neutral pH.

The apparent hydrophobicity of Syrian hamster interferon can be profoundly influenced by the pH of the solvent. At lower pH values, interferon binds to both octyl-agarose and phenyl-agarose. The midpoint of transition between binding of interferon and its release from these ligands is about the same, pH 6.0, suggesting the identity of the binding site on the interferon molecule for both aliphatic and aromatic hydrocarbons.     

Large scale purification of human fibroblast interferon

Human fibroblast interferon was partially purified, about 4,000-fold, on a chromatographic tandem of columns: concanavalin A-agarose leads to phenyl-agarose, to a specific activity of ca 4 x 10(7). The overall recovery of interferon activity was ca 60%.     

Physico-chemical characterization of hamster interferon.

The glycoprotein nature of Syrian hamster interferon was tested on several immobilized lectins. The specific retention of a small portion (20%) of interferon activity was observed only on concanavalin A-agarose; Component I of the interferon (not retained) has an apparent molecular weight of 23,500 whereas Component II (retained) is larger, 31,500. The apparent hydrophobicity of Syrian hamster interferon was probed by its chromatography on: (a) straight chain hydrocarbons of varied length; (b) aromatic ligands (aminobenzene, benzylamine, beta-phenylethylamine, gamma-phenyl-propylamine); ligands listed in (a) and (b) were immobilized to cyanogen bromide-activated agarose (isourea linkage); and (c) phenyl-agarose (Phenyl-Sepharose CL-4B), an aromatic ligand immobilized via a 2-hydroxypropyl arm to the agarose (ether linkage).     

Affinity chromatography of lysyl hydroxylase on concanavalin A-agarose.

Lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) has a high affinity for columns of concanavalin A-agarose, which was markedly reduced in the presence of alpha-methyl-D-mannoside, suggesting that the enzyme is a glycoprotein. Once bound, the enzyme could not be eluted with the glycoside alone, whereas an effective elution was achieved by a combination of alpha-methyl-D-mannoside and ethylene glycol. The data thus suggest that hydrophobic interaction stabilized the complex of the enzyme with the column. This information was applied to obtain a lysyl hydroxylase purification of about 3000-fold with a recovery of more than 10% from extract of chick embryos by relatively simple steps.     

Hydrophobic binding sites on human interferon.

Human interferon binds to a omega-carboxpentyl-agarose column at low ionic strength (0.15 M NaCl) and is still retained when the ionic strength is raised (to 1.0 M NaCl). The binding can be reversed, however, by ethylene glycol, indicating a hydrophobic interaction. The binding of human interferon to omega-aminohexyl-agarose is weak, even at a low ionic strength, and is probably exclusively electrostatic. This disparate binding behavior may be caused by the presence of a positive charge, adjacent to the hydrophobic binding site, on human interferon. The interaction of human interferon with omega-carboxypentyl-agarose is quite selective, inasmuch as the majority of proteins present in interferon preparations pass through the column unretained. Hydrophobic chromatography of human interferon may thus be useful in its purification.     

Interaction of thyroid peroxidase with concanavalin A covalently coupled to agarose.

We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 X g, 1-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with alpha-methyl-D-mannoside. The alpha-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. Peroxidase activity corresponds with a glycoprotein band. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: alpha-methyl-D-mannoside greater than D-mannose greater than alpha-methyl-D-glucoside greater than D-glucose greater than D-galactose. This order of specificity is typical of Con A-sugar interactions. Furthermore, inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding. Reactivation of the carbohydrate binding site by the addition of Ca2+ and Mn2+ to demetallized Con A-agarose restores thyroid peroxidase binding. These and other experiments suggest that htyroid peroxidase is, like several other peroxidases, a glycoprotein. In addition, the interaction between thyroid peroxidase and Con A-agarose may provide a new purification tool for thyroid peroxidase.     

Separation of human leukocyte interferon components by concanavalin A-agarose affinity chromatography and their characterization.

Human leukocyte interferon (HL-IF), produced by mixed leukocytes infected with Newcastle disease virus, was resolved into three distinct fractions when chromatographed on concanavalin A-agarose. The major portion (70--75%) of interferon appeared in the breakthrough (BT fraction). The bound interferon (25--30%) was displaced from the column as two peaks: the first was eluted with 0.01 M methyl alpha-D-mannoside, yielding 15-20% of the interferon activity (alpha-MM fraction), and the second by including ethylene glycol (70%) in the eluant, yielding the remaining 5--15% of the interferon (EG fraction). No interferon was retained when HL-IF produced in the presence of glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) was chromatographed on concanavalin A-agarose, suggesting that the fraction of interferon retained by this lectin is glycosylated. The three fractions of interferon (BT, alpha-MM, and EG) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cross-species antiviral activity, and neutralization by specific antisera. The BT fraction contains exclusively the 16 000 molecular weight component of human leukocyte interferon. The majority of the alpha-MM fraction (90%) is the 21 000 molecular weight component. However, the EG fraction contains the 16 000 and 21 000--23 000 molecular weight components in essentially equal proportions. On the basis of cross-species antiviral activity and neutralization by specific antisera, the BT and alpha-MM fractions are leukocyte-type interferon and the EG fraction seems to be primarily of fibroblast type.     

Effect of phospholipids on thyroid oligosaccharyltransferase activity and orientation. Evaluation of structural determinants for stimulation of N-glycosylation

Oligosaccharyltransferase solubilized by Nonidet P-40 was found to have a highly specific lipid requirement which is consistent with the lability of the enzyme when removed from its membrane association. Enzyme activity as measured by the N-glycosylation of a hexapeptide acceptor was greatly stimulated and stabilized by phosphatidylcholine (PC) while other naturally occurring phosphoglycerides had minimal effect. The quaternary ammonium group of PC was observed to be involved in the interaction with the enzyme as modification of the choline moiety by removal of methyl groups resulted in a progressive loss of the stimulatory effect (choline greater than N,N-dimethylethanolamine greater than N-monomethylethanolamine greater than ethanolamine) which was reflected primarily in the Vmax rather than the Km values. Evaluation of a number of PC and choline derivatives indicated that the nonpolar domain of the lipid also played an important specifying role. Two hydrophobic chains attached to the phosphoglycerol backbone were found to be essential, and furthermore the length and degree of unsaturation of the fatty acid substituents as well as their position of attachment on the glycerol moiety greatly affected the extent of activation. Since the L-isomer of PC brought about a 3-fold greater stimulation than the D-isomer the interaction of the enzyme with the phospholipid appears to be stereoselective. Upon chromatography of the PC-stabilized enzyme on concanavalin A-agarose almost complete retention occurred at 0.4% Nonidet P-40, while no binding took place at a detergent concentration of 0.075%; this suggested that upon dilution in the presence of PC, the oligosaccharyltransferase was reconstituted into vesicles in an asymmetric fashion with its N-linked carbohydrate located internally. Enzymatic assay of these vesicles demonstrated that the active site of the enzyme was also oriented toward the interior. These studies indicate that the activity as well as the membrane insertion of the oligosaccharyltransferase are to a large measure influenced by its interaction with PC.     

Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes

An NAD- and guanidine-dependent ADP-ribosyltransferase has been purified more than 500,000-fold from turkey erythrocytes with an 18% yield. The enzyme in the 100,000 X g supernatant fraction was bound to phenyl-Sepharose, eluted with 50% propylene glycol, and further purified by sequential chromatographic steps on carboxymethylcellulose, NAD-agarose and concanavalin A-agarose. The transferase was specifically eluted from concanavalin A-agarose with alpha-methylmannoside. The enzymatic activity was extremely labile following the first purification step. Both propylene glycol and NaCl stabilized the transferase; significant increases in enzyme recovery were obtained by conducting the NAD- and concanavalin A-agarose chromatography in buffer containing propylene glycol. The purified protein exhibits one predominant protein band on SDS-polyacrylamide gels with an estimated molecular weight of 28,300. On Ultrogel AcA54 chromatography, single coincident peaks of ADP-ribosyltransferase activity and protein were observed. Enzyme activity was independent of DNA; the highly purified transferase was inhibited by thymidine, nicotinamide, and theophylline. The specific activity of the purified enzyme (350 mumol of ADP-ribose transferred from NAD to arginine methyl estermin-1mg-1) is comparable to that reported for purified NAD glycohydrolases and poly(ADP-ribosyl)transferases.     

Antiviral and Antiproliferative Activities of Three Molecular Components of Leukocyte-Derived Human Interferon

Antiviral and antiproliferative activities of three naturally occurring components of leukocyte-derived human interferon (HuIFN-α) separated by concanavalin A-agarose affinity chromatography were studied in a variety of neoplastic cell lines. Significant differences were seen with these different components. The results strongly suggest that careful consideration must be given while selecting any one component of HuIFN-α for any of the antiviral or antiproliferative studies. There is no clear evidence that glycosylation of HuIFN-α has any significant influence on its in vitro antiviral or antiproliferative activities, although apparently glycosylated and non-glycosylated components gave different antiviral and antiproliferative responses in different tumor cells.     

疏水相互作用层析分离重组人干扰素α2b

目的采用疏水相互作用层析分离重组人干扰素α2b,去除干扰素样品中的二聚体,得到高纯度的干扰素用于进一步的研究。方法首先采用阳离子交换层析纯化复性重组人干扰素α2b,去除了大部分的杂蛋白,然后采用疏水相互作用层析纯化重组人干扰素α2b,去除复性过程中产生的错误折叠体和二聚体,并考察盐浓度、pH值、流速和洗脱液中尿素对疏水相互作用层析纯化效果的影响。结果硫酸铵初始浓度1.2 mol/L、缓冲液pH值6.0、流速2.5 mL/min、洗脱液中添加尿素浓度为2 mol/L时疏水相互作用层析纯化效果最佳。最终得到的重组人干扰素α2b非还原型SDS-PAGE电泳均呈单一条带。结论确定了疏水层析纯化重组人干扰素α2b的最优条件,成功提取到具有高活性、高纯度的重组人干扰素α2b纯品。     

The relaxin receptor-binding site geometry suggests a novel gripping mode of interaction

Relaxin has a unique, clearly identifiable, mixed function receptor-binding region comprising amino acid residues that evolve sequentially from the central portion of the B chain alpha-helix. Two arginine residues in positions B13 and B17 that project like forefinger and middle finger from the helix provide the electrostatic element opposed by the hydrophobic (thumb) element isoleucine (B20), offset from the arginines by about 40 degrees. The binding intensity of relaxin to its receptor decreases by 3 orders of magnitude if alanine is substituted for the newly discovered binding component isoleucine in position B20. The arginine residues cannot be replaced by other positive charges, nor can the guanidinium group be presented on a longer or shorter hydrocarbon chain. In contrast, the hydrophobic interaction is incremental in nature, and the contribution to the total binding energy is roughly proportional to the number of hydrocarbon units in the side chain. It appears that a hydrophobic surface exists on the receptor that offers optimal van der Waals' interaction with beta-branched hydrophobic amino acids. The binding energy increases roughly 10-fold with each methylene group whereby beta-branching is more effective per surface unit than chain elongation. Aromatic side chains appear to demarcate the extent of the binding region in so far as residues larger than phenylalanine decrease receptor binding. The exceptional clarity of binding site geometry in relaxin makes for an excellent opportunity to design peptido-mimetics.     

Purification and characterization of mouse interferon with novel affinity sorbents.

Several novel selective sorbents for mouse interferon are described that exploit the hydrophobic property and glycoprotein nature of this molecule. Low-molecular-weight ligands (hydrocarbons) and high-molecular-weight ligands (bovine serum albumin) immobilized on agarose bind selectively mouse L-cell interferon. The high selectivity of binding is due primarily to a hydrophobic effect, although electrostatic forces are also apparently involved. Mouse L-cell interferon binds to immobilized serum albumin and can be completely recovered by raising the ionic strength of the eluant. The specific activity of interferon preparations can be increased 2,000-fold to a value of 3 x 10(8) reference units per mg of protein in a single step with full recovery of the antiviral activity. A selective adsorption, although to a lesser degree, can be also obtained on hydrocarbon-coated agarose (Affi-Gel 202), resulting in 300-fold purification on desorption. The existence of two major components of mouse interferon was revealed upon its chromatography on the following sorbents: (i) bovine serum albumin-agarose, (ii) omega-carboxypentyl-agarose; and (iii) Bandeiraea simplicifolia lectin-agarose. This report thus provides for the first time a means for efficient and clear-cut separation of interferon components, thus enabling their further characterization.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.     

The involvement of O-linked oligosaccharide chains of the sea urchin egg receptor for sperm in fertilization

Recent investigations on the sea urchin egg receptor for spermhave led to its sequencing and the demonstration that it isa 350 kDa glycoprotein. In the current study, the N- and O-linkedoligosaccharide chains were cleaved from the protein fractionatedon concanavalin A-agarose. The putative O-linked oligosaccharidechains that did not bind to the lectin were further fractionatedby anion-exchange chromatography. Using a competition bioassaythat measured the ability of these oligosaccharide chains toinhibit fertilization, it was found that the N-linked chainswere devoid of inhibitory activity. Rather, the inhibitory activitywas localized to the O-linked chains, with the most highly charged,sulphated chains showing the highest inhibitory activity. Thebioactive oligosaccharides were labelled by reduction and assayedfor binding to sperm. The results of the binding assay, coupledwith the fertilization bioassay, indicate that the oligosaccharidesinhibit fertilization by binding to acrosome-reacted sperm.The bioactive oligosaccharide lacked species specificity infertilization bioassays, unlike the intact receptor and a recombinantaglyco protein containing only the extracellular domain of thereceptor. Since previous work showed that the recombinant proteininhibits fertilization species specifically and binds to acrosome-reactedsperm, a two-step model of sperm-egg interaction is proposed.The first step is postulated to be a low-affinity ionic interactionof the sulphated O-linked oligosaccharide chains of the receptorwith sperm that is not species specific. This is followed bya high affinity, species-specific interaction of the sperm withone or more binding sits on the polypeptide chain of the receptor. fertilization oligosaccharide receptor sea urchin egg sea urchin sperm     

Isolation of the Canavalia ensiformis seed alpha-mannosidase by chromatography on concanavalin A, the lectin from the same plant, without involving its sugar binding site

A method is described for the purification of the alpha-mannosidase from Canavalia ensiformis. By three consecutive steps, a more than 500-fold purification is achieved and the pure enzyme obtained in 75% yield. One of these steps utilizes the specific interaction of the alpha-mannosidase with concanavalin A, the lectin from the same plant. This interaction is dependent on pH and ionic strength but does not involve the sugar binding site of the lectin. The interaction between both proteins may be important also in vivo.     

Affinity of human leukocyte interferon for polyribonucleotides.

Human leukocyte interferon (HL-IF)binds to AGPOLY(A)TM, AGPOLY(U)TM and AGPOLY(I)TM. The bound interferon could be displaced from all three polyribonucleotides by including sodium chloride in the eluant. The nature of interaction of HL-IF with polyribonucleotides is electrostatic and not hydrophobic since its binding was not prevented in the presence of 50% ethylene glycol. The binding of HL-IF on AGPOLY(I)TM is stronger at lower pH since an increase in ionic strength is required to displace it.     

Purification of alpha-amylase isoenzymes from Scytalidium thermophilum on a fluidized bed of alginate beads followed by concanavalin A-agarose column chromatography

An alpha-amylase has been purified from the thermophilic fungus Scytalidium thermophilum. A ninefold purification was achieved in a single step using fluidized bed chromatography wherein alginate was used as the affinity matrix. There are at least two isoenzymes as shown by concanavalin A (Con A)-agarose column chromatography. The isoenzyme binding to Con A is stable for at least 3 h at 80 degrees C in the presence of calcium ions. The isoenzymes have similar molecular weights of around 45,000 Da as shown by SDS-PAGE analysis. The isoenzymes differ only slightly in their pH optima and temperature optima but the isoenzyme binding to Con A-agarose has slightly higher thermal stability.     

Evidence for the glycoprotein nature of kidney renin.

The glycoprotein nature of renin isolated from either rabbit or human kidney has been demonstrated by affinity chromatography on concanavalin A-Sepharose. The bulk of rabbit renin activity bound to concanavalin A is released by 20 to 50 mM alpha-methyl-D-mannoside. Adsorption of renin is prevented by periodate oxidation prior to chromatography. Mild acid treatment (pH 2.5) prior to chromatography does not alter the concanavalin A binding profile although the pI values of native rabbit renin (5.1-5.6) are shifted into a broader distribution (4.7-6.4). The molecular weight values of rabbit renin obtained by gel filtration and those from zone centrifugation are identical (37000 +/- 1000), consistent with a low percent of carbohydrate in the glycoprotein. A hydrophobic contribution to the binding of renin by concanavalin A is evident since, in the presence of mM Ca2+ and Mn2+, higher concentrations of alpha-methyl-D-mannoside are required to affect the same release of renin at 23 degrees C compared to that at 4 degrees C. Furthermore, 25% ethylene glycol releases renin in the absence of alpha-methyl-D-mannoside. It is concluded that renin contains a small number of carbohydrate residues in relatively close proximity to a hydrophobic surface which enhances the interaction with concanavalin A.