An affinity adsorbent, 5'-adenylate-aminohexyl-sepharose. II. Purification and characterization of multi-forms of RNase T2

RNase T2 bound to an affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4B, specifically at pH 4.5. The colorless enzyme was eluted only by the simultaneous addition of 2'(3')-AMP (1 mM) and NaCl (greater than 1 M) at pH 4.5. By applying this affinity chromatography to the purification of RNase T2, pure enzyme with a specific activity of 60 was obtained in only four steps and the yield was about 10 times higher than that of the previous purification method. This enzyme preparation was found to be heterogeneous in molecular weight and was separated into two fractions on Sephadex G-75 gel filtration. As the smaller enzyme with a molecular weight of 36,000 was identical with RNase T2 in every property examined, we tentatively designated the larger one with an apparent molecular weight of 80,000 as high molecular weight RNase T2 (RNase T2-L). RNase T2-L was still heterogeneous and was separated into five fractions, RNases T2-L 1-5, by repeated Sephadex G-150 gel filtration. The amino acid and carbohydrate analyses revealed that each of these fractions has a protein moiety in common with RNase T2 and the heterogeneities were due to the carbohydrate content, mainly galactose content.     

A new affinity adsorbent for guanyloribonuclease. Guanylyl-(2'-5')-guanosine coupled to aminohexyl-Sepharose.

Guanylyl-(2'-5')-guanosine binds to RNase T1 in 1:1 stoichiometry with a dissociation constant of 0.22 mM at pH 5.0 and 25 degrees C. This nucleotide, coupled to aminohexyl-Sepharose 4B, is able to serve as an affinity adsorbent for guanyloribonuclease [EC 3.1.4.8]. The strength of interaction between the adsorbent and various guanyloribonucleases at pH 5.0 was found to decrease in the following order: RNase N1 greater than RNase F1 greater than RNase T1 greater than RNase St. The bound enzymes can be released from the adsorbent either by increase of ionic strength or by increasing the pH from 5.0 to 7.5. The interaction between RNase T1 and the adsorbent is weakened by the presence of a low concentration of 2', 3'-, or 5'-GMP, which are competitive inhibitors of the enzyme. RNase F1 was purified to homogeneity by use of this affinity adsorbent.     

Purification of several bacteriolytic enzymes by affinity chromatography on lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with sepharose.

Using lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with Sepharose, several bacteriolytic enzymes were purified from crude preparations of animal and microbial origin. Quail egg-white, human milk and salivary lysozymes [EC 3.2.1.17] were adsorbed onto the adsorbent at pH 5-7 and eluted with 2M NaCl at pH 10. By means of these treatments, lysozymes were purified 20-250 fold with activity recoveries of 60-80%, and the quail lysozyme thus purified was shown to be discelectrophoretically homogeneous. Some bacteriolytic enzymes of microbial origin were also highly purified by using this affinity adsorbent. A bacterial lysozyme from Bacillus sp. ML-208 showed high affinity for the ligand and was not eluted under the conditions mentioned above, but was recovered by elution with 2M guanidine-HCl at pH 5.8, resulting in a 500-fold increase in the specific activity. A Pseudomonas-lytic enzyme from Streptomyces sp. P-51 was easily released from the adsorbent by elution with 0.5M NaCl at pH 5.0. A staphylolytic F2 enzyme from S. griseus S-35 and a chitinase [EC 3.2.1.14] from yam, both of which were completely inert toward M. lysodeikticus cell wall, passed through the adsorbent column. A modified ligand, in which muramic acid and glucosamine residues were N,O-acetylated, failed to adsorb any of these animal and bacterial lysozymes. Some of the enzymatic properties and bacteriolytic action spectra of these purified enzymes are also described in this paper in comparison with those of hen egg-white lysozyme.     

Isolation of the lymphocytosis promoting factor-haemagglutinin of Bordetella pertussis by affinity chromatography.

The lymphocytosis promoting factor-haemagglutinin of Bordetella pertussis was isolated from solutions obtained after cell disintegration by a novel affinity chromatographic method using an adsorbent composed of human haptoglobin covalently attached to a Sepharose 4B matrix. The haemagglutinin was bound to the adsorbent at pH 6.5 and eluted by a stepwise change to a pH 10 buffer. A 300--600-fold purification of the haemagglutinin was achieved by this one-step process. The chemical and biological properties of the haemagglutinin isolated by affinity chromatography were found to be similar to those of the protein isolated by other workers from culture supernatants. The affinity chromatographic method was found to be specific for the purification of the lymphocytosis promoting factor-haemagglutinin and no purification of the fimbrial-haemagglutinin of Bordetella pertussis was achieved by the method.     

Semisynthetic fluorohydrolases prepared by chemical modification of ribonuclease.

A process for conformational modification of protein, which we have previously reported, was investigated as a means of generating fluorohydrolase activity in bovine ribonuclease (RNase). The resulting modified RNase had catalytic activity that depended upon the chosen modifier. Bovine pancreatic ribonuclease, modified by addition of hexamethylphosphoramide (HMPA) at pH 3, was derivatized with diimidates of chain lengths from C1 to C8. The derivative with the highest activity was obtained when RNase was crosslinked with dimethyl pimelimidate (C5). This derivative, which was active over a pH range of 6.5 to 8.0 with an optimum pH of 7.4, hydrolyzed phenylmethylsulfonylfluoride (PMSF) and the potent acetylcholinesterase inhibitor, diisopropyl phosphorofluoridate (DFP). The mean fluorohydrolase activity for four preparations using dimethyl pimelimidate was 0.8 +/- 0.2 U mg-1. Gel filtration on G-75 Sephadex and SDS-polyacrylamide gel electrophoresis showed components having a molecular weight of 13,000 and 27,000, with activity restricted to the 27,000 molecular weight fraction. After gel filtration, the specific activity was 9.1 +/- 2.4 U mg-1, resulting in a molecular activity of 125 min-1. The mechanism of this unique transformation of RNase into a fluorohydrolase is not known, nor has the location of the active site been determined.     

Some radiometric microassays for butyrylcholinesterase.

The use of commercially available 5′-UTP-agarose as an affinity chromatography resin for RNase has been described. It was shown that at pH 5.3, 0.025 m piperazine-HCl buffer was effective for the adsorption of active RNase A and exhibited little nonbiospecific binding as has been shown earlier for SepharoseaPhpUp [Stewart, G. R., and Stevenson, K. J. (1973) Biochem. J.135, 427–441]. Phosphate buffer at either pH 3.0 or 5.45 eluted essentially all of the RNase activity added to the column; however, pH 5.45 was slightly more efficient. Competitive elution experiments with 2′(3′)-UMP yielded a linear plot of $\text{1}\text{(V ? V}{}_{\mathrm{o}}$ vs [I]. From this plot K_I and K_IM were calculated to be 70 and 130 μm, respectively. It is suggested that since this material is different from that which is used most often for RNase A affinity chromatography, it may prove useful for RNase binding studies.     

Citrate synthases from germinating castor bean seeds – I. Purification and properties

The mitochondrial and glyoxysomal citrate synthase (EC 4.1.3.7) from the endosperm of germinating castor beans ( Ricinus communis L., type Sanzibaricnsis) were purified to a final specific activity of 76 and 78 U (mg protein)⁻¹, respectively. Both citrate synthases could be bound to ATP-Sepharose. However, only the mitochondrial enzyme could be eluted by either 100 μ M oxaloacetate or 100 μ M coenzyme A (indicative of affinity chromatography), while the glyoxysomal enzyme was only eluted by 0.5 M KCI (indicative of ion-exchange chromatography). Many properties of the two isoenzymes were similar including the pH dependence and temperature dependence of activity, the pH stability, and the inactivation of the enzyme at elevated temperatures. The most pronounced differences between the two citrate synthases were the isolelectric points of pH 5.9 for the mitochondrial and of pH 9.1 for the glyoxysomal enzyme. Both citrate synthases are dimers in the native form with a molecular weight of 95000 each, as determined by gel filtration on Sepharose CL-6B and by polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. However, the glyoxysomal citrate synthase existed also as a tetramer with a molecular weight of 200000 in the presence of 10 m M MgCl₂.     

Separation of human renal renin and pseudorenin by affinity chromatography on hemoglobin-Sepharose-2B

Human renal renin (EC 3.4.99.19) and pseudorenin were easily separated in a single step by affinity chromatography on hemoglobin-Sepharose-2B. Renin and pseudorenin were monitored by their actions on crude and partially purified hog protein renin substrates at neutral and acidic pH and on synthetic labelled polymeric renin substrate. Under the conditions employed (0.1 M sodium acetate (pH 3.5)/1 M sodium chloride at 4 degrees C) renin does not bind to the affinity adsorbent while pseudorenin is effectively bound and can be eluted only after raising the pH to 6.5. Pseudorenin-free renin prepared by this method is devoid of proteolytic activity toward hemoglobin. The chromatographic behaviour of renal pseudorenin on hemoglobin-Sepharose-2B is similar to that of cathepsin D.     

Heterogeneity of alkaline ribonuclease in the mouse and Ehrlich ascites cells.

The specific activity of alkaline RNase II was l00 to 1800 times higher in mouse pancreas than in mouse liver, serum, ascites fluid, and Ehrlich ascites cell grown intraperitoneally. Ehrlich ascites cells grown in cell culture medium had a much lower alkaline RNase II activity than cells grown intraperitoneally. Chromatography on CM-52 cellulose of acid- and heat-treated preparations showned a considerable heterogeneity of the mouse enzymes. Depending on the source of the extract, two to six forms fo alkaline RNase were eluted. Pancreatic extract contained two RNase forms. These also seemed to be present as minor components in preparations from other sources except Ehrlich ascites cells grown in vitro. Ehrlich ascites cells grown in vivo contained forms of the RNase which were not present in other extracts. Possible reasons for this heterogeneity were investigated. In addition to their stability to acid and heat the different RNase forms were similar in that they were much more active at alkaline pH than at acidic pH, they did not require divalent metal ions for activity, and they degraded RNA 'endonucleolytically.' Also, native DNA, denatured DNA, and poly A were poor substrates compared with RNA. Some differences seemed to exist, however, with respect to their abilities to degrade poly U and poly C and their sensitivities to the endogenous RNase inhibitor.     

An agarose derivative containing an arsenical for affinity chromatography of thiol compounds

An adsorbent for affinity chromatography of thiol compounds was prepared by covalently linking p-aminophenylarsine oxide to CNBr-activated Sepharose 6B. The adsorbent retained mono- and dithiols from acid and neutral solutions. Monothiols and 1,4-dithiols were eluted at pH 12, whereas more alkaline conditions were necessary for elution of 1,2- and 1,3-dithiols. The latter could also be eluted at pH 12 by 0.01 m phenylarsine oxide. Alternatively, thiol compounds may be eluted at neutral or acid conditions by other thiol compounds. Monothiols and 1,4-dithiols were thus eluted by cysteine, whereas 1,2- and 1,3-dithiols required dithiopropylamine for elution.     

Affinity chromatography of alpha-chymotrypsin, subtilism, and metalloendopeptidases on carbobenzoxy-L-phenylalanyl-triehtylenetetraminyl-sepharose.

Carbobenzoxy-L-phenylalanyl-triethylenetetraminyl-Sepharose (Z-L-Phe-T-Sepharose) was found to be an effective affinity adsorbent for bovine pancreatic alpha-chymotrypsin [EC 3.4.21.1] as well as neutral [EC 3.4.24.4] and alkaline [EC 3.4.21.14] proteases of Bacillus species. These enzymes were adsorbed in the neutral pH range. alpha-Chymotrypsin was recovered by elution with 0.1 A acetic acid while neutral subtilopeptidase was eluted with 0.5 M NaCl at pH 0. Thermolysin and subtilisin were found in eluates with 1.5 and 2.0 M guanidine-HCl at pH 7.2, respectively. The resulting enzymes appeared homogeneous on disc-electrophoresis and showed higher specific activities than those of crystalline or highly purified preparations available commercially. Modifications of the active site serines of alpha-chymotrypsin and subtilisin by treatment with diisopropylfluorophosphate (DFP) or phenylmethanesulfonyl fluoride (PMSF) resulted in loss in their binding abilities to the adsorbent. Complexes of porcine alpha2-macroglobulin with each of these four enzymes and that of Streptomyces-subtilisin inhibitor (S-SI) with subtilisin were also found in nonadsorbed fractions.     

Contamination of ribosome inactivating proteins with ribonucleases,separated by affinity chromatography on red sepharose

Three preparations of type 1 ribosome inactivating proteins (RIPs), namely, agrostin, saporin, and luffin, were subjected to affinity chromatography on Red Sepharose and eluted with a linear concentration gradient of NaCl in 10 mM Tris-HCl buffer (pH 7.4). The eluate was assayed for ability to inhibit translation in a cell-free rabbit reticulocyte lysate system which measures RIP activity, and for ability to hydrolyze yeast transfer RNA which measures RNase activity. It was found that, in all three RIP preparations, the peak of RIP activity, which coincided with the peak of absorbance at 280 nm, was eluted earlier than the peak of RNase activity. It appears that RNase is a possible contaminant of ribosome inactivating protein preparations and that this contamination can be minimized by using Red Sepharose.     

Two ribonucleases H from cultured plant cells.

Two forms of enzyme with ribonuclease H (RNase H) [EC 3.1.4.34] activities, have been partially purified from cultured plant cells, strain GD-2, derived from carrot root. One is an Mn2+-dependent RNase H, and the second is an Mg2+-dependent RNase H. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at around 0.2 M and 0.4 M potassium chloride in phosphocellulose chromatography, and were further purified using blue Sepharose. Mg2+-dependent RNase H exhibits maximal activity at pH 9.0, and requires 10 to 15 mM Mg2+ for maximal activity, whereas the Mn2+-dependent enzyme is most active at pH 8.0, is maximally active at an Mn2+ concentration of 0.4 mM, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. The enzymes liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. The apparent molecular weight of the Mg2+-dependent RNase H was estimated to 18--20 X 10(4) and that of the Mn 2+- dependent RNase H was estimated to be 14 x 10(4) by gel filtration.     

The thrombin-like enzyme from Bothrops atrox snake venom. Properties of the enzyme purified by affinity chromatography on p-aminobenzamidine-substituted agarose.

The trhombin-like activities from the snake venoms of two subspecies of Bothrops atrox, moojeni (type I) and marajoensis (type II), were purified to homogeneity by affinity chromatography on a support consisting of the inhibitor, p-aminobenzamidine, linked to Sepharose 4B with a spacer of diaminodipropylaminosuccinate. At room temperature the enzyme was not bound to the affinity support but rather was retarded in relation to the unbound protein. As a result the thrombin-like activity eluted in a large volume following the main protein fraction. However, at 4 degrees the enzyme was absorbed to the affinity support and could be eluted specifically with the ligand benzamidine (0.15 M). Optimal conditions for column loading and washing were 0.05 M Tris.HCl/0.4 M NaCl, pH 9.0 at 4 degrees. The type I enzyme isolated in this manner showed a single major band on pH 8.9 disc gel electrophoresis as well as two minor bands. Further purification by isoelectric focusing yielded one major and two minor components. All three protein fractions had identical thrombin-like activities and amino acid composition. The major band had a specific activity of 210 to 230 NIH thrombin units/mg, a S20, w of 2.65 S, a molecular weight of 29,000, and an E1% 280 of 15.6. This protein has a carbohydrate content, measured as hexose, glucosamine, and sialic acid, of 27%. From the amino acid and carbohydrate composition a partial specific volume of 0.700 ml/g was calculated. The type I enzyme, purified on affinity chromatography only, did not activate Factor XIII and was free of thromboplastin-like activity. The type II enzyme behaved very differently from the type I on pH 8.9 polyacrylamide disc gels yielding two major bands and two minor bands. The relative amounts of these four bands were not a function of purity. The type II enzyme had a specific activity of 650 to 700 NIH thrombin units/mg, a S20, w of 2.60, and a molecular weight of 31,400.     

Affinity chromatography and separation of the molecular forms of monkey brain alpha-L-fucosidase on fucose-linked sepharose.

A simple affinity system which required coupling of alpha-L-fucose to Sepharose 4B by epichlorohydrin treatment of Sepharose 4B in the presence of alpha-L-fucose under alkaline conditions has been described. A partially purified preparation of monkey brain alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) was resolved at pH 5.0 into two major fractions: one bound and one retarded. The enzyme bound to the affinity column and specifically eluted by 2 mM alpha-L-fucose at pH 5.0 appeared to be homogeneous by polyacrylamide gel electrophoresis and was constituted mainly by the tetrameric form of the enzyme. The enzyme fraction retarded by the affinity column was found to contain mainly the monomeric form of the enzyme. Additional evidence for the different molecular forms of the enzyme in the bound and retarded fractions came from pH activity profiles and heat inactivation studies. The fucose-Sepharose appeared to bind the tetrameric form of the enzyme specifically and, further, alpha-L-fucose helped to retain the molecular integrity of the tetrameric enzyme.     

Ethoxyformylation of ribonuclease U2 form Ustilago sphaerogena.

RNase U2 was inactivated by incubation with ethoxyformic anhydride at pH 6.0 and pH 4.5. The absorbance of the RNase U2 increased at around 250 nm and decreased at around 280 nm. The inactivation occurred in parallel with the amount of modified histidine and plots of the relationship between the remaining activity and the modified histidine suggested that the modification of one of the two histidine residues totally inactivated the enzyme. The inactivated enzyme RNase U2 was reactivated by a low concentration of hydroxyamine, with removal of the ethoxyformyl group from the modified histidine residue. At pH 4.5, 2'-adenylate and 2'-guanylate protected RNase U2 from inactivation by ethoxyformic anhydride. The difference CD spectra showed that the ability of RNase U2 to form a complex with 2'-adenylate was lost on ethoxyformylation.     

Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers.

The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure from pairs of purified variants. Several chromatographically distinct peaks of differing enzymological properties were purified from each hybridization mixture in quantities of up to a few milligrams, and represented distinct species of hybrid hexamers differing in subunit ratio.     

Purification of the D-2 dopamine receptor from bovine striatum

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

Interaction of the estradiol receptor from calf uterus with its nuclear acceptor sites.

The specific interaction between 17 beta-estradiol-receptor complex and nuclear acceptors was analyzed by immobilizing various nuclear proteins to CNBr-activated agarose. The specific, high affinity sites identified in a fraction of basic proteins that can be solubilized from purified nuclei of calf uterus (Puca, G.A., Sica, V., and Nola. E (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 979-983) were chromatographed on Sephadex G-100 columns. Elution of the acceptor activity depends on the pH and ionic strength of the buffer used. With 5 mM HCl, however, a peak of acceptor activity with a molecular weight of about 70,000 was partially dissociated from the other basic nuclear proteins. The high affinity binding of the receptor to the acceptor proteins was estradiol-, but not progesterone-, cortisone-, or testosterone-dependent; it was very sensitive to ionic strength and showed a physiological pH optimum. Low affinity binding, such as that seen between receptor and histone, showed no estradiol dependence and little ionic strength and pH sensitivity. Native or heat-denatured DNA strongly modified the receptor-acceptor interaction, reducing the number of binding sites of acceptor for the receptor without changing the high affinity of the interaction. Heating of the acceptor protein before its covalent linkage to agarose considerably increased the affinity of the resulting agarose derivative. Free sulfhydryl groups of the receptor but not of the acceptor molecule play an important role in the acceptor-receptor interaction. When receptor and acceptor preparations were incubated in solution, the resulting complex was included on a Sephadex G-100 column and it eluted from DEAE-cellulose columns at lower ionic strength than the receptor alone. Even though not absolutely specific, these two properties allowed determination of the molecular weight (85,000) of the acceptor protein at neutral pH and more nearly physiological ionic strength. The apparent KD of the acceptor-receptor interaction was determined to be 2 x 10(-10) M at O degrees. Apparently similar, high affinity binding sites for estradiol receptors are also present in nuclei of other tissues.     

Affinity chromatography of amine oxidase from Aspergillus niger.

Omega-Aminohexyl-Sepharose 4B served as an excellent biospecific adsorbent for affinity chromatography of amine oxidase (monoamine:O2 oxidoreductase (deaminating), EC 1.4.3.4) from Aspergillus niger. The enzyme was completely adsorbed on this affinity resin when applied to a column in 0.1 M potassium phosphate buffer (pH 7.2). Although a small part of the enzyme was retained on the column through ionic interaction and eluted with 1.0 M potassium phosphate buffer (pH 7.2), most of the enzyme adsorbed was eluted with 0.5 M potassium phosphate buffer (pH 7.2) containing 10 mM butylamine. Essentially no retention of the enzyme on a column of epsilon-aminopentyl-Sepharose or delta-aminobutyl-Sepharose occurred under the same conditions, indicating that an appropriate length (more than approx. 12 A) of a hydrocarbon extension between the agarose matrix and the terminal amino group would be necessary for efficient adsorption of amine oxidase. The modification of the enzyme with 3-methyl-2-benzothiazolinone hydrazone (carbonyl inhibitor) or dithionite (reducing agent) resulted in loss of the ability to bind to omega-aminohexyl-Sepharose. It was also demonstrated that the affinity chromatography on omega-aminohexyl-Sepharose can be used as a powerful means of purifying this enzyme from crude extracts of Aspergillus niger. All of the three adsorbents were effective as a substrate in the amine oxidase reaction, but their substrate activities were as low as the corresponding free diamines.