Affinity purification of immunoglobulin M using a novel synthetic ligand

While monoclonal antibodies of the G class can be conveniently purified by affinity chromatography using immobilized protein A or G, even on a large scale, scaling up IgM purification still presents several problems, since specific and cost-effective ligands for IgM are not available. A synthetic peptide (TG19318), deduced from the screening of a combinatorial peptide library, was characterized previously by our group for its binding properties for immunoglobulins of the G class and its applicability as a synthetic ligand for polyclonal and monoclonal IgG purification, from sera or cell culture supernatants. In this study, we have examined the ligand recognition properties for IgM, immobilizing the synthetic peptide on different affinity supports and examining its ability to purify IgMs from serum, ascitic fluid and cell culture supernatants. TG19318 affinity columns proved useful for a very convenient one-step purification of monoclonal IgMs directly from crude sources, loading the samples on the columns equilibrated with saline buffers at pH values ranging from 5 to 7, and eluting adsorbed IgM by a buffer change to 0.1 M acetic acid or 0.05–0.1 M sodium bicarbonate, pH 9.0. Antibody purity after affinity purification was very high, close to 85–95%, as determined by densitometric scanning of sodium dodecyl sulfate–polyacrylamide gels of purified fractions, and by gel permeation analysis. Antibody activity was fully recovered after purification, as determined by immunoassays. Column capacity was related to the type of support used for ligand immobilization, and ranged from 2 to 8 mg of IgM/ml of support.     

Studies on Mold Protease

An acid protease of Rhizopus chinensis was purified by sequential chromatographies on columns of Duolite A-2, Sephadex G-100 and CM-cellulose, and crystallized from aqueous acetone solution. The preparation was shown to be monodisperse on column chromatography of ion-exchange sephadex and on ultracentrifugal analysis. The enzyme was most active at pH values between 2.9 and 3.3 and was stable over the range of pH 2.8 to 6.5. The protease was markedly inactivated by ferric ions and sodium lauryl sulfate, whereas it was affected by neither sulfhydryl reagents nor metal-chelating agents. In milk-clotting activity, the acid protease was shown to be one of the most potent enzymes among those of fungal origin. Substrate specificity experiments on several synthetic peptides indicated that the peptide bonds susceptible to the action of the enzyme were mainly those involving amino group of aromatic amino acids.     

The use of C8-octyl columns for the analysis of catecholamines by ion-pair reversed-phase liquid chromatography with amperometric detection

The chromatographic behavior of norepinephrine (NE), epinephrine, dopamine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylacetic acid on octylsilane (C₈) reversed-phase high-performance liquid chromatography columns was observed under various mobile phase conditions including manipulations of pH, pairing ion and methanol concentrations. The optimum isocratic conditions permitting quantitative resolution of these substances in minimum time and with maximum detector response were determined. Employing a pH 3.0–3.2 mobile phase comprising an aqueous buffer solution containing 0.1 M NaH₂PO₄, 0.1 mM EDTA, and 0.2 mM 1-octanesulfonate, admixed with a volume of methanol equal to 4% of the aqueous volume, the performance of the C₈ columns compares favorably to that of the more widely used C₁₈ columns. The column eluates were monitored with an amperometric detector utilizing a glassy-carbon flow-cell electrode. The detector response for NE was 1.5–2.0 nA/ng and the baseline noise was as little as 0.002 nA thereby permitting quantitation of 5-pg levels or more in the injected samples. By coupling the liquid chromatographic system to a procedure which eliminates non-catechol contaminants from the neuronal and body fluid specimens by alumina adsorption of the catechols, a sensitive and dependable method was developed and employed for the determination of catechol levels in discrete regions of rat brain, cat spinal cord, and in human plasma.     

Simple purification of aromatic -amino acid decarboxylase from human pheochromocytoma using high-performance liquid chromatography

We purified aromatic -amino acid decarboxylase (AADC) homogeneously and rapidly from human pheochromocytoma using high-performance liquid chromatography. HPLC with gel permeation and hydrophobic columns was highly effective, and the entire purification could be finished within 3 days. Purified AADC showed a single band with an M_r of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and decarboxylated -3,4-dihydroxyphenyl-alanine, -5-hydroxytryptophan, and -threo-3,4-dihydroxyphenylserine (a synthetic precursor of natural norepinephrine). Amino acid analysis of purified AADC was performed.     

Production,Purification and Properties of Ribonuclease from Aspergillus niger

Aspergillus niger NRC–A–1–233 was cultivated by the shaking method. The optimal cultural conditions for ribonuclease (RNase) production were: composition of medium: sucrose, 15%; NH₄NO₃, 0.2%; KH₂PO₄, 0.1%; MgSO₄·7 aq., 0.025%; initial pH, 2.2; shaking conditions: 50 ml of medium /500 ml flask; cultivation time, 120 hr. The RNase was purified by acid clay treatment and chromatography on DEAE-cellulose and Sephadex G–75 columns. The purified RNase was homogeneous by ultracentrifuge and disc electrophoresis.

The molecular weight of the RNase was estimated to be 28,500 on SDS-polyacrylamide gel and its isoelectric point was 2.8 by Ampholine electrofocusing method. Digestion rate of RNA by the RNase was 100%. The RNase did not have an exact base specificity and produced four kinds of 3′-nucleotides from yeast RNA.     

Extracellular Lytic Enzymes of Myxococcus virescens

An easy and rapid method for the purification of a bacteriolytic endopeptidase produced by Myxococcus virescens is described. The bacteria were grown in casitone media and the cells were sedimented by centrifugation. About 1.2 g of montmorillonite were added per liter of cell-free culture solution. The clay was sedimented by centrifugation and the enzyme was then eluted by 0.05 M Na-phosphate buffer pH 6.0, containing 0.4 M NaCl. The enzyme was diluted with water and chromatographed on carboxymethyl-cellulose columns. The purified enzyme liberated free amino groups but no reducing sugars or N-acetylhexosamines when acting on purified N-acetylated cell walls of Micrococcus lysodeikticus. Analysis of N- and C-terminal amino acids in the digestion products showed that the enzyme had liberated about 110 nmoles of lysine ε-amino groups and 60 nmoles of alanine carboxyl groups per mg of cell wall. When it acted on a bisdisaccharide pentapeptide dimer isolated from M. lysodeikticus cell walls, it cleaved about 30% of the alanyl-lysine linkages. Consequently the enzyme was an alanyl-lysine endopeptidase. It had no muramyl-alanine amidase activity.     

Separation of basic,hydrophilic peptides by reversed-phase ion-pair chromatography: II. Analytical applications with particular reference to phosphoserine peptides

Phosphoserine peptides, obtained by phosphorylation of synthetic precursors with cyclic AMP-dependent protein kinase, can be efficiently separated from the corresponding non-phosphorylated peptides and from each other by ion-pair high-performance liquid chromatography. All experiments were performed under isocratic conditions on a C₁₈ column, using phosphate buffers with pH 3.2–4.5, n-hexane sulfonic acid as counter ion, and ethanol as organic modifier.     

Qualitative Characterization of Some Peptides Inducing Morphogenesis in the Nematode- Trapping Fungus Arthrobotrys oligospora

Some peptides inducing capture organ formation in Arthrobotrys oligospora were characterized with regard to their amino acid contents. The N-terminal, C-terminal and total amino acids were determined as their dinitrophenyl-derivatives by thin layer chromatography on silica gel in two different solvents. The amino acid composition was further confirmed by gas-liquid chromatography. The peptides investigated had a high proportion of non-polar and aromatic residues. Thus, leucine, isoleucine, valine, proline, and tyrosine were present in all the peptides. In addition, phenyl-alanine, glycine, and alanine occurred in some preparations. Tyrosine, valine, and phenylalanine were found in N-terminal position, and leucine or isoleucine were C-terminals.     

Biochemical Characterization of Secreted Proteases During Growth in Tetrahymena pyriformis WH-14: Comparison of Extracellular with Intracellular Proteases

Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and α-N-benzoyl-DL-arginine-ρ-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000–24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3, and 1.4 mM for P-I, P-II. P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S^-1.M^-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.     

Syngamus trachea: infections produced by prenatal inoculations of chicken embryos.

(1) Extracts of Ascaridia galli, a nematode parasite of domestic fowl (Gallus gallus), contained potent inhibitors of trypsin and chymotrypsin. (2) These inhibitors extracted by TCA and heat treatment and partially purified by DEAE- and CM-cellulose chromatography were found to be proteins of low molecular weight. (3) These inhibitors were nondialyzable, stable up to 15 min at 100 C, and active over a wide pH range (3–10). 8.0 M urea and 0.1 M β-mercaptoethanol had no effect on the inhibitors. (4) Complex formation between the inhibitors and trypsin and chymotrypsin was complete within 60 and 30 sec of contact, respectively.     

The inductive role of casein on proteinase production from Lactobacilli

Summary The ability of casein to stimulate proteinase production in pH controlled fermentations of variousLactobacillus strains was examined in MRS and whey permeate medium. All examined strains have maximum growth rate in substrates containing 0.5% casitone as amino acid source. In all cases lactic acid production coincides with the growth. When casitone was replaced by total caseine, the growth rate was several fold lower, but the specific activity of the proteinase was at least three fold higher. These results clearly show the inductive role of casein in proteinase production by lactobacilli.     

Regulation of the synthesis of mucin glycoproteins in swine trachea explants

Summary Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin glycoprotein was about 0.035 mg per cm² per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal incorporation was observed at 200 μM glucosamine. A higher concentration of³⁵SO₄, 1000 μM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 μg/ml increased the rate of secretion twofold, whereas 0.1 to 100 μg/ml of hydrocortisone and 0.1 to 100 μg/ml of epinephrine significantly decreased the rate of secretion. Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10⁻⁵ M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10⁻⁹ M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats. Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified. The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified by chromatography on Sepharose CL-6B columns under dissociating conditions in 2M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually indistingushable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same molar ratio ofN-acetylgalactosamine,N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations. This investigation was supported by U.S. Public Health Service Grants HL 20868, HL 24688, and HL 24718 from the National Heart, Lung and Blood Institute, Bethesda, MD, and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, Bethesda, MD.     

Acid phosphatase synthesis inAspergillus flavus

Proteins with phosphatase activity were produced during the growth ofAspergillus flavus in a phosphate-supplemented liquid synthetic medium. The best carbon and nitrogen sources for the synthesis of phosphatase were glucose and ammonium sulfate, respectively. The proteins were separated by molecular exclusion and ion exclusion chromatography (IEC) into three components one of which showed phosphatase activity. The molar mass of the enzyme was approximately 62 kDa. The purified enzyme exhibited an optimum activity at pH 4.0 and at 45°C. The activity of the enzyme was stimulated by Ca²⁺ and Mg²⁺ but inhibited by fluoride, iodoacetic acid, ethylenediaminetetraacetic acid and 2,4-dinitrophenol, and exhibited an apparentK _M of approximately 420 μmol/L.     

Fractionation of trinucleotides from partial micrococcal nuclease digests of calf thymus DNA

The enzymic digest is first fractionated according to chain length by column chromatography on DEAE-Sephadex in 7 M urea at neutral pH. The trimer fraction is further resolved according to Gp content on DEAE-Sephadex in 50% MeOH and NH₄-formate buffers at pH 5.2. The three subgroups of trinucleotides containing 0, 1, and 2 Gp residues, respectively, are then separated according to Ap, Cp, and Tp content on DEAE-cellulose in 7 M urea and 0.1 M formic acid. Among the 23 trimers so obtained, sequence isomers such as TpGpAp and ApGpTp are resolved by partition chromatography on cellulose columns with 30% NH₄-sulfate at neutral pH.     

Purification and Characterization of an Extracellular Alkaline Serine Protease from Aspergillus terreus (IJIRA 6.2)

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37°C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60°C. With Na-caseinate, the K _m of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue. Received: 8 October 1999 / Accepted: 3 November 1999     

Purification and Some Enzymatic Properties of Acid Protease A and B of Scytalidium lignicolum ATCC 24568

Three kinds of acid proteases were purified from the culture filtrate of Scytalidium lignicolum ATCC 24568. About 3 mg of A–1, 6 mg of A–2 and 60 mg of B were obtained from one liter of culture broth. These purified enzymes were monodisperse by physicochemical criteria such as ultracentrifugal analysis and disc electrophoresis.

A–1 and A–2 were very similar to each other on their enzymatic properties except the small difference of isoelectric point. A–1 and A–2 were active between pH 3.0~3.5 toward casein, and stable between pH 2.5 and 5.5 for 20 hr at 37°C. Both enzymes were strongly inhibited by NBS, but not by EDTA, DFP and sulfhydryl reagents.

B was most active at pH 2.0, and stable at pH values between 1.5 and 5.0. This enzyme was also inhibited by NBS and KMnO₄, but not by EDTA, DFP and sulfhydryl reagents.

The molecular weights and isoelectric points of A–1, A–2 and B were 43,000, pH 3.6; 43,000, pH 3.8 and 22,000, pH 3.2, respectively.

A–1 and A–2 were not inhibited by S–PI and synthetic pepsin inhibitor such as diazoacetyl-dl-norleucine methylester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). B was inhibited by EPNP, but not by S–PI and DAN.     

Preparation and purification of pteroic acid from folic acid

Crude pteroic acid, obtained by microbiological degradation of folic acid, has been purified by column chromatography on cellulose CF11, eluting with 0.1 M glycine buffer of pH 10.0, containing 0.15% w/v ascorbic acid and saturated with isoamyl alcohol.     

Purification and Some Properties of Acid Invertase of Persimmon Fruits

Single cells were prepared from mesocarp tissue of ripe persimmon (Diospyros kaki cv. Fuyu) fruits, and inter- or intracellular localization of acid invertase (AI, EC 3.2.1.26) was studied. AI was localized in the intercellular fraction (cell wall fraction). AI was isolated and purified from the cell wall fraction of ripe persimmon fruits by column chromatography on SE-53 cellulose and Toyopearl HW 55F. The specific activity of purified AI was 570 units per mg protein at 30°C. The molecular mass of AI was estimated to be 44 kDa by gel filtration over Sephacryl S-200 and 70 kDa by SDS–PAGE. The optimum pH of the activity for sucrose was 4.25. The purified enzyme hydrolyzed sucrose and raffinose but not melibiose. The enzyme had a K_m of 3.2 mM for sucrose and a K_m of 2.6 mM for raffinose. Silver nitrate (5 μM), HgCI₂ (2 μM), p-chloromercuribenzoate (100mM), pyridoxamine (10mM), and pyridoxine (2.5mM) inhibited AI activity by 95, 85, 100, 41, and 300%, respectively.     

Purification and characterization of a novel exopolygalacturonase from Fusarium oxysporum f.sp. lycopersici

A novel exopolygalacturonase (EC 3.2.1.15) was purified to apparent homogeneity from cultures of Fusarium oxysporum f.sp. lycopersici on synthetic medium supplemented with polygalacturonic acid, using two steps of purification: preparative isoelectric focusing and cationic exchange chromatography. The enzyme designated PG3 had an apparent M_r of 63 000±3000 Da upon SDS-PAGE and a pI of 7.0. PG3 was active within a broad range of pH from 3.5 to 9. The temperature optimum was 55°C. PG3 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The enzyme was N-glycosylated. The production of PG3 was constitutive at low levels, and synthesis was increased following induction by PGA and partially repressed by glucose.     

Fractionation of polyclonal antibodies to fragments of a neuroreceptor using three increasingly chaotropic solvents

We have developed specific antibodies against fragments of anaplastic lymphoma kinase (ALK) in order to develop tools for characterizing the expression and biological function of this orphan receptor. The first fragment consisted of residues 280 to 480 of the murine extracellular domain, was expressed in Escherichia coli (E. coli), purified in the presence of urea from the pellet of mechanically lysed cells and injected into rabbits as an unfolded protein in urea. The second fragment consisted of residues 1519 to 1619 of the murine sequence, corresponding to the C-terminal side of the kinase domain. It was expressed in E. coli as a soluble glutathione-S-transferase fusion protein, purified from the supernatant of broken cells and injected into rabbits as a folded protein. Both antisera were purified using antigen affinity chromatography, with the polyclonal antibodies eluted stepwise using three different buffers, 0.1 M glycine, pH 2.9, followed by 7 M urea, pH 4, followed by 6 M guanidine–HCl (GdnHCl), pH 4. Antisera prepared against either antigen contained antibodies that eluted in each of the three pools, indicating that solvents more chaotropic than acid were required to elute antibody populations that were tightly bound to the antigen column. All three antibody pools were reactive towards their respective antigens upon Western blot analysis. Purified polyclonal antibodies (pAbs) to both fragments also recognized the full-length protein expressed in Chinese hamster ovary cells. In every case, the pAbs eluting in GdnHCl were the most sensitive for detecting full-length ALK.