Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

Hydrodynamic characterization of vasoactive intestinal peptide receptors extracted from rat lung membranes in Triton X-100 and n-octyl-beta-D-glucopyranoside

Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane.     

Purification of a 130-kDa T cell glycoprotein that binds human interleukin 4 with high affinity

The interleukin 4 (IL-4) receptor was purified from the gibbon T cell line MLA 144. These cells were found to express high numbers of human IL-4-binding proteins (5000-6000 sites/cell) with an affinity constant (Kd) similar to that measured in human cell lines (Kd = 40-70 pM). Affinity cross-linking of 125I-IL-4 to human cell lines and MLA 144 cells demonstrated the labeling of three proteins of approximately 130, 75, and 65 kDa. Human IL-4-binding sites were solubilized from MLA 144 cells using Triton X-100 and then purified by carboxymethyl chromatography, which removed 50% of the protein without loss of IL-4-binding activity. Then sequential affinity purification over wheat germ agglutinin and a single IL-4 Affi-Gel 10 column resulted in a final 8000-fold purification of the IL-4 receptor. When analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel, the purified receptor migrated as a single molecular species of 130 +/- 5 kDa. Identification of the 130-kDa protein as the IL-4 receptor was demonstrated by cross-linking experiments and specific binding of 125I-IL-4 to nitrocellulose membranes after electrophoretic transfer of the purified receptor on sodium dodecyl sulfate-polyacrylamide gel.     

Purification of human placental acid alpha-mannosidase by an immunological method

An immunoaffinity column was used for the purification of alpha-mannosidase from human placenta. The enzyme was purified to homogeneity by extraction in the presence of various protease inhibitors, immunoaffinity chromatography, Ultrogel AcA-34 gel filtration and hydroxyapatite chromatography. Two subunits were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their molecular weights were 65 kDa and 27 kDa. Heterogeneity of the molecular weight of the large subunit was not observed in our preparation. This method is relatively simple and rapid for obtaining the purified enzyme which is structurally not modified during purification procedures.     

Structural studies of Fc receptors. III. Isolation and molecular weight analysis of the component chain of Fc gamma receptor of macrophage

Surface receptors of guinea pig peritoneal macrophages specific for the Fc region of IgG (Fc gamma receptor) were isolated and identified as a surface-radioiodinated component with a molecular weight of 44,000 that bound in an Fc-specific manner to IgG2 of guinea pig immunoglobulin immobilized in any of the following three different ways: IgG2 antibody in insoluble immune complex, IgG2 antibody bound to antigen-coupled Sepharose, and IgG2 covalently coupled to Sepharose. In order to obtain the Fc gamma receptor retaining the binding activity, the Fc-binding component was isolated by IgG2 affinity chromatography in which mild acidic buffer (pH 5.0-4.0) was chosen to elute the component bound to the affinity column. Forty-five to sixty-two percent of the eluted radioactivity was shown to rebind to the IgG2-affinity column. The bound fraction showed a single radioactive peak of 44,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Fc-binding component isolated by the affinity chromatography behaved similarly in gel filtration in the presence of a detergent, as did the detergent-solubilized Fc gamma receptor before isolation by affinity chromatography. These results suggested that the Fc gamma receptor was isolated in a native form. Furthermore, it was confirmed that the isolated Fc gamma receptor is distinct from actin or the actin-like protein (DNase I-binding protein) which had been reported to bind to IgG-affinity column.     

Isolation and characterization of methionine synthetase from human placenta

The cobalamin-dependent enzyme, methionine synthetase, has been purified approximately 1000-fold to apparent homogeneity from human placenta with a 19% recovery. The final two steps of the purification utilized two different affinity columns. The first was a N5-methyltetrahydrofolate-cystamine-agarose column, and the second was a S-adenosylhomocysteine-agarose column. The enzyme was eluted from the first affinity column by buffer containing reducing agent which released the folate and the enzyme while elution from the second affinity column was accomplished with buffer containing 0.5 M sodium chloride. Criteria for purity were the observations that single peaks of enzyme activity, protein, and cobalamin with an apparent molecular weight of 160,000 were obtained by gel filtration and that holomethionine synthetase contained 1 mol of cobalamin/mol of protein. Furthermore, analysis by high performance liquid chromatography using a molecular weight sizing column demonstrated a single peak of protein with a corresponding cobalamin peak. This single peak of protein was progressively converted to a second protein peak that was enzymatically inactive, and this conversion was associated with a directly proportional loss of enzyme activity and cobalamin from the first peak. Methionine synthetase appeared to have a molecular weight of 160,000 on unreduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and subunits of Mr 90,000, 45,000, and 35,000 on reduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.     

Purification of a nocardicin A-sensitive LD-carboxypeptidase from Escherichia coli by affinity chromatography.

An LD-carboxypeptidase releasing the terminal D-Ala from UDP-MurNAc-L-Ala-D-Glu-m-A2pm-D-Ala (UDP-MurNAc-tetrapeptide) was purified from Escherichia coli to biochemical homogeneity and characterized biochemically. Final purification was achieved by nocardicin A-Sepharose affinity chromatography. An apparent molecular weight of 32,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme, which seems to be a monomeric protein as indicated by gel filtration. The optimum pH of the enzyme was 8.4, and the pI was 5.5. The Km for UDP-MurNAc-tetrapeptide was 1.5 x 10(-4) M, and the Vmax was 0.4 nmol/min. Nocardicin A inhibited the enzyme competitively, with a Ki of 5 x 10(-5) M. Benzylpenicillin, cephalosporin C, thienamycin, and D-alanyl-D-alanine did not affect the enzyme activity. Possible functions of the enzyme for growth and division of the murein sacculus are discussed.     

Two glutamyl-tRNA reductase activities in Escherichia coli

delta-Aminolevulinic acid (ALA) is the first committed precursor for tetrapyrrole biosynthesis. ALA formation in Escherichia coli occurs in a tRNA-dependent three-step conversion from glutamate. Glu-tRNA reductase is the key enzyme in this pathway. E. coli K12 contains two Glu-tRNA reductase activities which differ in their molecular weights. Here we describe the purification of one of these enzymes. Four different chromatographic separations yielded a nearly homogeneous protein. Its apparent molecular mass under denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation and gel filtration) is 85,000 +/- 5,000 Da. This indicates a monomeric structure for the active enzyme. Gel filtration and glycerol gradient centrifugation indicate that the other activity has a molecular mass of 45,000 +/- 5,000 Da. In the presence of NADPH both enzyme activities converted E. coli Glu-tRNA(2Glu) to glutamate 1-semialdehyde. Addition of GTP or hemin did not affect the reductase activity. Both enzymes display sequence-specific recognition of tRNA; E. coli Glu-tRNA(2Glu) is a good substrate while the Chlamydomonas reinhardtii, Bacillus subtilis, and Synechocystis Glu-tRNA(Glu) species are poorly recognized.     

Separation and purification of lymphocyte chemotactic factor (LCF) and interleukin 2 produced by human peripheral blood mononuclear cells

Two T-cell chemotactic factors, lymphocyte chemotactic factor (LCF) and interleukin 2 (IL-2), were separated and characterized from culture supernatants of concanavalin A-stimulated human peripheral blood mononuclear cells. LCF was purified approximately 7800-fold to homogeneity from culture supernatant using gel filtration and high-performance liquid chromatography (HPLC). LCF was found to be distinct from both IL-2 and interleukin-1. Sephadex G-100 gel filtration of crude supernatants from concanavalin A-stimulated mononuclear cells showed two molecular weight regions of T lymphocyte chemotactic activity. A 10,000- to 25,000-Da region contained both IL-2 and LCF and a 45,000- to 75,000-Da region contained only a high molecular weight form of LCF. Both high and low molecular weight species of LCF eluted with 40-44% acetonitrile from a reversed-phase C18 HPLC column. IL-2 present only in the low molecular weight region eluted from the C18 column with 65-75% acetonitrile. The migration of T lymphocytes to IL-2 was totally inhibited by anti-interleukin 2 receptor antibody while the response of T cells to LCF was unaffected. LCF eluting off the C18 column was purified to homogeneity by two subsequent cycles of gel filtration HPLC. The resultant protein showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 10,500. The data presented here demonstrate that IL-2 and LCF are distinct lymphocyte chemotactic factors and although they are not readily separable from crude supernatants by molecular sieve chromatography, they can easily be distinguished by reversed-phase HPLC.     

Human monocyte carboxylesterase. Purification and kinetics

Human peripheral blood monocytes were isolated by density gradient centrifugation and purified by counterflow centrifugation elutriation. Membrane-localized carboxylesterase (CBE) was extracted with nonionic detergent (Triton X-100) and purified by ion exchange (DEAE-cellulose), gel filtration (Sephacryl S-300), hydroxylapatite column, and high performance liquid chromatography. The purified enzyme migrated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single protein band with a molecular weight of 60,000. Under nondenaturing conditions, monocyte CBE formed a trimer and eluted from a gel filtration column as a protein with an approximate molecular weight of 200,000. Electrophoretic patterns of the enzyme on polyacrylamide gels run a neutral pH did not vary during enzyme purification. At least four major isoenzymes of human monocyte CBE were observed with isoelectric points between 7.5 and 7.8. Pure human monocyte CBE hydrolyzed short chain alpha-naphthyl, o-nitrophenyl, and p-nitrophenyl esters. Amide esters and thioesters were not hydrolyzed by the enzyme. Short chain alcohols activated the enzyme and organophosphorus compounds, diphenyl carbonate, sodium fluoride, and phenylmethylsulfonyl fluoride inhibited the enzyme. EDTA and sulfhydryl reagents had no effect on enzyme activity. The amino acid content of the enzyme was consistent with other CBEs. Inhibitors reacted either with the active or effector site of the enzyme. Purified enzyme now permits the characterization of CBE structure and regulation.     

Activation of the rat liver cytosol glucocorticoid receptor by sephacryl S-300 filtration in the presence and absence of molybdate. Physical properties of the receptor and evidence for an activation inhibitor

The DNA-binding and physical properties of the rat liver cytosol glucocorticoid receptor were determined before and after Sephacryl S-300 filtration in the presence or absence of molybdate. Cytosol was prepared and labeled with [3H]triamcinolone acetonide in buffer containing molybdate. Prior to gel filtration, only 5 +/- 3% (mean +/- S.E.) of labeled receptors bound to DNA-cellulose. After gel filtration in the presence and absence of molybdate, the per cent of labeled receptors binding to DNA-cellulose was 57 +/- 10% and 83 +/- 1%, respectively. Nonreceptor fractions from the Sephacryl S-300 column contained a heat-stable factor which blocked receptor activation but did not block the binding of activated receptors to DNA-cellulose. The activation inhibitor eluted from the column in the region of the albumin standard, but after heating its size was considerably reduced (Mr less than 3500). Receptors activated by Sephacryl S-300 filtration underwent the same size changes in the presence or absence of molybdate. Prior to gel filtration, the S20,w of labeled receptors in the presence of molybdate was 9.2 +/- 0.2 S. After filtration in the presence and absence of molybdate, the S20,w of labeled receptors was 4.2 +/- 0.2 and 4.4 +/- 0.1 S, respectively. The Stokes radius (Rs) of labeled receptors after gel filtration in either the presence or absence of molybdate was 65 +/- 1 A. From the Rs and S20,w values, the molecular weight (Mr) of activated receptors was calculated to be 115,000 to 121,000, which was in close agreement with the Mr of affinity-labeled receptors determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Purification and characterization of glutamate decarboxylase from Neurospora crassa conidia.

L-Glutamate decarboxylase, an enzyme under the control of the asexual developmental cycle of Neurospora crassa, was purified to homogeneity from conidia. The purification procedure included ammonium sulfate fractionation and DEAE-Sephadex and cellulose phosphate column chromatography. The final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gels with a molecular weight of 33,200 +/- 200. A single band coincident with enzyme activity was found on native 7.5% polyacrylamide gels. The molecular weight of glutamate decarboxylase was 30,500 as determined by gel permeation column chromatography at pH 6.0. The enzyme had an acidic pH optimum and showed hyperbolic kinetics at pH 5.5 with a Km for glutamic acid of 2.2 mM and a Km for pyridoxal-5'-phosphate of 0.04 microM.     

Cellular retinoic acid-binding protein from rat testis. Purification and characterization

Cellular retinoic acid-binding protein has been purified to homogeneity from rat testes. The procedures utilized in the purification included acid precipitation, gel filtration, and chromatography on DEAE-cellulose. The binding protein was purified approximately 12,000-fold, based on total soluble testicular protein. The protein is a single polypeptide chain with a molecular weight of 14,600, determined by information from gel filtration and sodium dodecyl sulfate-polyacrylamide electrophoresis. The protein binds retinoic acid with high affinity; the apparent dissociation constant was determined by fluorometric titration to be 4.2 X 10(-9) M.     

Solubilization of the Neuropeptide Y Receptor from Rat Brain Membranes

The neuropeptide Y (NPY) receptor was solubilized from rat brain membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The binding of 125I-NPY to CHAPS extracts was protein, time, and temperature dependent. Unlabeled NPY and the related peptides peptide YY (PYY) and pancreatic polypeptide inhibited 125I-NPY binding to solubilized receptors with relative potencies similar to those seen with membrane-bound receptors: NPY greater than PYY much greater than pancreatic polypeptide. Scatchard analysis of equilibrium binding data showed the CHAPS extracts to contain a single population of binding sites with a KD of 3.6 +/- 0.4 nM (mean +/- SEM) and a Bmax of 5.0 +/- 0.2 pmol/mg of protein. In addition the 125I-NPY binding to the soluble receptor was not inhibited by guanosine-5'-O-(3-thiotriphosphate), in contrast to the GTP sensitivity displayed by the membrane-bound receptor. Gel filtration chromatography using Sepharose 6B revealed a single peak of binding activity corresponding to a Mr of approximately 67,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after chemical cross-linking revealed a single band at Mr 62,000. After solubilization and gel chromatography a 50- to 100-fold purification of the NPY receptor was obtained.     

Purification and some properties of cytotoxin produced by Clostridium difficile

The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.     

Purification and properties of heme oxygenase from rat liver microsomes.

Heme oxygenase was purified to apparent homogeneity from liver microsomes of rats which had been treated with either cobaltous chloride or hemin to induce heme oxygenase in the liver and the purified preparations from either rats showed an apparent molecular weight of about 200,000 when estimated by gel filtration on a column of Sephadex G-200, and gave a minimum molecular weight of about 32,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hepatic heme oxygenase could bind heme to form a heme . heme oxygenase complex showing an absorption peak at 405 nm, and the extinction coefficient at 405 nm of the heme . heme oxygenase complex was 140 mM-1 cm-1. The heme bound to the hepatic heme oxygenase protein was easily converted to biliverdin when the complex was incubated with the NADPH-cytochrome c reductase system in air. The hepatic heme oxygenase appears to have characteristics essentially similar to those of the splenic heme oxygenase (Yoshida, T., and Kikuchi, G. (1978) J. Biol. Chem. 253, 4224 and 4230). The heme oxygenase preparation which was purified from the cobalt-treated rats contained a small amount of cobaltic protoporphyrin, indicating that cobalt protoporphyrin was synthesized in these rats.     

Glycogen phosphorylase from Neurospora crassa: purification of a high-specific-activity, non-phosphorylated form.

A highly active glycogen phosphorylase was purified from Neurospora crassa by polyethylene glycol fractionation at pH 6.16 combined with standard techniques (chromatography and salt fractionation). The final preparation had a specific activity of 65 +/- 5 U/mg of protein (synthetic direction, pH 6.1, 30 degrees C) and was homogeneous by the criteria of gel electrophoresis, amino-terminal analysis, gel filtration, and double immunodiffusion in two dimensions. The enzyme had a native molecular weight of 180,000 +/- 10,000 (by calibrated gel filtration and gel electrophoresis) and a subunit molecular weight of 90,000 +/- 5,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each subunit contained one molecule of pyridoxal phosphate. No phosphoserine or phosphothreonine was detected by amino acid analysis optimized for phosphoamino acid detection. The enzyme isolated from cells grown on high-specific-activity 32Pi (as sole source of phosphorus) contained one atom of 32P per subunit. All the radioactivity was removed by procedures that removed pyridoxal phosphate. Thus, the enzyme could not be classified as an a type (phosphorylated, active in the absence of a cofactor) or as a b type (non-phosphorylated, inactive in the absence of a cofactor). The level of phosphorylase was markedly increased in mycelium taken from older cultures in which the carbon source (glucose or sucrose) had been depleted. The polyethylene glycol fractionation scheme applied at pH 7.5 to mycelial extracts of younger cultures (taken before depletion of the sugar) resulted in co-purification of glycogen phosphorylase and glycogen synthetase.     

Purification by affinity chromatography of the glycine receptor of rat spinal cord

The glycine receptor of rat spinal cord was solubilized with the nonionic detergent Triton X-100 and subsequently purified by affinity chromatography on aminostrychnine-agarose and wheat germ agglutinin-Sepharose. An overall purification of 1950-fold was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed three glycine receptor-associated polypeptides of Mr = 48,000, 58,000, and 93,000. [3H]Strychnine was incorporated irreversibly into the Mr = 48,000 polypeptide upon UV-illumination. The dissociation constant (KD) of [3H]strychnine binding to the purified glycine receptor was 9.3 +/- 0.6 nM. The glycine receptor agonists glycine, beta-alanine, and taurine inhibited the binding of [3H]strychnine to the purified receptor. Gel filtration and sedimentation in sucrose/H2O and sucrose/D2O gradients gave a Stokes radius of 7.7 nm, a partial specific volume of 0.780 +/- 0.005 ml/g and a sedimentation coefficient s20,w of 8.2 +/- 0.2 S for the purified glycine receptor. From these data, a molecular weight of 246,000 +/- 6,000 was calculated for the glycine receptor protein.     

Rapid, simplified method for production and purification of tetanus toxin

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.