Efficient and rapid purification of recombinant human alpha-galactosidase A by affinity column chromatography

The lysosomal enzyme alpha-galactosidase A (alpha-Gal A) metabolizes neutral glycosphingolipids that possess alpha-galactoside residues at the non-reducing terminus, and inherited defects in the activity of alpha-Gal A lead to Fabry disease. We describe here an efficient and rapid purification procedure for recombinant alpha-Gal A by sequential Concanavalin A (Con A)-Sepharose and immobilized thio-alpha-galactoside (thio-Gal) agarose column chromatography. Optimal elution conditions for both columns were obtained using overexpressed human alpha-Gal A. We recommend the use of a mixture of 0.9 M methyl alpha-mannoside and 0.9 M methyl alpha-glucoside in 0.1 M acetate buffer (pH 6.0) with 0.1 M NaCl for the maximum recovery of glycoproteins with multiple high-mannose type sugar chains from Con A column chromatography, and that the Con A column should not be reused for the purification of glycoproteins that are used for structural studies. Binding of the enzyme to the thio-Gal column requires acidic condition at pH 4.8. A galactose-containing buffer (25 mM citrate-phosphate buffer, pH 5.5, with 0.1 M galactose, and 0.1 M NaCl) was used to elute alpha-Gal A. This procedure is especially useful for the purification of mutant forms of alpha-Gal A, which are not stable under conventional purification techniques. A protocol that purifies an intracellular mutant alpha-Gal A (M279I) expressed in COS-7 cells within 6h at 62% overall yield is presented.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Separation into two major forms of beta(1-3)N-acetylglucosaminyltransferase from human serum

The major beta(1-3)N-acetylglucosaminyltransferase [beta(1-3)GlcNAc-transferase] activity in human serum was isolated by DEAE- and CM-Sepharose column chromatography. This enzyme fraction consisted of two forms of the enzyme, which were separated from each other on a DEAE-Sepharose column and designated as GNAc-TI and GNAc-TII, respectively. They have the same molecular weights (about 90,000), optimum pH values (between pH 7.5 and 8.5) and apparent Km values for N-acetyllactosamine and lactose (7.1-8.8 mM and 10.9-11.5 mM, respectively). Glycosidase and methylation analyses of the reaction products demonstrated that both the enzyme catalyze exclusively the transfer of one N-acetylglucosamine to position C-3 of the terminal galactose of lactose in the beta linkage.     

Characterization of virus adsorption by using DEAE-sepharose and octyl-sepharoser

Viruses were characterized by their adsorption to DEAE-Sepharose or by their elution from octyl-Sepharose by using buffered solutions of sodium chloride with different ionic strengths. Viruses whose adsorption to DEAE-Sepharose was reduced most rapidly by an increase in the sodium chloride concentration were considered to have the weakest electrostatic interactions with the solids; these viruses included MS2, E1, and phiX174. Viruses whose adsorption to DEAE-Sepharose was reduced least rapidly were considered to have the strongest electrostatic interactions with the column; these viruses included P1, T4, T2, and E5. All of the viruses studied adsorbed to octyl-Sepharose in the presence of 4 M NaCl. Viruses that were eluted most rapidly following a decrease in the concentration of NaCl were considered to have the weakest hydrophobic interactions with the column; these viruses included phiX174, CB4, and E1. Viruses that were eluted least rapidly from the columns after the NaCl concentration was decreased were considered to have the strongest hydrophobic interactions with the column; these viruses included f2, MS2, and E5.     

Isolation and characterization of an antithrombin III variant with reduced carbohydrate content and enhanced heparin binding

Two distinct forms of antithrombin III were isolated by chromatography of normal human plasma on heparin-Sepharose. The predominant antithrombin species present (AT-III alpha), which eluted from the affinity column in 1 M NaCl, was identified as the antithrombin III form which has been previously characterized. Ionic strength of the buffer was increased to elute a variant form of antithrombin III, designated as AT-III beta. The molecular weight of AT-III beta is less than that of AT-III alpha, but physicochemical studies do not indicate measureable differences in the polypeptide portion of the proteins. Carbohydrate determination revealed the sole detectable structural difference in the two antithrombins: levels of hexosamine, neutral sugars, and sialic acid in AT-III beta were all 25-30% less than in AT-III alpha. Kinetic studies of thrombin inactivation by both antithrombins, in the presence of nonsaturating amounts of heparin, indicated that AT-III beta inhibited thrombin more rapidly. AT-III beta is also distinguishable from AT-III alpha on the basis of heparin-binding affinity estimated from titration of protein fluorescence with heparin. Thus, antithrombin III exists as two molecular entities in human plasma which differ both structurally, in carbohydrate content, and functionally, in their heparin-binding behavior.     

Co-purification of galactosyltransferases from chick-embryo liver.

Two galactosyltransferases with nearly identical Mr values were purified 5000-7000-fold from microsomal membranes of chick-embryo livers by using several affinity columns. One enzyme transfers galactose from UDP-galactose to form a beta-(1----4)-linkage to GlcNAc (N-acetylglucosamine) or AsAgAGP [asialo-agalacto-(alpha 1-acid glycoprotein)]. The other enzyme forms a beta-(1----3)-linkage to AsOSM [asialo-(ovine submaxillary mucin)]. Both enzymes were solubilized (85%) from a microsomal pellet by using 1% Triton X-100 in 0.1 M-NaCl. The supernatant activities were subjected to DEAE-Sepharose chromatography and four affinity columns: UDP-hexanolamine-Sepharose, alpha-lactalbumin-Sepharose, GlcNAc-Sepharose and either AsAgAGP-Sepharose or AsOSM-Sepharose. The AsAgAGP enzyme [(1----4)-transferase] and the AsOSM enzyme [(1----3)-transferase] behave identically on the DEAE-Sepharose and UDP-hexanolamine-Sepharose columns, and similarly on the alpha-lactalbumin-Sepharose column. Final separation of the two enzymes, however, could only be achieved on affinity columns of their immobilized respective acceptors. Both purified enzymes migrate as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after silver staining, and both have an apparent Mr of 68 000. The enzymes were radioiodinated and again subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioautographic analyses showed only one, intensely radioactive, band. Activity stains performed for both transferases after cellulose acetate electrophoresis indicate that, with this system too, both activities have identical mobilities, and co-migrate, as well, with the major, silver-stained, protein band. Kinetic studies with the purified enzymes show that the Km value for GlcNAc, for the (1----4)-transferase, is 4mM; for the (1----3)-transferase the Km value for AsOSM is 5mM, in terms of GalNAc (N-acetylgalactosamine) equivalents. Both enzymes have a Km value of 25 microM for UDP-galactose.     

Characterization of Virus Adsorption by Using DEAE-Sepharose and Octyl-Sepharose†

Viruses were characterized by their adsorption to DEAE-Sepharose or by their elution from octyl-Sepharose by using buffered solutions of sodium chloride with different ionic strengths. Viruses whose adsorption to DEAE-Sepharose was reduced most rapidly by an increase in the sodium chloride concentration were considered to have the weakest electrostatic interactions with the solids; these viruses included MS2, E1, and X174. Viruses whose adsorption to DEAE-Sepharose was reduced least rapidly were considered to have the strongest electrostatic interactions with the column; these viruses included P1, T4, T2, and E5. All of the viruses studied adsorbed to octyl-Sepharose in the presence of 4 M NaCl. Viruses that were eluted most rapidly following a decrease in the concentration of NaCl were considered to have the weakest hydrophobic interactions with the column; these viruses included X174, CB4, and E1. Viruses that were eluted least rapidly from the columns after the NaCl concentration was decreased were considered to have the strongest hydrophobic interactions with the column; these viruses included f2, MS2, and E5.     

IUB commission of editors of biochemical journals the citation of bibliographic references in biochemical journals recommendations (1971)

A pentose-rich acidic glycoprotein was isolated from protease digested bovine vitreous humor by fractionation on an AG1-X2 column using NaCl solution gradient.The material eluted at 0.35 M NaCl (glycoprotein) was electrophoretically heterogeneous at pH 8.6 after partial purification on Sephadex G-25. Gel filtration on G-100 resolved the glycoprotein into two fractions. These fractions differ in molecular weight; mol. wt approx. 95 000 material consisted of two components on electrophoresis and mol. wt approx. 28 000 material showed only a single component on electrophoresis. The lower molecular weight component was re-chromatographed on Sephadex G-100 yielding a single orcinol positive component which gave a homogeneous band on gel electrophoresis.Quantitative analysis of this material gave 30% protein, 7.0% pentose, 18.7% glucosamine, 9.2% galactosamine, 10.9% hexuronic acid and 16.1% hexose.Treatment with 0.5 M NaOH at 20°C for 24 h resulted in a 50% decrease in the threonine content suggesting the possible involvement of this amino acid in the protein-carbohydrate linkage group.Paper chromatography of the fraction hydrolysate demonstrated the presence of glucurone, xylose, arabinose, glucose and galactose.     

Developmental changes in sugar responses of the chorda tympani nerve in preweanling rats

To clarify developmental changes in the gustatory system of the rat, integrated taste responses from the chorda tympani (CT) nerve were recorded and analyzed at different postnatal ages. The response magnitude was calculated relative to the response to the standard, 0.1 M NH4Cl. Even at 1 week of age, the CT responded well to all tested 0.1 M chloride salts (NH4Cl, NaCl, LiCl, KCl, RbCl and CsCl). The responses to 0.1 M NaCl and LiCl increased with increasing age of the rat while response magnitudes to KCl, RbCl and CsCl did not change up to 8 weeks. At 1 week, the integrated response pattern was quite similar to that in adult rats for NaCl, HCl and quinine hydrochloride (QHCl). The concentration-response functions for NaCl, HCl, QHCl and sucrose at 2 weeks were essentially the same as those at 8 weeks. These results suggest that taste buds in the 2-week-old rat are functionally mature for the detection of the four basic taste stimuli. The relative magnitude of the responses to the various sugars was smaller at 1 week compared to the adult rat and reached a maximum at weeks 3-4, then decreased gradually with age. Among the six sugars, sucrose was the most effective followed by lactose. From weeks 1-4, the magnitude of the integrated taste response to fructose was smaller than that to lactose except at 3 weeks of age. Maltose, galactose and glucose were less potent stimuli than the other sugars tested. The response magnitude to lactose at 4 weeks had decreased compared to that for the other sugars. Taste responses to the sugars in preweanling and adult rats were not cross-adapted by the individual sugars. These results suggest that after 1 week of age during postnatal development in the rat, taste information from the CT rapidly increases in its importance for feeding behavior.     

Heterogeneity of heparan sulfate chains in a proteoglycan from bovine lung

A heparan sulfate proteoglycan from bovine lung gas-exchange tissue was isolated by extraction of the tissue with 4.0 M guanidine HCl in the presence of multiple protein inhibitors. The proteoglycan was purified by precipitation with cetylpyridinium chloride in 0.5 M KCl followed by CsCl isopycnic centrifugation (po = 1.45) in 4.0 M guanidine/HCl. Further purification was achieved by gel filtration on Sepharose CL-2B and by chromatography in DEAE-Sepharose CL-6B column. The proteoglycan had 14.9% protein and 22.4% uronate. Heparan sulfate chains from the proteoglycan were isolated after beta-elimination. Fractionation of heparan sulfate chains was achieved on Dowex-1 Cl- column, eluting with a stepwise increase in the concentration of NaCl, 1.0 to 2.0 M with 0.2 M increments. Of the total heparan sulfate recovered from the column, about 10% eluted by 1.2 M NaCl, 68% by 1.4 M NaCl, 18% by 1.6 M NaCl and 4% by 1.8 M NaCl. The fractions varied in their total and N-sulfate ester contents and iduronic acid to glucuronic acid ratios. The fraction that eluted from the Dowex-1 Cl- column at 1.6 M NaCl had the highest molecular weight, 37000, and the fraction that eluted at 1.8 M NaCl had the lowest molecular weight, 12000, as determined by gel filtration method, and the greatest sulfate content. The core protein, obtained by digestion of proteoglycan by heparan sulfate lyase, showed mostly a single band in SDS-polyacrylamide gel electrophoresis. The observations indicate a heterogeneity of the composition of heparan sulfate chains in the proteoglycan. This heterogeneity likely contributes to variations in biologic properties of different heparan sulfate proteoglycan preparations.     

Partial characterization of thymocyte-activating factor derived from MDP-stimulated guinea pig fibroblasts

The activity of fibroblast-derived thymocyte activating factor (FTAF) of the guinea pig was measured, and the factor was partially characterized. The FTAF activity was heat labile, and destroyed by treatment with trypsin, chymotrypsin, and Streptomyces griseus protease, suggesting the protein nature of FTAF. FTAF bound to DEAE-Sepharose CL-6B in Tris-HCl buffer at pH 8.0, and was eluted with 0.1-0.2 M NaCl. FTAF was absorbed with Blue Sepharose CL-6B. The factor bound to a hydroxylapatite column in 10 mM phosphate buffer and was eluted in two major fractions, one fraction with 40 mM phosphate buffer, the other with 70-110 mM phosphate buffer. Finally, FTAF did not have as much effect on the proliferation of lymph node T cells as T-cell-activating monokines which exhibited marked stimulating effects on both T lymphocytes and thymocytes.     

Complementable Fraction and Complemented Enzyme of Mutant Ml5 from Escherichia Coli: Partial Purification by Affinity Chromatography

The technique of affinity chromatography has been used in the partial purification of complementable fractions and complemented enzyme of β-galactosidase from Escherichia coli mutant M15. The crude extract of mutant ML5 was incubated with fragment CM-B. The complemented enzyme and complementable fractions were passed through a small column of p-amino-phenyl-β-D-thiogalactoside to which inhibitors had been covalently attached. A high percentage of the nonspecific protein passed directly through the affinity column while the specific enzymatic protein remained bound to the gel. Phosphate buffer with NaCl was used to elute the complementable fractions from the column. Sodium borate buffer was used to elute the bound complemented enzyme from the affinity support. The results of this study show that 100% of the complemented enzyme was bound to the column. The partially purified enzyme had the same position in disc gel electrophoresis as β-galactosidase from E. coli.     

Distributional and developmental variations of multiple forms of calpastatin in mouse brain

DEAE-cellulose chromatography of mouse brain extract demonstrated the occurrence of two calpastatin fractions, CS-0.1 and CS-0.2, with distinctly higher content of the latter. CS-0.1 emerged from the column at 0.1 M NaCl, inhibited calpain II more strongly than calpain I, and identified also immunologically with hitherto known calpastatin. CS-0.2 emerged at 0.2 M NaCl, inhibited calpain I more strongly than calpain II, and did not crossreact with anti-calpastatin antibody used. Fairly consistent amounts of CS-0.2 and calpain II were found in the brain of mice from 10 days to 10 weeks after birth, while CS-0.1 became measurable only after 4-week growth. In adult mice, CS-0.1 was highest in specific activity in brainstem, lower in cerebellum, and not detectable in cerebral hemisphere. Physiological significance of multiple forms of calpastatin and their variations found is not known.     

Separation of poly(A) RNA's synthesized by soybean embryos

A stepwise elution procedure in which the MAK column with 0.85M buffered NaCl is held in a stationary phase (2–3 psi,35?C, 1 hr) permits the separation of two tenaciously boundRNA fractions. Poly(A) RNA's elute with the ribosomal and tenaciouslybound RNA's. MAK fractions contain different percent of RNA'sthat bind to poly(dT) cellulose and have messenger RNA activity.The results suggest that the soybean embryonic tissue synthesized,at least, four poly(A) RNA's. (Received August 17, 1976; )     

裙带菜褐藻糖胶的分离纯化及其结构分析

裙带菜经水提法提取得到褐藻糖胶粗多糖,经DEAE-Sepharose FF离子交换层析和 Sepharose 4B层析后,得到Sl、S2两个单一组分,相对分子质量分别为550 808、38 335.基本结构及单糖组成分析表明,二者均含有岩藻糖、糖醛酸、硫酸基、半乳糖、甘露糖、葡萄糖、鼠李糖、木糖、阿拉伯糖,但含量差别较大,推测与二者的生理活性的差异有关.     

Sugar best single chorda tympani nerve fiber responses to various sugar stimuli in rat and hamster

1. Sugar best single chorda tympani nerve fiber of rat and hamster were tested with six sugars. 2. Fibers were selected for this experiment, only if they responded to 1.0 M sucrose or 1.0 M maltose and they responded poorly to 0.1 M NaCl. 3. In rat, some single fibers gave larger responses to maltose than to sucrose, while in hamster nearly all nerve fibers responded best to sucrose. 4. The order of effectiveness of sugars was maltose greater than fructose greater than or equal to lactose greater than sucrose greater than glucose greater than galactose in rat and sucrose greater than fructose greater than or equal to glucose greater than or equal to galactose greater than maltose greater than lactose in hamster.     

Human placenta alpha-N-acetylglucosaminidase: purification, characterization and demonstration of multiple recognition forms.

alpha-N-Acetylglucosaminidase (EC 3.2.1.50) was purified from human placenta by a four-step procedure including ammonium sulfate precipitation, affinity chromatography with immobilized antibodies against urinary alpha-N-acetylglucosaminidase, gel chromatography and discontinuous gel electrophoresis with a 30% recovery and 26 300-fold purification. Immunological methods revealed the contamination with about 10% non-alpha-N-acetylglucosaminidase protein. Isoelectric focusing led to a distribution of activity between 4.3 and 6.5 with maxima at pH 5.1 and pH 5.7. After treatment with neuraminidase, alpha-N-acetylglucosaminidase activity assembled at pH 5.7. The multiple isoelectric forms were endocytosed with different rates by cultured human skin fibroblasts. Placenta alpha-N-acetylglucosaminidase has an apparent molecular weight of 304 000 and contains 23.4% carbohydrate consisting of glucose, galactose, mannose, hexosamines and neuraminic acid. Gel electrophoresis in the presence of 0.1% sodium dodecylsulfate separated placenta alpha-N-acetylglucosaminidase into subunits with molecular weights of 86 500 and 81 000. The activity towards various substrates, the kinetics of hydrolysis, the pH optimum and the stability of the catalytic activity were assayed.     

Partial purification and characterization of a factor from a cloned thymic epithelium cell line

The culture supernatant from a cloned line of thymic epithelium (TEPI) is shown to enhance the response of thymocytes to alloantigen as measured by cell-mediated lympholysis. The supernatant has no effect on the spleen cell response to alloantigen as measured by cell-mediated lysis and does not contain interleukin 1, interleukin 2, interleukin 3, or interferon-gamma activity. The activity is shown to have an apparent m.w. of 160,000 by Sephacryl S-200 gel permeation chromatography, to have an isoelectric point of 6.5, and to elute from DEAE-Sepharose at 0.07 M NaCl.     

Orientation of the beta subunit polypeptide of (Na+ + K+)ATPase in the cell membrane

Although the animal cell (Na+ + K+)-ATPase is composed of two polypeptide subunits, alpha and beta, very little is known about the beta subunit. In order to obtain information about the structure of this polypeptide, the beta subunit has been investigated using proteolytic fragmentation, chemical modification of carbohydrate residues, and immunoblot analysis. The sialic acid moieties on the oligosaccharide groups on the beta subunit of (Na+ + K+)-ATPase were labeled with NaB3H4 after oxidation by sodium periodate, or the penultimate galactose residues on the oligosaccharides were similarly labeled after removal of sialic acid with neuraminidase and oxidation by galactose oxidase. All of the carbohydrate residues of the protein are located on regions of the beta subunit that are found on the non-cytoplasmic surface of the membrane. Cleavage of the galactose oxidase-treated, NaB3H4-labeled beta subunit by chymotrypsin at an extracellular site produced labeled fragments of 40 and 18 kDa, indicating multiple glycosylation sites along the polypeptide. Neither the 40 kDa fragment nor the 18 kDa fragment was released from the membrane by chymotrypsin digestion alone, but after cleavage the 40 kDa fragment could be removed from the membrane by treatment with 0.1 M NaOH. This indicates that the 40 kDa fragment does not span the lipid bilayer. The 40 kDa fragment and the 18 kDa fragment are also linked by at least one disulfide bond. The 18 kDa fragment also contains all of the binding sites found on the (Na+ + K+)-ATPase for anti-beta subunit antibodies. Both the 40 kDa fragment and the 18 kDa fragment were also generated using papain or trypsin to cleave the beta subunit. These data indicate that the beta subunit of (Na+ + K+)-ATPase contains multiple sites of glycosylation, that it inserts into the cell membrane near only one end of the polypeptide, and that one region of the polypeptide is particularly sensitive to proteolytic cleavage relative to the rest of the polypeptide.     

Isolation and characterization of an inhibitor-sensitive and a polycation-stimulated protein phosphatase from rat liver nuclei

Two protein phosphatases were isolated from rat liver nuclei. The enzymes, solubilized from crude chromatin by 1 M NaCl, were resolved by column chromatography on Sephadex G-150, DEAE-Sepharose and heparin-Sepharose. The phosphorylase phosphatase activity of one of the enzymes (inhibitor-sensitive phosphatase) was inhibited by heat-stable phosphatase inhibitor proteins and also by histone H1. This phosphatase had a molecular weight of approx. 35 000 both before and after 4 M urea treatment. Its activity was specific for the β-subunit of phosphorylase kinase. Pretreatment with 0.1 mM ATP inhibited the enzyme only about 10%, and it did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as the catalytic subunit of phosphatase 1. The other phosphatase (polycation-stimulated phosphatase) was insensitive to inhibition by inhibitor 1, and it was stimulated 10-fold by low concentrations of histone H1 (A_0.5 = 0.6 μM). This enzyme had a molecular weight of approx. 70 000 which was reduced to approx. 35 000 after treatment with 4 M urea. It dephosphorylated both the α- and β-subunits of phosphorylase kinase. The enzyme was inhibited more than 90% by preincubation with 0.1 mM ATP and did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as phosphatase 2A.