α-D-Mannosidase from the aleurone layers of resting wheat grains: Sugar-depleted forms

α-D-Mannosidase from the aleurone layers of resting wheat grains has been purified to homogeneity by a procedure involving Con A-Sepharose chromatography. The enzyme has been shown to be a glycoprotein containing D-glucose, D-mannose, D-glactose and N-acetyl-D-glucosamine. A minor component, showing the characteristics of sialic acid has also been detected by gas chromatography. Studies dealing with the effect of Endo-H treatment on the affinity of the enzyme for Con A-Sepharose, indicate that the sugar moiety contains both high-mannose chain(s), accessible to Endo-H and interacting strongly with Con A-Sepharose, and oligosaccharide chain(s) resistant to Endo-H and interacting with Con-A-Sepharose to a lesser extent. Removal of sugar by Endo-H and periodate treatments affects the enzyme stability to heat and protease degradation.     

The role of sialic acid and galactose residues in determining the survival of human plasma alpha-antitrypsin in the blood circulation.

Human plasma α₁-antitrypsin (α₁-AT) is a glycoprotein known to contain terminal sialic acids (N-acetylneuraminic acids) in the carbohydrate units. These residues were converted to a radioactive seven-carbon analog (NANA-7) by sequential periodate oxidiation and tritiated borohydride reduction. Modified α₁-AT prepartions, namely, (a) periodate oxidized α₁-AT, (b) asialo α₁-AT (neuraminidase-treated α₁-AT), (c) (NANA 7)-α₁-AT (periodate-oxidized, -tritiated, borohydride-reduced α₁-AT), (d) (NANA-7)-α₁ AT (partially desialylated by neuraminidase), and (e) partially desialylated (NANA 7)-α₁-AT oxidized with galactose oxidase, all retained the following properties attributable to native α₁-AT: trypsin-inhibitory and chymotrypsin-inhibitory activities, immunological reactivity to antibody against native α₁-AT, and the ability to bind to concanavalin A-Sepharose 4-B columns. After intravenous injection of intact (NANA-7)-α₁-AT into rats, the labeled material had a circulating half-life of 18 h. When (NANA-7)-α₁-AT was partially desialylated (four residues of NANA-7 out of a total of six were removed, thus exposing an equivalent number of galactose residues at the terminal positions) by neuraminidase, injection into rats of this material resulted in a rapid and almost complete disappearance of the label from the circulation in 30 min. There was a concomitant accumulation of radioactivity in the liver. The rate of this rapid transfer depended on the presence of intact galactose residues as the terminal, nonreducing sugar in the carbohydrate units. Galactose oxidase treatment of the partially desialylated (NANA-7)-α₂-AT, which presumably oxidized the primary alcohol of galactose at C-6 to an aldehyde group, caused a reversion of its survival time in the circulation to that of the intact (NANA-7)-α₁-AT.     

Specific binding of sulfated proteoglycans to concanavalin A - Sepharose 4B.

More than 90 % of [³⁵S]proteoglycans isolated from the secretions of human skin fibroblasts bind to Concanavalin A-Sepharose 4B (Con A-Sepharose) in the presence of 1 M NaCl. Above pH 5.0 1 M concentrations of methyl-α-D-mannoside and other haptenic inhibitors for Con A-sugar interaction prevent binding of [³⁵S]proteoglycans, whereas equimolar concentrations of non-haptenic carbohydrates do not effect binding. Below pH 5.0 [³⁵S]proteoglycans bind to Con A-Sepharose in the presence of both methyl-α-D-mannoside and galactose. About 60 % of the proteoglycans bound at pH 4.0 are eluted at pH 7.5 in the presence of 1 M methyl-α-D-mannoside. [³⁵S] Glycosaminoglycans prepared from [³⁵S] proteoglycans do not bind to Con A-Sepharose in the presence of 1 M NaCl.These results indicate a [³⁵S]proteoglycan-Con A interaction via the protein core of the proteoglycan and the sugar binding sites of Con A.     

Purification and kinetic characterization of polygalacturonase - 3 from palmyrah palm (Borassus flabellifer L)

Polygalacturonase-3 was isolated and purified to homogeneity from palmyrah palm (Borassus flabellifer L.) fruit using Con A-Sepharose affinity column. The purified enzyme migrated as a single band on native and SDS–polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme was estimated to be 66 kDa by size elution chromatography. Optimum polygalacturonase activity as a function of pH and temperature was determined using polygalacturonic acid as substrate. Optimum pH and temperature values ranged between the pH?4.0–5.0 and temperature 30–40 °C. At the optimum pH and temperature, the K_m and V_max values were determined by Lineweaver–Burk method. The value K_m (0.33 mM) reveals that polygalacturonase has significant reactivity towards polygalacturonic acid. The enzyme showed varied responses towards divalent and monovalent metal ions. Ca²⁺ activated the polygalacturonase-3 enzyme protein. Both teepol and cetyltrimethylammonium bromide inhibited polygalacturonase-3 activity by 44 %, while 2-mercaptoethanol stimulated the enzyme marginally.     

Partial purification and further characterization of the novel endoglucosaminidase from human serum that hydrolyses 4-methylumbelliferyl-N-acetyl-β-d-chitotetraoside (MU-TACT hydrolase)

A novel endoglucosaminidase, originally described by Den Tandt et al. [Int. J. Biochem.20 (1988), 713–719] and bearing the provisional name MU-TACT hydrolase, was purified from human serum 56,000-fold by means of ammonium sulphate precipitation, anion-exchange chromatography, Con A-Sepharose chromatography and gel filtration on Sepharose CL-6B followed by Superose 12HR. Based on the latter technique the native apparent molecular weight of the enzyme appeared to be equal to that of myoglobin, being approx. 17 kD. The enzyme eluted clearly at a different volume than lysozyme. MU-TACT is a commercially available substrate for lysozyme. For unknown reasons two major peptides co-purify that give bands on SDS-PAGE of 55–60 and 31 kD, respectively.     

Alpha1-antitrypsin synthesis by human lymphocytes

$\text{De}$$\text{novo}$ synthesis of alpha₁-antitrypsin (α₁AT) by human peripheral lymphocytes has been demonstrated in the present study. Treatment of the mononuclear cells with concanavalin A(Con A) resulted in a triple increase in the amount of α₁AT synthesized by the untreated cells. A small amount of α₁AT, equivalent to that synthesized by the unstimulated mononuclear cells, was observed in cultures of monocyte-depleted lymphocytes, with or without Con A stimulation. Monocytes treated with or without Con A scarcely synthesized α₁AT. Conditioned media derived from monocyte enriched mononuclear cells treated with Con A enhanced about threefold α₁AT synthesis by the Con A-stimulated lymphocytes. α₁AT is suggested to be synthesized by lymphocytes assisted by monocytes.     

Purification of phosphotransacetylase by affinity chromatography.

Phosphotransacetylase from Clostridium kluyveri was purified using a C⁸-(6-aminohexyl)-amino-desulfo-coenzyme A-Sepharose column. The method of synthesis of the affinity matrix is described. A crude extract was treated with ammonium sulfate and chromatographed on the desulfo-coenzyme A-Sepharose column. Using this method the enzyme was purified 83-fold and was found to be 73% pure. A new method for the determination of the purity of phosphotransacetylase by activity staining of polyacrylamide gels with 5,5′-dithiobis(2-nitrobenzoic acid) is described.     

Preparation of high-density Concanavalin A adsorbent and its use for rapid, high-yield purification of peroxidase from horseradish roots

Preparation of Concanavalin A-adsorbents by immobilization on Sepharose activated with 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP-reagent) is reported. High immobilization yields of lectin (above 90%) were attained using an optimized CDAP-activating protocol. The effect of ligand density on the performance of the adsorbent for specific binding of glycoproteins was studied using horseradish peroxidase (HRP) as a model. Adsorption yields of pure HRP exceeding 90% were obtained with Con A-derivatives containing not < 20 mg of immobilized Con A/ml of packed gel. With lectin content of 2 mg/(ml of packed gel), only 20% of HRP was adsorbed. Purification of peroxidase from horseradish roots extract was successfully accomplished on Con A-Sepharose with high Con A content.     

Human placental sialidase: partial purification and characterization

A sialidase [EC 3.2.1.18] has been partially purified from human placenta by means of procedures comprising Con A-Sepharose adsorption, ammonium sulfate precipitation, sucrose density gradient centrifugation, and high-pressure liquid chromatography on a Shim pack Diol 300 column. On high-pressure liquid chromatography, most of the beta-galactosidase that comigrated with the sialidase on sucrose density gradient centrifugation was removed. The sialidase was purified 3,600-fold from the preparation obtained by Con A-Sepharose adsorption. The enzyme liberated the sialic acid residues from (alpha 2-3) and (alpha 2-6) sialyllactose, colomic acid, fetuin, and transferrin, but not from bovine submaxillary mucin. The enzyme also hydrolyzed gangliosides GM3, GD1a, and GD1b in the presence of sodium cholate as a detergent, but GM1 and GM2 were less susceptible to the enzyme. The optimum pHs for 4-methylumbelliferyl-N-acetylneuraminate, sialyllactose, fetuin, and GM3 lay between 4.0 and 5.0.     

Identity of the HL-A common portion fragment and human beta2-microglobulin

Glycogen synthetase D from the 17,000 × g supernatant of a homogenate of human polymorphonuclear leukocytes has been purified to a specific activity of 7,4 units/mg protein in a single step, chromatography on Concanavalin A bound to agarose (Con A-Sepharose). The overall recovery of the enzyme was 66% and the entire procedure requires only 3–4 hours. After an in vitro D to I conversion, glycogen synthetase I was purified to a specific activity of 11,5 units/mg protein in a similar procedure.     

Isolation and partial characterization of a proteinase inhibitor from human colorectal adenocarcinoma

A proteinase inhibitor has been isolated from human colorectal adenocarcinomas by extraction with a low-ionic-strength buffer and a combination of Con A-Sepharose, Sephadex G-200, DEAE-cellulose and chromatofocusing steps. The preparation appeared to be homogeneous upon gel exclusion chromatography and SDS-polyacrylamide gel electrophoresis and had an estimated molecular weight of 66 000. The inhibitor was able to bind and inhibit urokinase, plasmin, trypsin, tissue plasminogen activator and thrombin. The binding appeared to be stoichiometric and relatively fast. The isoelectric point of the protein was 4.6–4.7. The inhibitor did not crossreact with antisera elicited against α₂-macroglobulin, α₂-antiplasmin, antithrombin III or C₁-inhibitor, but it did crossreact with an antiserum against α₁-antitrypsin in double immunodiffusion. The antiserum only partially attenuated the activity of the inhibitor. Whereas α₁-antitrypsin completely inhibited the amidolytic activity of elastase, the tumor inhibitor had no effect on elastase under the same conditions.     

慈菇（Sagittaria safittifolia）α－半乳糖苷酶的纯化与性质

以对硝基苯糖苷基为底物,测定了慈菇的１２种糖苷酶,其中α－甘露糖苷酶、α－和β－半乳糖苷酶活力较高;经硫酸铵分级沉淀,ＳｅｐｈａｄｅｘＧ－１５０分子筛层析,ＣｏｎＡＳｅｐｈａｒｏｓｅ４Ｂ亲和层析,ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ离子交换层析,从慈菇抽提液纯化了α－半乳糖苷酶。纯化酶的比活提高１０７２倍,活力回收１５．６％,在圆盘聚丙烯酰胺凝胶电泳和ＳＤＳ－ＰＡＧＥ上均显示１条蛋白质带,在α－半乳糖苷酶浓度为１５０ｍＵ／ｍｌ的溶液中测不到其他糖苷酶的活力。慈菇α－半乳糖苷酶的分子量用ＳｅｐｈａｄｅｘＧ－１００凝胶过滤柱测定或在ＳＤＳ－ＰＡＧＥ上测定均为６０ｋＤ,酶反应的最适ｐＨ在５．８附近,最适温度为６０℃。该酶分解对硝基苯基－α－半乳糖苷的Ｋ＿ｍ值为３．７×１０￣（－４）ｍｏｌ／Ｌ,Ｖ＿ｍ值为２．１×１０￣（－４）ｍｏｌ／Ｌ。银离子、汞离子显著抑制酶活力,Ｄ－半乳糖和密二糖均竞争性地抑制该酶水解对硝基苯基α－Ｄ－半乳糖苷的活力,根据Ｄｉｘｏｎ作图求得其Ｋ＿ｉ值分别为０．９２×１０￣（－３）ｍｏｌ／Ｌ和１．９８×１０￣（－３）ｍｏｌ／Ｌ。２－脱氧－Ｄ－半乳糖和Ｌ－岩藻糖为酶活力的非竞争性抑制剂。化学修饰     

Catabolism of desialylated human plasma alpha1-antitrypsin and its trypsin complex in the rat.

Human plasma α₁-antitrypsin (α₁-AT) was labeled with either ³H [³H-labeled NANA (N-acetyl-neuraminic acid)-7] residues in the carbohydrate moiety) or ¹⁴C (?-N-methyl-[¹⁴C]lysyl residues in the protein backbone) or with both isotopes in the corresponding residues. After intravenous injection into rats of the doubly labeled partially (50%) desialylated (methyl-[¹⁴C]·[³H]NANA-7)-α₁-AT, the rates of disappearance from the plasma of both isotopes were very rapid and yielded essentially the same circulatory half-life of 5 min. The rapid disappearance of the doubly labeled glycoprotein from the plasma was accompanied by concomitant fast and equal accumulations of ¹⁴C and ³H in the liver which constituted about 70% of the administered dose 15 min after the injection. The asialo (methyl-[¹⁴C])-α₁-AT·trypsin complex or methyl-[¹⁴C]-α₁-AT·trypsin complex had a plasma survival time (45 min) that was intermediate between methyl-[¹⁴C]-α₁-AT and its desialylated derivative. These complexes were removed from the plasma by the liver (45% of the injected dose 60 min after injection), although not as rapidly as asialo (methyl-[¹⁴C])-α₁-AT. Blockade of the reticuloendothelial (Kupffer) cells by simultaneous injection of heat-denatured albumin inhibited the liver uptake of the inhibitor·trypsin complexes but not that of the uncomplexed asialo α₁-AT. Radioactive ?-N,N-dimethyllysine, ?-N-monomethyllysine, methionine, choline, and betaine were separated and identified from the trichloro-acetic acid-soluble fraction of rat livers 25 min after injection of asialo (methyl-[¹⁴C])-α₁-AT.     

Preparation of steroid radioligands for ultrafiltration assays by a solid-phase transport globulin method using concanavalin A-Sepharose

Concanavalin A-Sepharose (Con A-Sepharose) was applied to separate non-protein-bound and albumin-bound radioactive impurities from steroid radioligands. Con A-Sepharose gel, plasma, and steroid radioligand were mixed, incubated, and then washed with buffer. This method was compared with an affinity diafiltration method which separates non-protein-bound radioactive impurities with a filter membrane. 3H-Water and 3H-estrone sulfate, chosen to serve as molecules representative of non-protein-bound and albumin-bound impurities, were removed quite effectively by the Con A-Sepharose method, while 85% of 3H-estrone sulfate could not be removed by the diafiltration method. Plasma unbound cortisol (F) and testosterone (T) values determined by ultrafiltration using 3H-F and 3H-T prepared by the Con A-Sepharose method were significantly lower than those determined using the radioligands unprocessed or prepared by the diafiltration method. The whole procedure of the Con A-Sepharose method takes only 3-4 hours. This method is a simple, rapid, and effective technique for preparation of steroid radioligands for plasma protein binding studies.     

Structure of a β-galactan isolated from the nuclei of Physarum polycephalum

A sulfated and phosphorylated β-D-galactan ([α]_D + 8°) was isolated from the nuclei of the acellular slime mould Physarum polycephalum. The polysaccharide was isolated from cesium chloride gradients during the preparation of ribosomal DNA and purified. The purified galactan contained 89% galactose, 2.5% phosphate and 9.6% sulfate groups and had an average degree of polymerisation of 560. Periodate degradation and permethylation studies indicated the presence of mainly (1 → 4)-, but also of (1 → 3)-, and (1 → 6)-linked galactose units with one branch every 13 units. These results suggested that the intranuclear galactan, apart from its higher sulfate content, is similar to the extra-cellular polysaccharide produced by P. polycephalum.     

F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein

It has been claimed (Andreu, J.M., Warth, R. and Muñoz, E. (1978) FEBS Lett. 86, 1–5) that the F₁-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F₁-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F₁ preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F₁-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F₁-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (, β, γ, δ and ε) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F₁-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F₁-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan.     

Evidence supporting the identity of beef heart mitochondrial chloroform-released adenosine triphosphatase (ATPase) with coupling factor I

Highly purified mitochondrial chloroform-released beef heart ATPase had molecular weight 330 000, five bands (α, β, γ, δ, ε) in sodium dodecyl sulfate gel electrophoresis and could restore the oxidative-phosphorylation function of A particles. Maximal inhibition (90%) of the enzyme by N,N′-dicyclohexylcarbodiimide was achieved at a molar ratio of inhibitor to protein of 30 : 1. Chloroform introduced into an aqueous solution of beef heart coupling factor I protected it from cold inactivation.     

Chemical modifications of lysyl and arginyl residues of human plasma α1-antitrypsin

Reactions of human plasma α₁-antitrypsin (α₁-AT) with reagents known to modify the lysyl residues [citraconic anhydride, acetic anhydride, 2,4,6-trinitrobenzenesulfonic acid (TNBS)] and arginyl residues [1,2-cyclohexanedione (CHD) and phenylglyoxal (PGO)] in proteins have been studied. Native and modified human plasma α₁-AT preparations were tested for their inhibitory activities against trypsin and α-chymotrypsin. TNBS was utilized to modify and quantitate free amino groups (?-NH₂ groups of lysine residues) in human plasma α₁-AT. The number of lysine residues determined by the TNBS spectrophotometric procedure agreed well with that found by amino acid analyses. Both the trypsin-inhibitory and chymotrypsin-inhibitory activities of α₁-AT were destroyed by modification with TNBS. CHD was employed to modify the arginyl residues of α₁-AT. Neither the trypsin-inhibitory nor the chymotrypsin-inhibitory activity of α₁-AT was affected by modification of its arginyl residues. Amino acid analyses of the CHD-treated α₁AT revealed that only the arginine residues were modified. PGO was also utilized for the modification of the arginyl residues in α₁-AT. Both the trypsininhibitory and chymotrypsin-inhibitory activities of α₁-AT were destroyed after modification. However, amino acid analyses showed that not only the arginyl, but also the lysyl residues of the PGO-treated inhibitor were modified. The side reaction of PGO with the lysyl residues could explain the loss of inhibitory activities. Reaction of a α₁-AT with citraconic anhydride resulted in an extensive modification of the amino groups accompanied by a 100% loss in inhibitory activity against both trypsin and α-chymotrypsin. Comparable results were observed when acetic anhydride was utilized as the acylating reagent. With the exception of the citraconylated α₁AT, all of the other chemically modified α₁-AT derivatives studied presently retained their immunological reactivities against antisera to native α₁-AT. Regeneration of about 60% of the PGO-blocked arginyl residues in α₁-AT did not lead to any recovery of the proteinase inhibitory activities. Full recovery of trypsin-inhibitory and immunological activities were achieved when about 50% of the citraconylated amino groups were deblocked. The CHD-treated α₁-AT still retained the capacity to form complexes with both trypsin and chymotrypsin. On the other hand, the other chemically modified α₁-AT derivatives have completely lost the ability to form complexes with the enzymes. Recovery of the ability to form complexes with the enzymes was, however, recovered when about 50% of the citraconylyl groups was removed from the α₁-AT molecule. Based on these modification studies, it is concluded that α₁-AT is a lysyl inhibitor type (i.e., the reactive site is Lys-X bond) and that the interaction of α₁-AT with trypsin or chymotrypsin very likely involves or requires the same site as in the case of the soybean trypsin inhibitor (Kunitz).     

Opiate Receptors from Different Tissue Sources: Solubilization and Characterization

Abstract: Membrane-bound opiate receptors from neuroblastoma-glioma hybrid cells and from different parts of the rat brain (whole brain minus cerebellum, cortex, thalamus-hypothalamus and cerebellum) were labeled with the methionine-enkephalin analogue, D-[³H]Ala²-Met-enkephalinamide, and solubilized with the nonionic detergent Brij 36T. The protease inhibitors bacitracin, phenylmethylsulfonyl fluoride, Trasylol, and leupeptin were included in the solubilization buffer to minimize proteolysis. Two simple techniques, ammonium sulfate precipitation and activated charcoal absorbence, were adapted to separate the free and the macromolecule-bound ligands. The solubilized receptor-[³H]enkephalin complexes were partially purified by consecutive passages through Sephadex G-75 and Sepharose 6B columns. Of the three peaks of radioactivity that were observed in the effluent of the Sepharose column, two contained proteins, and one of them, with a Stokes radius of 59 Å, seemed to contain the specific opiate receptor, as evidenced by additional experiments. This peak was further purified on thiol-Sepharose or diethylaminoethanol-Sephadex columns that were eluted with a gradient of 0–50 mM dithiothreitol or with 1.0 M KCI, respectively. The receptor-[³H]enkephalin complex from neuroblastoma-glioma cells (apparent δ-type receptors) binds less to the thiol-Sepharose beads than receptor-(³H]enkephalin prepared from the hypothalamus-thalamus, which is rich in μ receptors. The [³H]enkephalin receptor complexes of the various sources also differed in their stability. The dissociation of the ligand from the neuroblastoma-glioma receptor was monophasic, with a half- life of 250 min, whereas that of two brain regions was biphasic, with half-lives of 195–330 min and 10,000 min. The methods described may be of use for further purification of soluble opiate receptors, either active or cross-linked to the ligand.     

Preparation of Homogeneous Concanavalim A

Abstract

Concanavalin A (Con A), obtained either commercially or by affinity chromatography, was further purified by incubating at 6–8°C for 16–18 hr at pH 3.0–3.2 in 1 M MaCl, 0.08 M glycine and 3 mM each Ca⁺² and Mn⁺², heat treating at 45°C for 2 hr and centrifuqing. The supernatant was neutralized to pH 5 and stored in the cold. The overall yield was 70–80% Some of the properties of Con A at pH 5 are: The absorption coefficient of a 1 g/dl solution is 13.7 at 280 nm; the mean residue elliptic-ity at 224.5 nm is ?9,300° to ?9,800°; by sedimentation equilibrium, its molecular weight is 53,000 between pH 3.0 and pH 5.2. Con A solutions standing at room temperature at pH 7 for ten days lose through precipitation only 5–8% of the protein in 0.2 M NaCl and 15% of the protein in 0.1 M NaCI. In the solution conditions of SDS and urea-SDS gels, Con A not only unfolds slowly and incompletely, but it also forms high molecular weight aggregates. Thus, electrophoresis of Con A in such gels is unsuitable for tests of homogeneity. However, as judged by sedimentation equilibrium in 6.5 M quanidine at pH 8.1, purified Con A was monodisperse.