Mannose-specific isolectins with different hemagglutinating potencies isolated from Chinese daffodil (Narcissus tazetta var. chinensis) leaves

Three mannose-specific lectins exhibiting considerable similarities in NH₂-terminal amino acid sequence were isolated from leaves of the Chinese daffodil Narcissus tazetta (Family Amaryllidaceae). The purification protocol involved extraction with an aqueous buffer, anion exchange chromatography on DEAE-cellulose using stepwise elution with increasing salt concentrations, affinity chromatography on mannose-agarose, and FPLC-gel filtration on Superose 12. From the peak unadsorbed on DEAE-cellulose, and two peaks adsorbed on the ion exchanger and eluted respectively with 0.2 M Tris-HCl buffer and 0.5 M NaCl, were prepared fractions which yielded isolectins 1, 2, and 3 after adsorption on mannose-agarose and FPLC-gel filtraton. All three isolectins were homodimers with a molecular weight of 26 kDa. The lectin unadsorbed on DEAE-cellulose had the lowest, while the most strongly adsorbed lectin had the highest hemagglutinating activity.     

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

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

Purification of P0 Myelin Glycoprotein by a Cu2+-Immobilized Metal Affinity Chromatography

P0 is an abundant myelin glycoprotein of peripheral nerves of vertebrates. Various point mutations of this protein are responsible for hereditary neuropathies. In this paper we described purification of P0 glycoprotein using SDS and a metal chelate affinity chromatography. Purified myelin fraction from bovine spinal roots in 0.5% SDS, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.4 is filtered and applied directly to the Cu²⁺-immobilized affinity chromatography column, equilibrated with the same buffer. After eluting a void volume (or pass through) fraction, P0 protein was eluted by the same buffer but without salt. To remove contamination from the eluent, further purification is continued on a Concanavalin-A coupled agarose column. We purify within two days, 30 mg of P0 protein of apparent molecular weight 27 kDa. The method can be used to purify recombinant or mutated P0 protein found in severe pathologies.     

Isolation of lactoperoxidase, lactoferrin, alpha-lactalbumin, beta-lactoglobulin B and beta-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. beta-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, alpha-lactalbumin could not be adsorbed onto the resin. alpha-Lactalbumin and beta-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. alpha-Lactalbumin and beta-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. alpha-Lactalbumin was eluted using a linear (0-0.15 M) concentration gradient of NaCl in 0.05 M Tris-HCl buffer (pH 8.5). Subsequently, beta-lactoglobulin B and beta-lactoglobulin A were eluted from the column with 0.05 M Tris-HCl (pH 6.8), using a linear (0.1-0.25 M) concentration gradient of NaCl. The yields were 1260 mg alpha-lactalbumin, 1290 mg beta-lactoglobulin B and 2280 mg beta-lactoglobulin A from 1 l rennet whey.     

Isolation of lectin and albumin from Pisum sativum var. macrocarpon ser. cv. sugar snap

A mannose- and glucose-binding lectin bearing considerable sequence similarity to other legume lectins was isolated using a simple procedure, from legumes of the sugar snap Pisum sativum var. macrocarpon. The lectin was unadsorbed on Affi-gel blue gel and Q-Sepharose in 10 mM Tris-HCl buffer (pH 7.2) and adsorbed on SP-Toyopearl in 50 mM NaOAc buffer (pH 5). An albumin could also be purified at the same time. It was unadsorbed on Affi-gel Blue gel, adsorbed on Q-Sepharose and unadsorbed on SP-Toyopearl under the aforementioned chromatographic conditions. The lectin was almost identical in N-terminal sequences of its alpha- and beta-subunit to lectin from P. sativum L. var. Feltham First except for the 19th N-terminal residue of the beta-subunit. The lectin was devoid of antifungal activity. Out of the 15 N-terminal amino acids examined in pea albumin, three were different between the two varieties of P. sativum.     

Adsorption chromatography of nucleic acids on silicone-coated porous glass

Bovine liver tRNA was adsorbed on silicone-coated porous glass in 5 M NaCl, 10 mM Tris-HCl (pH 7.6) and fractionated by elution with decreasing NaCl concentrations. tRNAPro, tRNAVal, tRNAIle, tRNAThr, tRNASer, and tRNAPhe were eluted in this order. tRNA which had been digested with ribonuclease A was not adsorbed. Q beta RNA (adsorbed onto the glass in 5 M NaCl) was eluted with 1.5 M NaCl. RNA species in a crude rRNA fraction from Escherichia coli were separated into tRNA, 5S rRNA, and high molecular weight rRNA on siliconized porous glass. A half of calf thymus DNA was adsorbed on the glass in 5 M NaCl and the residual part passed through the column. The CD spectra showed that DNA and tRNA took the C-form and the A-form in 5 M NaCl, respectively. Therefore, the discrepancies of behavior of the DNA and RNA on siliconized porous glass may be related to the occurrence of these forms. The recovery of these nucleic acids from the column was 83-100%. Adsorption chromatography on siliconized porous glass may be a useful method for the separation of tRNA, rRNA, and mRNA.     

Purification of the iron-sulfur protein, ubiquinone-binding protein, and cytochrome c1 from a single source of mitochondrial complex III

By detergent-exchange chromatography using a phenyl-Sepharose CL-4B column, Complex III of the respiratory chain of beef heart mitochondria was efficiently resolved into five fractions that were rich in the iron-sulfur protein, ubiquinone-binding protein, core proteins, cytochrome c1, and cytochrome b, respectively. Complex III was initially bound to the phenyl-Sepharose column equilibrated with buffer containing 0.25% deoxycholate and 0.2 M NaCl. An iron-sulfur protein fraction was first eluted from the column with buffer containing 1% deoxycholate and no salt after removal of phospholipids from the complex by washing with the buffer for the column equilibration, as reported previously (Y. Shimomura, M. Nishikimi, and T. Ozawa, 1984, J. Biol. Chem. 259, 14059-14063). Subsequently, a fraction containing the ubiquinone-binding protein and another containing two core proteins were eluted with buffers containing 1.5 and 3 M guanidine, respectively. A fraction containing cytochrome c1 was then eluted with buffer containing 1% dodecyl octaethylene glycol monoether. Finally, a cytochrome b-rich fraction was eluted with buffer containing 2% sodium dodecyl sulfate. The fractions of the iron-sulfur protein and ubiquinone-binding protein were further purified by gel chromatography on a Sephacryl S-200 superfine column, and the cytochrome c1 fraction was further purified by ion-exchange chromatography on a DEAE-Sepharose CL-6B column; each of the three purified proteins was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Chromatographic Separation and Purification of Secretory IgA from Human Milk

Defatted and decaseinated human milk was concentrated and was fractionated on a preparative DEAE cellulose column. Elution with various concentrations of sodium chloride in Tris-HCl buffer (pH 8.0, 0.01 M) resulted in fractions that were rich in either secretory immunoglobulin A (SIgA) (0.1 M Nad) or free secretory component (SC) (0.05 M NaCl). The fractions, which were eluted with 0.10 M NaCl from the preparative column, were further fractionated on a G-200 Sephadex column. Repeated fractionation on this column resulted in a single purified fraction, which contained very high SIgA activity and showed immunological cross-reaction with both SC and serum IgA. Additional studies indicated that this fraction was homogeneous as shown by immunoprecipitin and disc gel electrophoresis. Injection of this purified SIgA into rabbits resulted in the production of monospecific antiscil.     

Purification of glycomacropeptide from non-dialyzable fraction of sweet whey by hydrophobic interaction chromatography on phenyl-agarose

Non-dialyzable fraction of sweet whey was chromatographed on a column of phenyl-agarose equilibrated with 0.01 M sodium phosphate buffer, pH 6.8 containing 5 M NaCl. Most whey proteins were adsorbed on the column, while the glycomacropeptide (GMP) was not. Amino acid analysis of the GMP fraction showed presence of traces (each < 1 residue/peptide) of arginine, histidine and phenylalanine which are not found in GMP. The estimated yield of GMP fraction was approximately 1.6 g l^-1 of sweet whey.     

D-alanine carboxypeptidase activity of Micrococcus lysodeikticus released into the protoplasting medium.

Conversion of whole cells of Micrococcus lysodeikticus to protoplasts allowed the release of a soluble form of a D-alanine carboxypeptidase into the protoplasting medium. The enzyme cleaves the terminal D-alanine from the radioactively labelled UDP-N-acetylmuramyl-pentapeptide containing L-lysine as the diamino acid. However, the enzyme is only minimally active in this fraction so that it had to be enriched and partially purified before its properties could be studied. Chromatography on carboxymethyl-Sephadex removed the lysozyme used in the protoplasting of the cells. The material which was unadsorbed to the column was applied to an affinity chromatography column of Ampicillin-Sepharose. Most of the contaminating protein was washed from the column while the D-alanine carboxypeptidase adhered to the resin and could be eluted with 0.5 M Tris-HCl buffer pH 8.6. Some of the properties of the enzymic activity were studied using this preparation. The enzyme was activated by Mg2+ ions with a broad optimum from 15--35 mM. It was maximally active when NaCl at a concentrations of 0.06--0.08 M was added to the assay, and the pH curve was biphasic with an alkaline optimum. The Km for substrate was found to be 0.118 mM. Enzymic activity was completely inhibited by low concentrations of Ampicillin and penicillin G.     

An amylase from fresh fruiting bodies of the monkey head mushroom Hericium Erinaceum

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn²⁺ and Fe³⁺ ions, but inhibited by Hg²⁺ ions.     

A simple method for preparation of snake venom phosphodiesterase almost free from 5'-nucleotidase.

A simple method, involving NAD+-Sepharose chromatography, was developed for the preparation of snake venom phosphodiesterase (EC 3.1.4.1) almost free from 5'-Nucleotidase (EC 3.1.3.5). Using an NAD+-Sepharose 4B column, phosphodiesterase was eluted in the unadsorbed fraction, whereas 5'nucleotidase was strongly adsorbed. The latter enzyme was desorbed when 0.2 M sodium bicarbonate buffer containing 1mM beta-NADH was used as a solvent. The affinity column could be used at least four times without any decrease of potency, and the method was applicable for the preparation of phosphodiesterase from the venoms of rattlesnake (Crotalus adamanteus) and Japanese mamushi (Agkistrodan halys blomhoffi).     

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.     

Cibacron Blue亲和层析及应用

以Ｆ３ＧＡ（Ｃｉｂａｃｒｏｎ　Ｂｌｕｅ　Ｆ３ＧＡ）为配基建立了一种可用于免疫毒素（ＩＴ）分离纯化的亲和层析方法。实验中用三种不同来源的核糖体灭活蛋白（ＲＩＰ）,即蓖麻毒素Ａ链（ＲＴＡ）,苦瓜毒素（ｍｏｍｏｒｄｉｎ,ＭＴ）和Ｓａｐｏｒｉｎ,以探讨ＲＩＰ与Ｆ３ＧＡ的相互作用。分析显示三种ＲＩＰ均能引起Ｆ３ＧＡ吸收光诸明显红移,提示ＲＩＰ均可与Ｆ３ＧＡ发生特异结合。将Ｆ３ＧＡ与Ｓｅｐｈａｄｅｘ交联可获得Ｂｌｕｅｄｅｘ。Ｂｌｕｅｄｅｘ亲和层析是一种经济有效,简单易行,便于在各类实验室中使用的蛋白质亲和层析技术。结果表明：在低盐溶液中ＲＴＡ和ＭＴ均可迅速地与Ｂｌｕｅｄｅｘ结合,而在高盐溶液中（０．６５ｍｏｌ／ＬＮａＣｌ）又极易被洗脱回收。这一技术用于免疫毒素的研究可有效地去除游离抗体,而不影响其杀伤活性。     

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.     

Isolation of a new heterodimeric lectin with mitogenic activity from fruiting bodies of the mushroom Agrocybe cylindracea

From the dried fruiting bodies of the mushroom Agrocybe cylindracea a heterodimeric lectin with a molecular weight of 31.5 kDa and displaying high hemagglutinating activity was isolated. The molecular weights of its subunits were 16.1 kDa and 15.3 kDa respectively. The larger and the smaller subunits resembled Agaricus bisporus lectin and fungal immunomodulatory protein from Volvariella volvacea respectively in N-terminal sequence. The lectin was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and was eluted by the same buffer containing 150 mM NaCl. It was adsorbed on SP-Sepharose in 10 mM NH4OAc (pH 4.5) and eluted by approximately 0.19 M NaCl in the same buffer. The lectin was obtained in a purified form after the mushroom extract had been subjected to (NH4)2SO4 precipitation and the two aforementioned ion exchange chromatographic steps. The lectin exhibited potent mitogenic activity toward mouse splenocytes. The hemagglutinating activity of the lectin was inhibited by lactose, sialic acid and inulin.     

Isolation of pleuturegin, a novel ribosome-inactivating protein from fresh sclerotia of the edible mushroom Pleurotus tuber-regium.

A ribosome-inactivating protein (RIP) designated pleuturegin, which inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 0.5 nM, was purified from fresh sclerotia of the edible mushroom Pleurotus tuber-regium. Pleuturegin was distinguished from most plant and previously reported mushroom RIPs in that it was adsorbed on DEAE-cellulose and unadsorbed on SP-Sepharose, although all of them were adsorbed on Affi-gel blue gel. Pleuturegin demonstrated an N-terminal sequence that was different from those of RIPs from Flammulina velutipes (flammulin and velutin), Hypsizygus marmoreus (hypsin), and Lyophyllum shimeji (lyophyllin), the only mushroom RIPs with known N-terminal sequences. The molecular mass of pleuturegin was 38 kDa, similar to that of flammulin (40 kDa) but considerably larger than those of velutin (13.8 kDa), hypsin (20 kDa), and lyophyllin (20 kDa). Pleuturegin was devoid of ribonuclease activity.     

A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus

An 18-kDa ribonuclease (RNase) with a novel N-terminal sequence was purified from fresh fruiting bodies of the mushroom Hypsizigus marmoreus. The purification protocol comprised ion exchange chromatography on DEAE cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose and Q-Sepharose and gel filtration by fast protein liquid chromatography on Superdex 75. The starting buffer was 10 mM Tris-HCl buffer (pH 7.2), 10 mM Tris-HCl buffer (pH 7.2), 10 mM NH(4)OAc buffer (pH 5), 10 mM NH(4)HCO(3) buffer (pH 9.4) and 200 mM NH(4)HCO(3) (pH 8.5), respectively. Absorbed proteins were desorbed using NaCl added to the starting buffer. A 42-fold purification of the enzyme was achieved. The RNase was unadsorbed on DEAE cellulose, Affi-gel blue gel and CM-cellulose but adsorbed on Q-Sepharose. It exhibited maximal RNase activity at pH 5 and 70 degrees C. Some RNase activity was detectable at 100 degrees C. It demonstrated the highest ribonucleolytic activity (196 U/mg) toward poly C, the next highest activity (126 U/mg) toward poly A, and much weaker activity toward poly U (48 U/mg) and poly G (41 U/mg). The RNase inhibited [(3)H-methyl]-thymidine uptake by leukemia L1210 cells with an IC(50) of 60 microM.     

Existence of Mg2+-dependent, neutral sphingomyelinase in nuclei of rat ascites hepatoma cells

A sphingomyelinase, which specifically hydrolyzes sphingomyelin into ceramide and phosphocholine, was solubilized from nuclear matrix fraction of rat ascites hepatoma, AH7974 cells. The solubilized enzyme was subjected to Mono Q column chromatography in an FPLC system. The sphingomyelinase which was adsorbed on the column and eluted at 0.25-0.5 M NaCl was characterized. The enzyme required 10 mM MgCl2, 0.01% Triton X-100, 1 mM dithiothreitol, and a higher concentration of buffer than 1 M for its maximal activity, and the optimal pH was 6.7-7.2 in 2 M Tris/acetic acid or 7.5 in 2 M potassium acetate/acetic acid. N-Ethylmaleimide completely inhibited the enzyme activity at 0.2 mM. Therefore, this enzyme is classified as a Mg2+-dependent, neutral sphingomyelinase. The sphingomyelinase sedimented at 4.3S through a 10-30% glycerol gradient containing 2 M potassium acetate. This enzyme was highly specific to sphingomyelin and did not hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Various characteristics of the nuclear sphingomyelinase were similar to those of the plasma membrane enzyme except its requirement for a high concentration of buffer and SH-reagent.     

两步串联层析法纯化鼠抗人CD80单克隆抗体4E5

采用阴离子交换与凝胶过滤两步串联层析法,纯化了小鼠腹水来源的CD80阻断型单克隆抗体4E5。腹水样品经离心、过滤预处理后,在Tris-HCl缓冲溶液(pH8.0, 50mmol/L)条件下上阴离子交换柱对目的单抗进行捕集,采用0-0.5 mol/L NaCl浓度分步洗脱;含目的单抗的洗脱馏分再上凝胶过滤柱纯化,用PB缓冲溶液（pH7.2, 20mmol/L）洗脱,获得目的单抗4E5,其生物学活性高、纯度大于95%,抗体总回收率达61%。