Isolation and characterization of fourteen ribosomal proteins from small subunits of yeast.

A method for preparation of a large amount of ribosomal subunits from Saccharomyces cerevisiae by a Ti-15 zonal rotor is described. The proteins of the small subunits (ca. 50 000 A260 units) were separated into 22 fractions by chromatography on carboxymethylcellulose columns. Fourteen proteins were then purified from the ten chromatographic fractions by filtration through Sephadex G-100 or Sephacryl S--200. The isolated proteins are YP 6, YP 7, YP 9, YP 12, YP 14', YP 14', YP 28, YP 38, YP 45, YP 50, YP 52, YP 58, YP 63, and YP 70. The molecular weight and amino acid compositions of these proteins are presented.     

球形芽孢杆菌Ts—1毒蛋白的分离纯化

Bacillus sphaericus strain Ts-1 is highly insecticidal to larvae of the mosquito. It's insecticidal component is toxic proteins. The toxin was extracted from spore-crystal complexes by disruption in a Sonicator Cell Disruptor Model W-220F followed by treatment with 0.05 mol/L NaOH. Fraction recovered from chromatography of the spore-crystal complexes on column of Sephadex G-200 were assayed against mosquito larvae and the toxic fractions from gel chromatography were subjected to SDS-PAGE. The toxic proteins in B. sphaericus Ts-1 spore-crystal complex migrated in position corresponding to 42kD and 43kD. Bioassay of the two purified proteins prepared by PAGE indicated that they were all toxic to mosquito larvae. Toxic protein was further purified by DEAE-cellulose chromatography. The toxic protein with a molecular weight of 42kD was obtained.     

Heterogeneity of streptococcus group A cell wall protein components

Cell wall surface proteins of group A streptococcus (M 29) were isolated by mild chemical extraction with 1 M hydroxylamine pH 6.0 (37 degrees C). The proteins were purified by ammonium sulfate fractionation, gel filtration on Sephadex G-150 and ion-exchange chromatography on DEAE-Trisacryl M. Using two independent methods (disc electrophoresis in 7.5% PAAG pH 8.9 and high pressure gel filtration), it was shown that after chromatography on Sephadex G-150 the original protein fraction contains up to 8 protein components, while SDS-PAAG electrophoresis performed according to Laemmli revealed up to 25 protein components in the same fraction. During SDS-PAAG electrophoresis six protein fractions performed after ion-exchange chromatography were resolved into 40 protein components whose molecular masses vary from 13 to 80 kDa. Possible reasons for the heterogeneity of surface proteins of group A streptococcus cell wall are discussed.     

Induction of oxidative stress in leukocytes by an Enterobacter cloacae toxin able to form oligomers and binding to proteins

A leukotoxic and hemolytic toxin was purified from cultures of Enterobacter cloacae. Stimulation of oxidative stress was observed and the production of reactive oxidant species was measured in leukocytes treated with toxin by means of nitroblue tetrazolium and chemiluminescence assays. Molecular weight of toxin was estimated by chromatography and SDS-PAGE. Two protean peaks with toxic activity were found in Sephadex G-100 (P1, 42.0 kDa; and P2, 13.3 kDa). The relative amounts between the peaks (P1/P2 = 0.36) changed when 2-mercaptoethanol was employed (P1/P2 = 0.59). When Sephadex G-200 chromatography was performed, a protean peak of Ve = 113 mL (100 kDa) was found; its was dissociated with 3 M urea in toxic proteins of lower mass: 42, 27, and 13.3 kDa. SDS-PAGE (15%) showed a single toxin band of purified monomer (13.3 kDa), but electrophoresis of a 42-kDa toxin with urea presented three bands of trimer, dimer, and monomer. An increase of casein hydrolysate and albumin molecular weight was observed by chromatography after incubation with toxin due to the binding of both proteins with toxin.     

Isolation and characterization of a neurophysin from ostrich neurohypophyses

A neurophysin has been isolated from ostrich neurohypophyses using acid acetone extraction, salt fractionation and Sephadex G-75 chromatography. The crude neurophysin eluting from the Sephadex G-75 column was subjected to a) reverse-phase HPLC followed by Sephadex G-75 chromatography, b) DEAE-Sephadex A-50 chromatography or c) isoelectric focusing. The different homogeneous ostrich neurophysin fractions so obtained were compared i.t.o. amino acid composition, spectral properties, N-terminal amino acid residues and PAGE. They all revealed a single N-terminal Ala residue and displayed spectral properties (A280/A260 less than 1) which are typical of mammalian neurophysin-like polypeptides. Ultracentrifugation studies on purified ostrich neurophysin over a range of concentrations revealed a reversible concentration dependent association behaviour characterized by the presence of dimeric complexes at higher concentrations. Partial sequencing from the N-terminus revealed the molecule to be VLDV-like. The purified molecule was also submitted to CNBr fragmentation.     

An affinity adsorbent, 5'-adenylate-aminohexyl-sepharose. II. Purification and characterization of multi-forms of RNase T2

RNase T2 bound to an affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4B, specifically at pH 4.5. The colorless enzyme was eluted only by the simultaneous addition of 2'(3')-AMP (1 mM) and NaCl (greater than 1 M) at pH 4.5. By applying this affinity chromatography to the purification of RNase T2, pure enzyme with a specific activity of 60 was obtained in only four steps and the yield was about 10 times higher than that of the previous purification method. This enzyme preparation was found to be heterogeneous in molecular weight and was separated into two fractions on Sephadex G-75 gel filtration. As the smaller enzyme with a molecular weight of 36,000 was identical with RNase T2 in every property examined, we tentatively designated the larger one with an apparent molecular weight of 80,000 as high molecular weight RNase T2 (RNase T2-L). RNase T2-L was still heterogeneous and was separated into five fractions, RNases T2-L 1-5, by repeated Sephadex G-150 gel filtration. The amino acid and carbohydrate analyses revealed that each of these fractions has a protein moiety in common with RNase T2 and the heterogeneities were due to the carbohydrate content, mainly galactose content.     

Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties

6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)(-1), was purified 344-fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and KM and Vmax values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G-200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Ki values and inhibition types were estimated by means of Lineweaver-Burk graphs obtained for NADPH and CO2 products.     

The binding in vitro of colchicine to axoplasmic proteins from chicken sciatic nerve

1. Axoplasmic proteins were fractionated by means of Sephadex G-200 chromatography followed by isoelectric focusing. Nine groups of proteins were separated. 2. The binding of colchicine to these groups of proteins was examined and it appeared to associate most strongly with one protein group, of pI value 4.9-5.0, which is the major (14)C-labelled component of slow-transport protein. 3. Other fractions also bind colchicine. It is not clear whether these are separate proteins or subunits of the major colchicine-binding fraction.     

Substances with alpha-melanotropin-like immunoreactivity in bovine brains.

1. Bovine cerebral hemispheres were extracted with an acidic medium (acetone-water-hydrochloric acid mixture, 40:5:1 by volume, pH 1.8). The precipitate which formed upon addition of a copious volume of cold acetone to the extract was designated acid acetone powder (AAP). 2. The AAP was then subjected to ion exchange chromatography on carboxymethyl (CM)-cellulose, gel filtration on Sephadex G100 and Sephadex G25, second ion exchange chromatography on CM-cellulose and high performance liquid chromatography. The absorbance of all fractions was measured at 280 nm and their alpha-melanotropin-(alpha-MSH)-like immunoreactivity was monitored with radioimmunoassay. 3. It was found that alpha-MSH-like immunoreactivity and bioactivity (lipolytic activity) was due to low molecular weight materials as evidenced by their retardation on Sephadex G-100 and Sephadex G-25. The immunoreactivity was distributed among fractions adsorbed and fractions unadsorbed on CM-cellulose and also among high performance liquid chromatographic fractions signifying the presence of multiple alpha-MSH-like molecules.     

RNA associated with nonhistone chromosomal proteins of dog liver

The nonhistone chromosomal proteins were separated on Sephadex G-200 into 3 fractions of which two were associated with 3S RNA. The RNA eluted with fraction I (guanine + cytosine content 54%) is tightly bound to the proteins from which it can be separated only after digestion with pronase. The RNA associated with fraction III (guanine + cytosine content 64%) can be separated from the proteins directly by chromatography on DEAE-Sephadex A 25. No dihydropyrimidines have been detected in any of the two RNAs.     

Growth inhibitors in plasma derived human serum

Summary It was reported previously that plasma derived human serum (PDS) inhibited the growth of cells established from malignant human breast tissues and the MCF-7 cell line but did not inhibit the growth of cells from nonmalignant mammary tissues, including the HBL-100 cell line. Plasma derived human serum was fractionated in the current study by molecular sieve chromatography on Sephadex G-100 in an effort to characterize the factor(s) responsible for inhibition. Plasma derived human serum contained several growth inhibitory fractions, which were designated G-1, G-2, G-3, and G-4. The G-1 was associated with the lipoproteins and immunoglobulins of serum. The lipid portion of G-1 inhibited the growth of both MCF-7 and HBL-100 cells, whereas the protein fraction contained a low activity factor directed against MCF-7 cells only. The G-2 also inhibited MCF-7 cell growth at a low specific activity and was separated in the serum albumin fraction. The MCF-7 inhibitory activity in the G-3 fractions from individual donors fluctuated with the level of activity in the starting sera. The cell specific G-3 components were purified further by Sephadex G-100 superfine chromatography and gel electrophoresis. A tentative molecular weight of 50,000 was assigned to the G-3 inhibitor. The G-4 fraction consisted of small molecular weight materials migrating in advance of the column volume, which inhibited the growth of both cell lines. This investigation was supported by Grant PDT-140 from the American Cancer Society, Inc., and PHS Grant CA30284 awarded by the National Cancer Institute, Bethesda, Maryland.     

Beta-endorphin-like and adrenocorticotropin-like materials in turtle heart and intestine

1. Turtle heart and intestine acetone powders were extracted with an acetone-water-HCl mixture. An acid acetone powder resulted by adding a copious volume of acetone to the extract. The powder was subjected to gel filtration on Sephadex G-25 and ion exchange chromatography on CM-cellulose. 2. In turtle heart, corticotropin-like bioactivity was distributed among chromatographic fractions (derived from material unretarded on Sephadex G-25) unadsorbed and adsorbed on CM-cellulose. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were adsorbed on CM-cellulose. 3. In turtle intestine, corticotropin-like bioactivity was absent. Opiate receptor binding activity was present in fractions unretarded as well as in fractions retarded on Sephadex G-25, indicating a molecular weight of greater and smaller than 5000 respectively. 4. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were found in a fraction adsorbed on CM-cellulose.     

Evidence of the requirement of a full complement of manganese and calcium ions for optimal binding of carbohydrates to electrophoretically homegeneous Concanavalin A

Affinity chromatography on Sephadex G-50 is a very simple and rapid method for invetigating the carbohydrate-binding ability polyacrylamide gel electrophoretically pure Concanavalin A. When highly purified concanavalin A is further fractionated on Sephadex, three protein-containing peaks A, B and C are often observed. The isolated pooled fractions are indistringuishable by polyacrylamide gel electrophoresis at pH 4. In addition they show identical properties when examined by ultracentrifugation or by gel filtration. These proteins differ in their content of Mn²⁺ and Ca²⁺ and in their ability to bind carbohydrates. Only those preparations which had the full complement of metal ions bound to Sephadex and had the ability to bind other polysaccharides.     

Proteinase inhibitors in aggregated forms of boar seminal plasma proteins

Boar seminal plasma proteins were separated by gel chromatography on Sephadex G-75 into five fractions (I–V). Serine proteinase inhibitors were found mainly in the protein fraction with relative molecular weight 5–25 kDa. Small amounts of these inhibitors were also found in the high molecular weight protein fraction (M_r>100 kDa). The protein fraction containing most of the proteinase inhibitory activity was further separated by RP HPLC. Isolated proteins were characterized by SDS electrophoresis and immunoblotting, N-terminal amino acid sequencing and by determination of the proteinase inhibitory activity. In the fraction containing proteinase inhibitors, also β-microseminoprotein (β-MSP), AQN 1 and lactoferrin were identified. The possible existence of complexes of protein components in the fraction with relative molecular weight 5–25 kDa was studied in detail using gel chromatographic separation on Sephadex G-50. A part of proteinase inhibitors with M_r 8 kDa was eluted together with AQN 1 spermadhesin. An interaction of isolated spermadhesin AQN 1 and proteinase inhibitor was shown.     

Antigen-independent leukocyte random locomotion inhibitory activity in human ultrafiltrated leukocyte extracts

Human ultrafiltrated leukocyte extracts (MW < 5000) were fractionated by Sephadex G-10 column chromatography and the effects of these fractions on leukocyte random locomotion were investigated in vitro. Fr-4, one of these fractions, had significant leukocyte random locomotion inhibitory activity, independent of the presence of mononuclear leukocytes. This inhibitory activity was not due to cytotoxic effects on leukocytes. As seen by scanning electron microscopy, the number of cell surface pseudopods on leukocytes incubated with Fr-4 was reduced. Fr-4A, one of three fractions separated from Fr-4 by Sephadex G-25 column chromatography, significantly inhibited leukocyte random locomotion. Fr-4A contained numerous components, one of which was identified as 2-deoxyribose, on the basis of thin-layer chromatography. Biologically 2-deoxyribose showed an inhibitory effect on leukocyte locomotion and a reduction of the extrusion of pseudopods on the surface of leukocytes, at the range of assayed concentrations. This inhibitory activity is probably derived from 2-deoxyribose.     

Density-Gradient and Chromatographic Fraction of Leptospiral Lipase

Fractionation of leptospiral lipase by CsCl density gradients and G-200 Sephadex chromatography yielded five active protein peaks. Two were obtained from the density gradients and three from G-200 Sephadex columns. Esterase activity of these fractions was demonstrated by electrophoretic examination. Several protein bands were visible when disc electrophoresis was performed on the respective fractions. Lipolytic and esterolytic activities were both present, and the overlapping of these activities was discussed.     

Effect of dialyzate fractions of spinach on growth of human-derived cells

Aqueous dialyzate of spinach was separated by Sephadex G-100, G-25 gel-filtrations and DEAE-cellulose ion exchange chromatography, and the effects of the fractions on growth of human-derived normal and cancer cell lines were studied. One of the fractions (SPW2) from a Sephadex G-100 gel-filtration of dialyzate promoted growth of a hybridoma cell line (HB4C5). Sephadex G-25 gel filtration of the SPW2 fraction produced four main fractions; SPW2-1, SPW2-2, SPW2-3 and SPW2-4. Among them, the SPW2-1, SPW2-3 and SPW2-4 fractions promoted the growth of a histiocytic lymphoma cell line (U-937) and hybridoma cell lines (HB4C5 and SI102). Both SPW2-3 and SPW2-4 fractions inhibited the growth of a breast cancer cell line (MCF-7). The SPW2-3 fraction, especially, was found to inhibit growth of cancer cell lines such as MCF-7, a differentiated hepatoma (HuH-7), a lung adenocarcinoma (PC-8), a lung squamous carcinoma (QG-56), and a lung anaplastic carcinoma (QG-90) more preferentially than that of normal cell lines. It was also found that a constituent of the SPW2-3 fraction caused the morphological alteration of U-937 cells in serum-free medium.     

Antigenic polysaccharides of bacteria of the genus Shigella. Determination of the structure of polysaccharide chains of Shigella boydii type 9 lipopolysaccharide and detection of unusually high molecular weight glycolipid

Specific acidic polysaccharide has been isolated from the Shigella boydii type 9 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and L-rhamnose. From the results of methylation analysis, partial acid hydrolysis and 13C NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [----4)DGlcp(alpha 1----4)DGlcAp(beta 1----3)DGlcNAcp(alpha 1----3)LRhap(alpha 1----]n. The lipopolysaccharide from Sh. boydii 9 was fractionated by gel chromatography on the Sephadex G-200 column in a buffer containing sodium deoxycholate into three fractions. PAGE-SDS of the fractions obtained, 13C NMR- and chromato-mass-spectrometry data indicated that the three fractions contained the O-specific polysaccharide as the only carbohydrate component. The substance from the most high-molecular weight fraction contained unusually long O-specific chains (60,000 dalton). In the fat acid composition this fraction differed from other lipopolysaccharides by absence of beta-hydroxymyristic acid.     

舟山眼镜蛇毒抗肿瘤有效组分分离及其抑瘤作用研究初探

目的从舟山眼镜蛇(Naja atraCantor)蛇毒(snake venom,SV)中分离得蛇毒组分,探讨SV及其分离组分的LD50和抑制肿瘤的作用。方法采用凝胶柱层析方法从蛇毒中分离得到了前Ⅰ1、Ⅰ1、Ⅱ1、Ⅱ2、Ⅲ1、Ⅲ2及Ⅳ等7种组分。采用急性毒性实验、MTT法,研究SV及其7种SV分离组分的LD50和抑制肿瘤的作用。结果 SV经Sephadex G-50层析,可分离为前Ⅰ、Ⅰ、Ⅱ、Ⅲ及Ⅳ5个组分,根据峰面积大小排列:ⅢⅡⅠⅣ前Ⅰ。5个组分再经Sephadex G-25柱层析,可获得7个脱盐组分:前Ⅰ1、Ⅰ1、Ⅱ1、Ⅱ2、Ⅲ1、Ⅲ2及Ⅳ。通过急性毒性实验,明确Ⅳ的毒性最大,其次为Ⅲ2及Ⅲ1,三者的LD50值均低于SV;而Ⅰ1、Ⅱ1、Ⅱ2的毒性均小于SV,前Ⅰ1几乎无毒。SV组分Ⅲ2和Ⅳ的抑瘤作用最强,在高浓度(20μg/ml)时对实验中的2种人肿瘤细胞的抑制率均达到60%以上。结论从SV中分离得到了前Ⅰ1、Ⅰ1、Ⅱ1、Ⅱ2、Ⅲ1、Ⅲ2以及Ⅳ7种组分;组分Ⅳ毒性最强,依次为Ⅲ2Ⅲ1SVⅡ2Ⅱ1Ⅰ1前Ⅰ1;SV及其7种分离组分对2种人肿瘤细胞株(SGC-7901、A549)的生长抑制有一定的特异性,而不同的SV分离组分对同一肿瘤细胞抑制作用亦有差异。     

Antioxidant Properties of the Peptides Isolated From Ganoderma lucidum Fruiting Body

Ganoderma lucidum is widely used as traditional medicine for centuries particularly in China, Japan and Korea. Many bioactive metabolites isolated from G. lucidum were therapeutically active against various diseases. The peptide isolated from water extract of G. lucidum was purified by employing Sephadex G-25, Sephadex G-50 and reverse phase HPLC column chromatography. The antioxidant property of the peptide fractions was determined by various in vitro methods. All fractions obtained from Sephadex G-25 and fraction G from Sephadex G-50 are effective antioxidants and comparably fraction C has the highest antioxidant activity. The molecular weight of purified peptide determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography and Matrix-assisted laser desorption ionization time-of-flight-mass spectrometer was found to be 2.8, 3.34 and 3.35?kDa respectively. The amino acid composition of the peptide was rich in phenylalanine, aspartic acid, proline, histidine and isoleucine. Peptide isolated in the present investigation suggests that has beneficial antioxidant properties may be due to its low molecular weight and specific amino acid composition.