Macrophage Activation by Tetrahymena pyriformis II. Active Protein Fractions from Tetrahymena

ABSTRACT. Water-soluble and 0.6 M KCl-soluble protein fractions prepared from Tetrahymena pyriformis , when inoculated into mice, could effectively induce activated macrophages having the ability to kill Toxoplasma gondii in vitro. This effect was not induced by other proteins tested, such as bovine serum albumin, pepsin from porcine stomach mucosa and chicken egg-white lysozyme, nor by muramyl dipeptide (MDP), a potent immunoadjuvant. Five fractions obtained by DEAE-Sephadex chromatography of the water-soluble protein fraction were compared with regard to induction of toxoplasmacidal activity in macrophages. The first peak was most effective for activation of macrophages. Five fractions obtained by chromatography of the 0.6 M KCl-soluble protein fraction were also examined and it was found that the first peak had the activity. No marked difference in activity was observed between the active fractions of water-soluble and 0.6 M KCl-soluble protein fractions. For practical use, we focused on the water-soluble active fraction. The minimum effective dose of the active fraction was 100 μg and the fraction could activate macrophages directly in vitro. Four fractions obtained by gel filtration of the active fraction on Sephadex G-200 were compared and the first peak had the activity. The first peak contained a single protein, revealed by SDS-polyacrylamide gel electrophoresis; its apparent molecular weight was 64,000.     

Physiological-biochemical aspects of omission of the spawning period in the Barents Sea cod Gadus morhua

Using histological and biochemical methods, ovaries of the Barents Sea cod Gadus morhua were studied in different physiological states (immature fish, normal maturation, omission of spawning). Among the fish missing spawning, two categories are identified: without signs of occyte maturation and with massive resorption of maturing oocytes. The maximal activity of lysosomal and cytoplasmic proteolytic enzymes was revealed in the fish ovaries with histological signs of massive oocyte resorption. In the ovaries of maturing fish the highest concentrations of water-soluble proteins was found. The molecular-mass protein composition and the fraction responsible for the cytosol proteinase activity were determined by electrophoresis and high-performance liquid chromatography (HPLC). The obtained results allow some suggestions to be made about mechanisms of degradation of cellular proteins and functions of proteinases in various physiological states of the cod gonads.     

Interactions of KB-cell glycoproteins with an adenovirus capsid protein.

Glycoprotein material extracted from human KB cells with a flurocarbon, trichlorotrifluoroethane (Arklone P), into a waste-soluble fraction binds to fibre, a structural protein of the adenovirus type-5 capsid. The fibre-binding glycoprotein(s) were purified by ion-exchange chromatography on DEAE-Sephadex and affinity chromatography on a fibre-Sepharose support. The purification procedure also includes a trypsinization step which eliminates the bulk of contaminating KB cell proteins present in the aqueous fraction without appreciably affecting the activity of the fibre-binding glycoprotein(s). Some comparison is made of the membrane-bound receptors for adenovirus and the water-soluble fibre-binding glycoprotein(s).     

Purification of a Synaptic Membrane Na+/Ca2+ Antiporter and Immunoextraction with Antibodies to a 36-kDa Protein

The conditions for optimal solubilization and reconstitution of bovine brain synaptic plasma membrane Na+/Ca2+ exchange activity were examined and a series of chromatographic procedures were used for the isolation of a protein involved in this transport activity. The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of 20% (vol/vol) glycerol led to optimal solubilization, and soybean phospholipids in low-pH medium were found to produce optimal reconstitution of activity after dialysis to remove the detergent. Sequential chromatography steps involving the use of gel filtration on Sephacryl S-400 HR, ion exchange on diethylaminoethyl-Sephacel, and metal chelate chromatography on tris-(carboxymethyl)ethylenediamine loaded with LaCl3 led to the isolation of a fraction highly enriched in both Na+/Ca2+ exchange activity and two protein bands identified by denaturing electrophoresis. The estimated molecular masses of the two proteins were 50 and 36 kDa. Development of polyclonal antibodies to the 36-kDa protein permitted immunoextraction of greater than 95% of the antiporter activity from solubilized synaptic plasma membranes. These antibodies cross-reacted with the electroeluted 50-kDa protein on enzyme-linked immunosorbent assays, suggesting a close relationship between the two proteins. These results indicate that the 36-kDa protein is at least a component of the brain membrane Na+/Ca2+ antiporter.     

Two-Dimensional Electrophoretic Studies of the Proteins and Polypeptides in Mature Pollen Grains and the Male Germ Unit of Plumbago zeylanica

Three fractions (male germ unit [MGU]-rich, cytoplasmic-particulate and water-soluble proteins) were isolated from pollen of Plumbago zeylanica L. Proteins were extracted using a phenol procedure and polypeptide patterns were compared on one- and two-dimensional polyacrylamide gels. The MGU-rich fraction contains the sperm and vegetative nucleus of the pollen grain and yielded 427 spots >33 kilodaltons. The cyto-plasmic-particulate fraction contained 515 spots >33 kilodaltons. The third fraction consisted of water-soluble proteins and polypeptides from the pollen cytoplasm, in which 285 spots (>33 kilodaltons) were identified. Of 133 polypeptide spots suitable for comparison, 18 were unique to the MGU-rich fraction, 3 to the cytoplasmic-particulate fraction, 14 to the water-soluble fraction, 65 were common to two different fractions (and absent in one), and 33 were common to all three of the fractions examined.     

Polyphenolic compounds from flowers of Hibiscus rosa-sinensis Linn. and their inhibitory effect on alkaline phosphatase enzyme activity in vitro

Graded concentrations (0.1-100 mg/mL reaction mixture) of the methanolic extract of the flowers of Hibiscus rosa-sinensis Linn., its water-soluble fraction as well as compounds isolated from this fraction were tested for their inhibitory effect on alkaline phosphatase enzyme activity in vitro. Both the methanolic extract and its water-soluble fraction showed significant inhibitory effects on the enzyme activity in vitro. On screening the activity of the compounds isolated from the water-soluble fraction, its high inhibitory activity was attributed to the presence of quercetin-7-O-galactoside which showed a high potent inhibition of the enzyme activity reaching 100% at 100 mg/mL reaction mixture. Phytochemical investigations of the water-soluble fraction were also carried out and afforded ten polyphenolic compounds including two new natural compounds, namely kaempferol-7-O-[6'-O-p-hydroxybenzoyl-beta-D-glucosyl-(1-->6)-beta-D-glucopyranoside] and scutellarein-6-O-alpha-L-rhamnopyranoside-8-C-beta-D-glucopyranoside). The chemical structure of the isolated compounds was elucidated on the basis of chemical and spectral data.     

Isolation and Characterization of the Multiple 7S Globulins of Soybean Proteins

Two major proteins (the 7S and 11S globulins) of soybean (Glycine max) were simultaneously isolated by a simple method based on their different solubilities in dilute tris (hydroxymethyl) aminomethane buffers. The purified 7S globulins, which represented essentially the entire 7S soybean protein fraction capable of dimerization at 0.1 ionic strength, were fractionated into five components by diethylaminoethyl Sephadex A-50 column chromatography. The five 7S components were characterized by disc-electrophoresis.     

大豆磷酸烯醇式丙酮酸磷酸酯酶研究Ⅲ、非专一性酸性磷酸酯酶的纯化与特性

从大豆叶片中分离能水解磷酸烯醇式丙酮酸(PEP)的酸性磷酸酯酶,通过硫酸胺分部(20%～50%饱和度)沉淀、DEAE纤维素层析、刀豆球蛋白琼脂糖凝胶亲和层析将酶纯化了422.47倍,活性达78.16U/mg蛋白。该酶对PEP专一性不强,Km为1.09mmol/L(PEP),最适pH5.8,在pH4.8～7.0范围内及60℃以下较稳定,水解PEP的活性被Mg2+、Mn2+激活,F、Cu2+、Zn2+、PO43、MoO42及3磷酸甘油酸(3PGA)、三磷酸腺苷ATP等代谢物抑制,受异柠檬酸等有机酸影响较小。     

On rat liver mitochondrial monoamine oxidase activity and lipids

Following earlier observations on the retention of 5-hydroxytryptamine oxidizing activity by a purified preparation of monoamine oxidase from rat liver mitochondria, this fraction has been obtained in a water-soluble form by Triton X-100 gradient gel filtration and DEAE-Bio-Gel A chromatography. The soluble fraction appears to depend on Triton X-100 and phospholipids for its activity. The results seem to implicate membrane lipid components in the expression of rat liver mitochondrial monoamine oxidase activity.     

Alpha-aminoadipate and kynurenine aminotransferase activities from rat kidney. Evidence for separate identity

alpha-Aminoadipate aminotransferase and kynurenine aminotransferase activities from rat kidney are reportedly associated with the same protein. We observed that when the supernatant fraction was maintained at pH 4.5 for 75 min, 100% of kynurenine aminotransferase activity was lost, whereas only 40% of aminoadipate aminotransferase activity was lost. We purified alpha-aminoadipate aminotransferase and kynurenine aminotransferase from rat kidney supernatant fraction to electrophoretic homogeneity by ammonium sulfate fractionation, DEAE-Sephacel, and hydroxylapatite chromatography. Kynurenine aminotransferase activity was precipitated by pH treatment. The remaining aminoadipate aminotransferase activity was concentrated and injected into rabbits to raise antibodies that were used to prepare an affinity column. A mixture of aminoadipate aminotransferase and kynurenine aminotransferase activities obtained after hydroxylapatite chromatography was subjected to affinity chromatography. Aminoadipate aminotransferase and kynurenine aminotransferase activities resolved as separate peaks, providing evidence that the two activities are associated with two different proteins.     

两株海洋真菌的鉴定及其次级代谢产物抑制烟草花叶病毒及抗肿瘤活性

摘要：【目的】通过对2株活性海洋真菌发酵产物提取物抑制烟草花叶病毒和抗肿瘤活性进行研究,为进一步得到活性纯品化合物作为抗病毒及抗肿瘤的先导化合物奠定基础。【方法】菌株发酵产物的粗提物是通过甲醇浸取并在真空条件下蒸干得到的。粗提物中溶于水的部分为水溶性部分,不溶于水的部分为脂溶性部分。通过间接酶联免疫法检测样品抑制烟草花叶病毒的活性,通过四甲基偶氮唑盐微量酶反应比色法（MTT法）检测样品抗肿瘤活性,通过形态及ITS rDNA序列法进行菌株鉴定。【结果】两株海洋真菌抑制烟草花叶病毒活性和抗肿瘤的活性均较高。分子鉴定结果显示,两株真菌分别与Penicillium oxalicum 和 Neosartorya fischeri 的同源性极高。菌株0312F1发酵液的水溶性部分具有抗病毒及抗肿瘤活性,菌株1008F1发酵液的脂溶性部分具有抑制烟草花叶病毒活性,而水溶性部分具有抗肿瘤活性。【结论】菌株0312F1和菌株1008F1发酵液的提取物抑制烟草花叶病毒的活性部位不同,而抗肿瘤活性部位相同。菌株0312F1发酵液提取物的水溶性活性部位对肝癌细胞BEL-7404的抑制效果比对胃癌细胞SGC-7901的抑制效果明显,而菌株1008F1发酵液提取物的水溶性活性部位对胃癌细胞SGC-7901的抑制效果比对肝癌细胞BEL-7404的抑制效果明显。     

Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.

A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.     

p-nitrophenyl butyrate hydrolyzing activity of hormone-sensitive lipase from bovine adipose tissue

The "esterase" activity of hormone-sensitive lipase (HSL) was studied using water-soluble p-nitrophenyl butyrate (PNPB) as a substrate. Bovine adipose tissue HSL was purified to near homogeneity by precipitation at pH 5.0, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, and high performance ion-exchange columns on Mono Q and Mono S. The purified preparation hydrolyzed emulsified triolein and cholesteryl oleate (CO), and water-soluble PNPB. In the two last steps of purification, the elution profile of the CO-hydrolyzing activity coincided with that of PNPB-hydrolyzing activity. The HSL was adsorbed to heparin-Sepharose and the CO- and PNPB-hydrolyzing activities were eluted together in the same peak. Diisopropylfluorophosphate (DFP) strongly inhibited the HSL activities and the inhibition profiles of the triolein-; CO-, and PNPB-hydrolyzing activities were essentially identical. Only one polypeptide of Mr 84,000 in partial purified HSL fraction was labeled by affinity labeling with [3H]DFP. On digestion of the enzyme with trypsin, the triolein- and CO-hydrolyzing activities were lost more rapidly than the PNPB-hydrolyzing activity. Phosphorylation increased the triolein-hydrolyzing activity to 40% more than that of the control, but did not affect the CO- and PNPB-hydrolyzing activities.     

Multiple aflatoxin B1 binding proteins exist in rat liver cytosol

The in vitro binding of aflatoxin B1 to rat liver cytosolic proteins was investigated. Aflatoxin B1 binding activity was assayed with protein purified by gel permeation chromatography, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Twenty-five percent of the total binding activity was associated with proteins eluted by 0 and 0.1 M NaCl. Over 50% of the total binding activity was associated with protein present in the 0.2 M NaCl fraction. Glutathione S-transferase activity was also monitored and found only in the low salt (less than 0.2 M NaCl) fractions. The proteins eluted by 0.2 M NaCl were further purified by hydroxylapatite column chromatography and binding was found predominantly in a single fraction. The protein purification steps resulted in a 20-fold increase in the specific binding activity over that initially observed in the cytosol. These results indicate that multiple proteins are capable of binding aflatoxin B1 in rat liver cytosol.     

Purification of nodule-specific uricase from soybean by arginine-sepharose affinity chromatography

A method to purify uricase from soybean root nodules is described. The separation uses a single affinity chromatography step on Arginine-Sepharose, which was constructed by coupling L-Arginine to Activated CH-Sepharose 4B. Crude extracts were loaded onto small columns of Arginine-Sepharose and a significant retardation of uricase was observed. With a re-run of the fraction containing maximal uricase activity on the same column highly purified enzyme was obtained. Analysis by SDS-polyacrylamide gel electrophoresis revealed two protein bands. Analytical isoelectric focusing showed two isoforms of uricase, one dominating with pI 9.0 and a minor with pI 7.0.     

Studies on Sulfhydryl Groups during Cell Division of Sea Urchin Egg : V. Change in contractility of the thread model in relation to cell division

The contractility of the thread model prepared from the KCl-soluble proteins of the egg and in vivo factors for the contraction are investigated in Hemicentrotus, Anthocidaris, and Pseudocentrotus eggs. The contractility of the thread model induced by metal ions or cystine changes during development in the characteristic pattern of high at the metaphase and low at the monaster and the interkinetic stages. The change in contractility is paralleled by the change in the —SH content of the protein. The water-soluble fraction of the eggs has activity in causing contraction of the thread model. This activity changes during development in the same way as the contractility itself. The contraction of the thread induced by the water-soluble fractions is accompanied by a decrease in the —SH content of the thread. The activity of the water-soluble fraction in inducing the contraction is proportional to its ability to decrease the number of —SH groups. On boiling, the activity is largely destroyed. The activity is due to two components, one being non-dialyzable and the other dialyzable. Separately each component has little effect, but when mixed, the activity of the original sample is completely restored.     

Composition of Acid-sensitive Fraction in Soybean Proteins

An acid-sensitive fraction (ASF) was prepared from defatted soybean meals by two procedures. ASF1 was prepared by precipitation at pH 4.5 followed by removal of 1 m NaCl-soluble materials from the precipitate. ASF2 was prepared by precipitation in solution containing 1 m NaCl at pH 4.5. The protein components of the two fractions were analyzed by gel electrophoresis in a dissociating-buffer system and found to contain β-conglycinin, glycinin and whey proteins. In addition to these, several other bands appeared.

Appreciable amounts of lipid (8.2% in ASF1 and 8.8% in ASF2) were also found in the fractions. They were separated by column chromatography and thin-layer chromatography. Glycolipids were the major components of the lipids. Both glycolipid and phospholipid fractions contained slower-moving materials on thin-layer chromatography.     

Liposomal formulations of protein proteinase inhibitors: Preparation and specific activity

Possibility of encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) was investigated. The influence of the PC/PE ratio (w/w) on efficiency of incorporation of the Bowman-Birk soybean proteinase inhibitor (BBI) and aprotinin (BPTI) into liposomes was studied. Protein encapsulation did not affect liposome sizes. Confocal laser scanning microscopy demonstrated that proteins were located in the central part of the spherical particle and also between bilayers. The study of biological (antitrypsin and antichymotrypsin) activity demonstrated partial spatial shielding of active sites of proteins entrapped in liposomes. The effect of an ionic detergent on the activity of the encapsulated BBI and BPTI is consistent with this hypothesis and suggests that this shielding is reversible. Stability of liposomes was examined using three various media modeling gastrointestinal fluids (gastric and intestinal juices and fluids). Data obtained indicate that the prepared liposomes seem to be promising formulations for BBI and BPTI delivery.     

Y4xP, an open reading frame located in a type III protein secretion system locus of Sinorhizobium fredii USDA257 and USDA191, encodes cysteine synthase

Sinorhizobium fredii USDA257, a soybean symbiont, exports several nodulation outer proteins (Nops) into the rhizosphere. These proteins, which are exported by a type III secretion system (TTSS), have a pivotal role in host-specific nodulation. The entire TTSS of S. fredii lies within a 31-kb region that includes conserved genes that code for secretion machinery proteins, Nops, and several open reading frames (ORF) of unknown function. Identifying the functions of these ORF is essential to understand fully the role of TTSS in nodulation. Here, we report the characterization of y4xP, an ORF of previously unknown function. Southern blot analysis revealed that USDA257 contains two copies of y4xP, while a sibling, USDA191, contains a single copy. The amino acid sequence of Y4XP is homologous to both eukaryotic and prokaryotic cysteine synthase, a key enzyme in sulfur assimilation. The coding region of USDA257 y4xP under control of T7 promoter was expressed in Escherichia coli, and the recombinant protein was purified by nickel-affinity chromatography. Antibodies generated against soybean cysteine synthase cross-reacted with the recombinant protein. A nonpolar mutant of y4xP of USDA191 showed a marked reduction in cysteine synthase activity. Enzyme activity was completely restored when the mutant was complemented with a plasmid containing the y4xP sequence. Cysteine synthase activity was confined to the cell cytosol. Extracellular protein fraction from genistein-induced USDA191 showed no cysteine synthase activity. This observation indicates that cysteine synthase, which is located in the TTSS locus, is not a type III secreted protein. A nonpolar cysteine synthase mutant was able to export all the Nops to the rhizosphere, albeit in reduced amounts compared with the wild-type USDA191. Interestingly, USDA191 cysteine synthase mutant was able to initiate nodules on 'McCall' soybean more efficiently than the wild-type. Our results demonstrate that y4xP encodes a cysteine synthase and inactivation of this gene enhances the ability of USDA191 to form nodules on 'McCall' soybean by regulating Nops production.     

Characterization of a plasma membrane-associated plasminogen activator on thymocytes

Plasma membranes isolated from normal thymocytes of hamster and rats were found to exhibit neutral protease activity toward 125I-labeled casein. The plasma membrane-associated proteases were completely inhibited by the serine protease inhibitors, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and p-nitrophenyl-p-guanidinobenzoate, partially inhibited by soybean trypsin inhibitor and antipain, but were only weakly inhibited by L-1-tosylamino-2-phenylethyl chloromethyl ketone. The plasma membrane-associated proteases were also completely inhibited by ZnCl2 (75--100 mu M), but they were not affected by several other divalent cations. The plasma membrane fraction contained a plasminogen activator activity which was specifically localized in this fraction. The plasma membrane-associated plasminogen activator activity was inhibited by all of the inhibitors which inhibited plasma membrane-associated proteases except L-1-tosylamido-2-phenylethyl chloromethyl ketone. Labeling of plasma membrane-associated serine esterases with [3H] diisopropyl fluorophosphate followed by separation of the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that this fraction contained a single major 3H-labeled protein of Mr 105 000. Both the plasminogen activator and the Mr 105 000 esterase were shown to be glycoproteins by affinity chromatography on lentil lectin-Sepharose. These results indicate that the plasminogen activator of thymocytes is a glycosylated serine protease with an active site-containing subunit of Mr 105 000 which is specifically localized in the plasma membrane.