The preparation and chemical composition of fractions from Aspergillus fumigatus wall and protoplasts possessing antigenic activity.

A detergent-soluble fraction was prepared from the fragmented wall of Aspergillus fumigatus mycelium using the non-ionic detergent Triton X-100, and a wall-free extract was prepared from the same source in the form of protoplasts, released by a lytic enzyme system from Trichoderma harzianum. These extracts were examined by polyacrylamide gel electrophoresis and their detailed chemical composition was established. They were compared with the water-soluble fraction prepared from total mycelium, which is used routinely in this laboratory for serological tests. All fractions had immunological reactivity towards an antiserum prepared in rabbits against this water-soluble fraction of the mycelium, as shown by double diffusion. Both protein and carbohydrate moieties appear to be involved in the antigenic sites, with carbohydrate reactivity predominantly associated with the protoplast fraction. The fact that all preparations contained at least one common antigenic determinant, as judged by lines of identity to a single antiserum, is discussed in relation to antigen location.     

On the heterogeneity of chromatin fractions after gel filtration

Partially salt-extracted nucleoproteins in the eluted fractions after gel filitration in 0.6 to 1.0 m-NaCl are heterogeneous in terms of the protein to DNA ratio. This heterogeneity is due to a redistribution within the gel column of the proteins associated with the DNA. If the fractions of the nucleoprotein peak are pooled before the NaCl is removed, then the resulting material is homogeneous.     

Schistosoma mansoni: antigenic heterogeneity of excretions and secretions

The excretory-secretory antigens of adult Schistosoma mansoni were obtained by in vitro cultivation of worms in a chemically-defined medium. The protein output in this system was low, 0.2–0.4 μg of proteinworm/48 hr. The composition of the crude culture antigen (CA) was approximately 80% protein, 15% carbohydrate and 5% nucleic acid. Disc gel electrophoresis of CA revealed the presence of at least 15 protein components, many with carbohydrate moieties. Three major fractions were obtained by gel filtration on Sephadex G-200. Fraction I contained the bulk of the glycoprotein material. Immunoelectrophoresis of CA with hyperimmune rabbit serum indicated the presence of at least 6 antigens, most of which eluted in Fraction II. Serum from infected mice and monkeys, but not from rabbits and rats, reacted with CA and its fractions, especially Fraction II, on immunodiffusion analysis. Comparison of CA with other adult worm extracts by immunodiffusion techniques showed that most of the excretory-secretory antigens could be obtained by either freezing and thawing or by extraction with 3 M KCl. The P.K.-type activity of CA was considerably greater than that of a lipid-free adult worm antigen. Both Fractions I and II had the P.K.-type activity. An antigen capable of eliciting macrophage migration inhibition factor from infected rat lymphocytes was detected in CA, although the lymphocyte toxicity of CA was high at concentrations above 10 μg/ml.     

Evaluation of equilibrium gel filtration chromatography for the study of protein binding of aluminum in normal and uremic sera

Normal and uremic serum samples were chromatographed on a gel filtration column and fractions were analyzed for protein and aluminum content. Whereas much of the aluminum in the serum samples eluted with protein, a significant fraction appeared to be associated with low molecular weight species. Analysis of column fractions for aluminum binding by a competitive binding assay indicated no aluminum-binding serum constituents in these later eluting fractions. Uremic serum appeared, however, to contain aluminum-binding constituents of a lower molecular weight than in normal serum.     

Inhibition of lymphocyte proliferation by uterine fluid from the pregnant ewe

Immunosuppressive molecules in uterine fluid from the nonpregnant uterine horn of unilaterally pregnant ewes at Days 60, 100, and 140 of gestation were examined. Uterine fluid from all days inhibited [3H]thymidine incorporation into phytohemagglutinin-stimulated lymphocytes. Inhibitory activity increased with advancing gestation. Uterine fluid also inhibited lymphocyte proliferation caused by other mitogens or by mixed lymphocyte reactions. Inhibitory activity was found in both salt volume (Mr less than 1000) and void volume (Mr greater than 5000) fractions of uterine fluid resolved by Sephadex G-25 desalting columns. Only activity in the void volume was sensitive to pronase. Several fractions containing inhibitory activity were resolved when dialyzed uterine fluid was fractionated into acidic and basic components by cation-exchange chromatography and further resolved by gel filtration using Sepharose CL-6B. The most active fractions (inhibition/micrograms protein) for both acidic and basic components eluted at the void volume of Sepharose CL-6B (Mr greater than 4 x 10(6). The inhibitory factor in the basic component that eluted at the void volume of Sepharose CL-6B was rich in carbohydrate, slightly cytotoxic, and partially sensitive to digestion with trypsin or oxidation with periodate. In conclusion, uterine fluid of unilaterally pregnant ewes is enriched in molecules that inhibit lymphocyte proliferation in vitro. Among these are low molecular weight, non proteinaceous factors and a very high molecular weight (Mr greater than 4 x 10(6) fraction that contains protein and carbohydrate.     

Characteristics and behavior during partial purification of estrogen sulfotransferase of guinea pig liver and chorion

Some characteristics of estrogen sulfotransferases from guinea pig liver and chorion were compared. Liver cytosolic activity was stimulated 10-fold by 25 mM monothiolglycerol and 2-fold by 15 mM MgCl2 or CaCl2, similar to that found previously for chorion. Liver and chorion activities were each eluted as a single peak from fast protein liquid chromatography (FPLC) gel filtration columns at apparent molecular weights of 52,300 and 50,000, respectively. Each was eluted during FPLC anion exchange under single, wide peaks with low recoveries. Liver sulfotransferase activity was eluted from Affi-gel Blue columns in the form of several peaks whereas the chorion activity behaved as a single species. The enzymes from both tissues, when partially purified by gel filtration followed by anion exchange, acted upon estrone and estradiol at the 3-position but activity toward dehydroepiandrosterone and testosterone was minimal or undetectable. Affi-gel Blue chromatography followed by FPLC gel filtration resulted in increases in specific activity of 26- and 90-fold for liver and chorion, respectively. Both enzymes were eluted from agarose-hexane-adenosine 3',5'-diphosphate (PAP-agarose) columns as single peaks. Average increases in specific activity for this column step were 40-fold and 96-fold for the entire eluted peaks of liver and chorion enzyme, respectively. Individual fractions from the PAP-agarose column indicated a specific activity increase of as much as 60-fold for liver and 208-fold for chorion. These latter were markedly unstable and it was not possible to obtain further purification by additional steps. Velocity versus substrate concentration curves for the partially purified enzymes showed complex kinetics, particularly with estradiol as substrate.     

Isolation of lipoxygenase isoforms from Glycine max embryo axes based on apparent cross-reactivity with anti-myosin antibodies

Three lipoxygenase isoforms were isolated from Glycine max embryo axes. A number of proteins around 97 kDa cross-reacted with several anti-actin and anti-myosin antibodies and these were used to follow their purification through gel filtration, hydroxyapatite and anion exchange columns. The 97-kDa cross-reactive material eluted in the unbound fractions of the last anion exchange column, and displayed two components of pI's 6.2 and 6.3. Further phase partition of this fraction in TX-114 yielded a hydrophobic 97 kDa protein. Additionally, a 95-kDa protein was retained and eluted from this last column. Partial peptide sequences indicated that the 95 kDa protein was soybean lipoxygenase-1, the first 97 kDa protein was lypoxygenase-3, and the hydrophobic 97 kDa protein was lipoxygenase-2. Several possible reasons for the cross-reactivity with the antibodies are discussed. To our knowledge, this is the first example of individual lipoxygenase isoforms isolated from soybean embryo axes.     

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.     

Electrophoretic elution of macromolecules from polyacrylamide or agarose gels

A method for elution of micrograms of macromolecules from polyacrylamide and agarose gels is described. The recoveries were greater than 90% with three different macromolecules tested (28 to 360 kDa). An amount as small as 1 microgram of human serum albumin was eluted from polyacrylamide gel with 90% recovery. The eluted material is collected into a small chamber the size of which can be changed as required. Elution and concentration are achieved simultaneously and in one step under mild conditions. Sterile eluates can be obtained, if the apparatus is constructed under sterile conditions.     

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.     

Restoration by phospholipids of dolichol pyrophosphate N-acetylglucosamine synthesis in delipidated rat lung microsomes

The effects of phospholipids on the reaction catalyzed by UDP-GlcNAc:dolichol phosphate GlcNAc-1-phosphate transferase have been studied with delipidated rat lung microsomes. Deoxycholate-solubilized enzyme was depleted of measurable phospholipid by either gel filtration on Sephadex G-100 or affinity chromatography on pentyl-agarose. The latter procedure also removed nucleotide and sugar nucleotide hydrolases. Delipidated protein fractions were devoid of GlcNAc-1-phosphate transferase activity unless supplemented with phospholipids. Maximal recovery of enzyme activity was obtained with an approximate 1:1 weight ratio of phosphatidylglycerol:phosphatidylcholine, with the observed rate being synergistic as compared to rates observed for each individual phospholipid. Variable recoveries of enzyme activity were obtained with mixtures containing other acidic phospholipids and phosphatidylcholine. Enzyme activity in the fraction eluted from pentyl-agarose could be recovered, after removal of Triton X-100, with sedimented phospholipid vesicles. Significant stabilization of enzyme activity associated with the phospholipid vesicles was obtained by the inclusion of dolichol phosphate.     

Transformation in pneumococcus: protein content of eclipse complex.

A two-step purification of pneumococcal eclipse complex is described, which uses sucrose gradient sedimentation followed by agarose gel permeation chromatography. Purified complex contains, in addition to donor DNA single strands, macromolecular material that can be labeled with methionine or leucine during development of competence. This material co-chromatographed with eclipse complex DNA on hydroxylapatite, was dissociated from the DNA by sodium dodecyl sulfate, and was completely digested by Pronase. The sodium dodecyl sulfate-released material eluted as a single peak in sodium dodecyl sulfate chromatography. These properties were consistent with the noncovalent association with eclipse complex of a protein or class of proteins with a narrow range of polypeptide sizes. Evidence for the specific association of this protein with transforming DNA is eclipse was also obtained from parallel purification from 35S-labeled nontransformed cells; the amount of methionine label in the corresponding fractions in such cells was only 5% of that in transformed cells.     

Affinity purification and chemical analysis of the interleukin-1 receptor

Interleukins-1 alpha and -1 beta regulate the metabolism of cells through a common plasma membrane receptor protein. In this study, it is demonstrated that the interleukin-1 (IL-1) receptor from detergent solutions of EL-4 cells can be stably adsorbed to nitrocellulose with full retention of IL-1 binding activity. This assay system was used to monitor the purification of the IL-1 receptor and to investigate the effects of several chemical modifications on receptor binding activity. IL-1 receptors extracted from EL-4 6.1 C10 cells can be bound to and specifically eluted from IL-1 alpha coupled to Sepharose. The affinity chromatography method resulted in the identification by polyacrylamide gel electrophoresis and silver staining of a protein of Mr 82,000 that was present in fractions exhibiting IL-1 binding activity. Experiments in which the cell-surface proteins of EL-4 cells were radiolabeled and 125I-labeled receptor was purified by affinity chromatography suggested that the Mr 82,000 protein was expressed on the plasma membrane. N-Glycanase treatment of this material showed that 23-35% of the total Mr (82,000) of the receptor is N-linked carbohydrate.     

以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白

发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。     

Purification and biologic properties of fibroblast somatomedin

Cultured human fibroblasts produce a peptide growth factor that cross-reacts with antisera to human somatomedin-C (Sm-C). To determine the identity of this species and compare its molecular properties to pure Sm-C, 2 liters of conditioned medium derived from human fibroblast monolayers were concentrated (X10) by ultrafiltration. The concentrated conditioned medium was purified further by CM-Sephadex ion-exchange chromatography. Following elution in 1.0 M NaCl, pH 8.0, the active material was purified by gel filtration on Sephadex G-150. The active fractions which eluted at Kd 0.45 (Mr estimated at 32,000) were further purified by isoelectric focusing. Two peaks of activity electrofocused at pI 5.4 and 7.2, respectively. The pI 5.4 peak contained only binding protein activity. The active fractions from the neutral pool were further purified by reverse-phase high pressure liquid chromatography on a C-18 Bondapak with a linear gradient of acetonitrile (10-60%). The active single peak which eluted at 55% acetonitrile gave a single band when analyzed by polyacrylamide gel electrophoresis. This material stimulated [3H]thymidine incorporation into human fibroblast DNA with approximately 3.2 times the potency of pure Sm-C but was equipotent in stimulating BALB/c 3T3 fibroblasts. It was degraded by fibroblast cultures at a slower rate compared to Sm-C, although it had a similar affinity for Sm-C-binding protein. We conclude that human fibroblasts produce two peptides that react with anti-Sm-C antibody but are chemically distinct from Sm-C. The greater response to fibroblast somatomedin may be due to its affinity for somatomedin-binding protein and slower degradation. These findings may have implications for understanding the regulation of human fibroblast replication.     

A Simple Device for Assuring High Reproducibility in Fluorometric Measurement of Deoxyribonucleic Acid in Yeast Cells

The heterogeneity and chemical composition were investigated in κ-casein from colostrum. The acid casein was obtained from four different Holstein cow colostra. The yield of acid casein from colostrum was higher than that from normal milk. κ-Casein from colostrum was prepared by the gel filtration method of Yaguchi et al. The gel filtration profiles differed among the four colostrum acid caseins.

Colostrum κ-casein was fractionated on a DEAE-cellulose column into one nonadsorbed and six adsorbed fractions with increasing salt concentration. Six adsorbed fractions had the same molecular weight and stabilizing ability for α_s1-casein in the presence of calcium ion. The amino acid composition and the phosphorus content of the adsorbed fractions were identical, but fractions eluted with high salt concentrations had more carbohydrates (galactose, sialic acid, glucosamine, galactosamine). Colostrum κ-casein was characterized by a higher content of carbohydrate moiety in comparison with normal κ-casein. Also glucosamine which has not been found in normal κ-casein was detected in colostrum κ-casein. The κ-casein component from colostrum contained at least one molecule of carbohydrate, though the carbo hydrate-free component was detected in normal κ-casein.     

Aspergillus fumigatus catalases: cloning of an Aspergillus nidulans catalase B homologue and evidence for at least three catalases

The presence of catalases in the water soluble fractions of three Aspergillus fumigatus strains was investigated using non-denaturing and denaturing polyacrylamide gel electrophoresis and Western analysis. Using non-denaturing polyacrylamide gel electrophoresis and staining for catalase activity, three separate catalases were identified. An A. fumigatus catalase gene (catB) was cloned from genomic DNA using the Aspergillus niger catR gene as a probe. Polyclonal antibodies were raised to a glutathione S-transferase-CatB fusion product expressed in Escherichia coli. Western analysis indicated that, under denaturing conditions, the polyclonal antibody recognised a 90-kDa band and under non-denaturing conditions, two separate bands were identified. These results indicate that A. fumigatus in addition to CatB, produces at least two other catalases, one of which is similar in size to CatB. The polyclonal antibody was also used to observe catalase expression in mice, experimentally infected with A. fumigatus. Staining was observed heterogeneously throughout the fungal hyphae. This result indicates that catalase is produced by A. fumigatus during invasive aspergillosis.     

Antigenic characterization of some potentially pathogenic mucoraceous fungi

The antigenic profiles of 10 mucoraceous fungi--Absidia corymbifera, Mortierella wolfii, Mucor miehei, M. pusillus, M. racemosus, Rhizopus arrhizus, R. microsporus, R. oryzae, R. rhizopodiformis, R. stolonifer,--Candida albicans and Aspergillus fumigatus were compared by crossed immunoelectrophoresis (XIE). Antigen-rich material was obtained from homogenized hyphae (or yeasts in the case of C. albicans), and antisera by multiple subcutaneous innoculation of rabbits with macerated but viable hyphal fragments of Ab. corymbifera, M. pusillus, R. oryzae or Asp. fumigatus. Unique and common antigens were demonstrable amongst the mucoraceous species although Mort. wolfii revealed little antigenic similarity with the others. Considerable sharing of antigens between Ab. corymbifera and M. pusillus was evident. Little or no cross reactivity was seen between extracts of C. albicans and Asp. fumigatus and the mucoraeceous antisera. R. oryzae and R. arrhizus, now regarded as synonymous, revealed close antigenic similarity. On the other hand, the distinction between both M. pusillus and M. miehei--which are regarded by some as belonging to a separate genus Rhizomucor--and less thermotolerant M. racemosus was reflected in their antigenic dissimilarity. Partial separation and characterization of antigens from the crude Absidia extract was achieved by concanavalin A-Sepharose chromatography. Antigens with and without affinity for concanavalin A could be demonstrated. Cross reactivity between Absidia antigens and M. pusillus antiserum appeared to be contained predominantly in material (possibly carbohydrate) which bound to concanavalin A and could be eluted with alpha-methyl-D-mannoside.     

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.