Sephacryl S-200 Gel Chromatography Research Articles

Purification, physicochemical and thermodynamic studies of antifungal chitinase with production of bioactive chitosan-oligosaccharide from newly isolated Aspergillus griseoaurantiacus KX010988

In our search for chitinase and chitosanase producer from unconventional sources, the marine-derived fungus Aspergillus griseoaurantiacus KX010988 was obviously the best producer of the highest chitinase and chitosanase activities by solid state fermentation of potato shells. Chitinase was purified in three steps involving ammonium sulphate precipitation, DEAE-cellulose ion-exchange chromatography and Sephacryl S-300 gel chromatography. 12.55 fold increase in purity with a recovery of 17.6 was obtained. The molecular mass of the purified chitinase was found to be 130kDa. It was optimally active at pH 4.5 and 40°C. Km and Vmax values were 0.22mgmL−1 and 19.6μmolemin−1mg−1 respectively. Mn2+ and Zn2+ ions lead to increased chitinase activity. While Fe2+and Cu2+ions strongly inhibited the chitinase activity. The thermodynamics of pure chitinase including activation energy for thermal denaturation (Ea,d), change of free energy (ΔGd), enthalpy(ΔHd), entropy(ΔSd) and half life values (T1/2) at 40, 50 and 60°C were determined. Chitinase showed antifungal activity against pathogenic fungus Fusarium solani. Chitosanase was partially purified by acetone precipitation (50–75%) v/v concentration. The hydrolytic products of moderate molecular weight of chitosan by chitosanase were analyzed by thin layer chromatography (TLC) after 12 and 24h respectively. Chitosan-oligosaccharides showed good antibacterial and antioxidant activities.

Identification of Horse Anti-Ferritin Autoantibody

Ferritin-binding proteins (FBPs) in horse serum were characterized by immunoblotting and ferritin-binding experiments. FBPs purified from horse serum by horse spleen ferritin-Sepharose 4B affinity chromatography were separated into two fractions by Sephacryl S-300 gel chromatography: FBP 1 and FBP 2. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, FBP 1 separated into 79.0- and 25.3-kDa bands, and FBP 2 separated into 62.7- and 25.3-kDa bands. Immunoblotting analysis using antibodies specific for horse IgM and IgA heavy chains and IgG F(c) fragment showed that the 79.0- and 62.7-kDa bands were IgM and IgG heavy chains, respectively. After forming complexes of horse FBPs with horse spleen ferritin, rabbit anti-horse ferritin antibody was used to form immune complexes against ferritin, allowing co-precipitation and subsequent identification by monoclonal antibodies to horse immunoglobulin (IgM, IgA, IgGa and IgGb). Horse serum was positive for IgM, IgGa, IgGb and IgA complexes and affinity-purified FBPs for IgM and IgGb complexes. FBP 1 was identified as an IgM complex that forms with ferritin but FBP 2 did not form any complexes with ferritin. These results suggest that circulating horse serum ferritin is in the form of IgM, IgG (IgGa and IgGb) and IgA complexes.

New endo-beta-1,4-glucanases from the parabasalian symbionts, Pseudotrichonympha grassii and Holomastigotoides mirabile of Coptotermes termites.

Abstract. An endo-beta-1,4-glucanase (EG) was purified from the hindgut of an Australian mound-building termite, Coptotermes lacteus. The hindgut extract had a peak separate from those for extracts obtained from the salivary glands and the midgut based on sephacryl S-200 gel chromatography, and also demonstrated an origin different from the endogenous EGs of the termite itself. The recovery was further purified by SDS-PAGE, and its N-terminal amino acid sequence analyzed. This showed high homology to EGs from glycoside hydrolase family (GHF) 7. PCR-based cloning methods were applied to the hindgut contents of C. lacteus and individual protozoan symbionts from C formosanus. cDNAs encoding putative EGs homologous to GHF7 members were then identified. The functionality of one of the putative proteins was confirmed by its expression in Escherichia coli.

Isolation and characterization of pepsinogen from Trimeresurus flavoviridis (Habu snake).

Pepsinogen was isolated from the gastric mucosa of Trimeresurus flavoviridis (Habu snake) by DEAE-cellulose and DEAE-Sepharose ion-exchange chromatographies, and Sephacryl S-200 gel-chromatography. The yield calculated from the crude extract was 29% with 6.2-fold purification. The purified pepsinogen gave a single band on both native- and SDS-PAGE. As no other active enzyme was detected on the chromatographies, it was concluded that the Habu snake has one major pepsinogen. The molecular mass of the pepsinogen was estimated to be 38 kDa by SDS-PAGE. The sequence of the N-terminal 26 amino acid residues was determined and compared with those of other pepsinogens. The N-terminal structure of Habu snake pepsinogen was more homologous with those of mammalian pepsinogens C than those of mammalian pepsinogens A. The pepsinogen was rapidly converted to pepsin by way of an intermediate form induced by acidification. The optimum pH of Habu snake pepsin for bovine hemoglobin was 1.5-2.0, and it retained full activity at pH 6.2 and 30 degrees C on incubation for 30 min. The optimum temperature for the snake pepsin was 50 degrees C and it was stable at 40 degrees C on incubation for 10 min. The proteolytic activity of the pepsin toward bovine hemoglobin was about two times higher than that of porcine pepsin A, however, the activity toward oxidized bovine insulin B-chain was lower than that of porcine pepsin A, and it did not hydrolyze oligopeptides. The specificity for oxidized bovine insulin B-chain of the pepsin was different from that of porcine pepsin A. Habu snake pepsin was inhibited by pepstatin A but not by serine, cysteine, or metallo protease inhibitors.

Isolation and characterization of a neutrophil chemotactic factor from Tritrichomonas foetus organisms.

A neutrophil chemotactic factor (TfNCF) was isolated from the crude extract of Tritrichomonas foetus organisms by a combination of anion-exchange chromatography on DE52 and gel filtration on Sephacryl S200. TfNCF showed homogenicity by both PAGE and SDS-PAGE. The molecular weight of TfNCF was estimated to be 22 and 24 kDa, by Sephacryl S200 gel chromatography and by SDS-PAGE under reducing conditions. Immunization of TfNCF caused almost complete protection against T. foetus infection in mice. Western blot analysis probed with anti-TfNCF antibody showed that the epitopes on TfNCF were not commonly shared on the other components of T. foetus organisms nor other helminthous parasite-derived components. Furthermore, pre-incubation of neutrophils with antigens of other helminthous parasites or N-formylmethionyl-leucyl-phenylalanine did not affect the neutrophil chemotactic activity for TfNCF. These results suggest that TfNCF is a novel NCF consisting of unique epitopes for both antigenicity and neutrophil chemotactic activity. The role of NCF in the initiation of the immune response is discussed.

Urokinase-type plasminogen activator in human eccrine sweat.

The presence of urokinase-type plasminogen activator (uPA) in human eccrine sweat has not been reported previously. Clean sweat was obtained from the upper trunk and arms of subjects which had been painted with white petrolatum to minimize epidermal contamination. Sweat was concentrated x 50 by ultrafiltration, and its PA activity determined by the two-step assay method (conversion of plasminogen to plasmin with the subsequent assay of plasmin activity using the substrate S-2251). PA activity was detectable in nine of 17 subjects by this method, which probably represents an underestimate of the true activity because of possible loss of the enzyme during concentration. Scraped (crude) sweat samples contained less PA activity. Sephacryl S-200 gel chromatography of the PA-positive pooled sweat showed a major peak of PA activity at M(r) 55,000. Gelatin-polyacrylamide enzymography revealed a major PA band at M(r) 55,000 and a minor band at 33,000. Sweat PA activity was 94% inhibited by epidermal PA inhibitor and anti-uPA IgG, but not by anti-tPA IgG. We conclude that the PA activity in sweat is derived from the sweat gland and is most likely of the urokinase type. The physiological significance of sweat uPA remains to be determined.

Isolation and characterization of a novel hen egg white lysozyme inhibitor from Bacillus subtilis I-139.

A novel lysozyme inhibitor, hen egg white lysozyme inhibitor (Hewli) was isolated from the culture broth of a bacterium (strain I-139) identified as Bacillus subtilis. Maximum lysozyme inhibitory activity was obtained when the bacterium was grown aerobically in a medium consisting of 0.65 % glucose, 0.5 % sodium glutamate, 1% meat extract, and 4% Polypepton (pH 7.0), at 30°C after 30 to 35hr. Hewli was purified 69-fold from the culture supernatant of B. subtilis I-139 by ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, Sephacryl S-200 gel chromatography, DEAE-Toyopearl 650M column chromatography, affinity chromatography on a column of immobilized egg white lysozyme, and acetone extraction. The purified lysozyme inhibitor has an amino acid composition of aspartic acid, glutamic acid, valine, and leucine in a 1:1:1:4 molar ratio and fatty acid composition of palmitic acid and/or vaccenic acid. Hewli inhibited B. subtilis I-139 gh canase and animal lysozymes such as the hen, turkey, ...

Production of an interleukin-1 inhibitor by cell line P388D1 murine macrophages stimulated with Haemophilus actinomycetemcomitans lipopolysaccharide

Murine macrophages of the P388D1 cell line stimulated with lipopolysaccharide (LPS) from Haemophilus actinomycetemcomitans Y4 released an interleukin-1 (IL-1) inhibitor, as well as IL-1. Maximal IL-1 activity in culture supernatants was detected after 24 h of culture. On the other hand, IL-1 inhibitor activity reached a maximum level after 72 h of culture. An IL-1 inhibitor was partially purified from the culture supernatant of P388D1 cells stimulated with Y4 LPS for 72 h by ammonium sulfate precipitation, followed by Sephacryl S-200 gel chromatography. A 160-kilodalton peak inhibitory to IL-1 and a 14-kilodalton peak showing IL-1 activity were separated by Sephacryl S-200 column chromatography. The partially purified IL-1 inhibitor significantly suppressed the proliferation of C3H/HeJ murine thymocytes that had been induced with murine and human IL-1 in the presence of a submitogenic dose of concanavalin A. The IL-1 inhibitor more strongly suppressed human recombinant IL-1 beta than human recombinant IL-1 alpha. This inhibitory activity of the partially purified preparation was unaffected by the presence of trypsin inhibitor and the protease inhibitor aprotinin. The IL-1 inhibitor did not exhibit either IL-2 or IL-2 inhibitor activity. The inhibitor suppressed C3H/HeJ thymocyte proliferation induced by IL-1 in the presence of a saturated concentration of IL-2 instead of a suboptimal concentration of concanavalin A. These results indicate that prolonged culture of Y4 LPS-stimulated murine macrophages releases a specific inhibitor of IL-1.

Production of platelet-derived growth factorlike protein by rat mesangial cells in culture.

Rat mesangial cells (MC) release a factor that competes in a dose-dependent manner with 125I-labeled platelet-derived growth factor (PDGF) for binding to human foreskin fibroblasts (HFF). The competitor activity in mesangial cell conditioned medium (MCCM) is reversible, trypsin sensitive, and inhibited by anti-PDGF IgG. MCCM also expresses potent mitogenic activity to HFF. Anti-PDGF IgG, in concentrations that completely abolished the mitogenic activity of pure PDGF and the competitor activity of MCCM, only partially (33-41%) inhibits this mitogenic activity. The PDGF receptor competing activity as well as the total mitogenic activity, coelutes with labeled pure PDGF on Sephacryl S-200 gel chromatography. Cation exchange chromatography of concentrated MCCM yields a major mitogen peak with little competitor activity and a smaller mitogenic peak with comparable competitor activity, suggestive of the presence of other mitogens in MCCM besides the PDGF-like protein. PDGF is a potent mitogen and may play a role at inflammatory sites. The production of PDGF-like protein by MC may provide insights for understanding the pathogenesis of glomerular diseases.

乾せん表皮のカリクレイン様酵素

Kinin-liberating activities were investigated in scales from the inflammatory skin diseases, psoriasis vulgaris and psoriasis pustulosa, using competitive enzme immunoassay for kinins. Tris-buffered saline extracts of both psoriatic scales liberated the same level of kinins from heated plasma (approximately 11ng/mg protein). In psoriasis vulgaris, three peaks of kinin-liberating activities were shown at Mr 90, 000 (VK-I), 65, 000 (VK-II) and 35, 000 (VK-III) by Sephacryl S-200 gel chromatography. They were further separated by DEAE Sepharose chromatography at pH 7.5. Kinin-liberating activity of VK-I was eluted in nonadsorbed fraction. However, this fraction liberated kinins only from the heated plasma but not from the purified low molecular weight kininogen (LMW-Kng), indicating VK-I is a plasma kallikrein-like enzyme. Kinin-liberating activity of VK-II was separated into two peaks. VK-IIa in nonadsorbed fraction liberated kinins only from the heated plasma, whereas VK-IIb in adsorbed fraction liberated kinins from both heated plasma and LMW-Kng, showing VK-IIa is plasma kallikrein-like and VK-IIb is glandular kallikrein-like. Kinin-liberating activity of VK-III eluted at the same position to VK-IIb, and was found to be glandular kallikrein-like, as well. In psoriasis pustulosa, majority of kinin-liberating activity peaked at Mr 90, 000 by Sephacryl S-200 gel chromatography (PK-I). PK-I demonstrated essentially the same elution profile as VK-I by the following DEAE Sepharose chromatography. VK-I, VK-IIa and PK-I were inactivated by soybean trypsin inhibitor, but only partially by aprotinin, confirming plasma kallikrein-like characteristics. VK-IIb and VK-III were inhibited by aprotintn but not by soybean trypsin inhibitor, confirming glandular kallikrein-like characteristics.

Purification and some properties of acid-stable .ALPHA.-amylases from shochu koji (Aspergillus kawachii).

Aspergillus kawachii α-amylase [EC 3.2.1.1] 1 and II were purified from shochu koji extract by DEAE Bio-Gel A ion exchange chromatography, Sephacryl S-300 gel chromatography (pH 3.6), ω-amino dodecyl agarose column chromatography and Sephacryl S-200 gel chromatography. By gel chromatography on a Sephacryl S-300 column, the molecular weights of the purified α-amylase I and II were estimated to be 104, 000 and 66, 000, respectively. The isoelectric points of α-amylase I and II were 4.25 and 4.20, respectively. The optimal pH range of α-amylase I was 4.0 to 5.0, and the optimum pH of α-amylase 'II was 5.0. The optimum temperatures of both α-amylases were around 70°C at pH 5.0. Both α-amylases were stable from pH 2.5 to 6.0 and up to 55°C, retaining more than 90% of the original activities. Heavy metal ions such as Hg2+ and Pb2+ were potent inhibitors for both α-amylases.

Antithrombin antigen of high molecular weight associated with neoantigen in hemophilic plasma after factor IX concentrate therapy

These studies were performed to investigate the cause(s) of the cathodal shift of mobility seen in crossed immunoelectrophoresis of antithrombin antigen in plasma of hemophilic patients after factor IX concentrate therapy. These plasmas were shown to contain antithrombin neoantigen with apparent identity to the neoantigen present in normal serum but not present in normal plasma. Sephacryl S-200 gel chromatography of serum demonstrated that the neoantigen eluted with the first two, early eluting protein peaks; thus the neoantigen had a higher molecular weight than native antithrombin. When the chromatographic fractions containing the neoantigen were studied by crossed immunoelectrophoresis, they were found to contain antithrombin antigen of more cathodal mobility than normal. Sephacryl S-200 chromatography of factor IX concentrate-treated hemophilic plasma also showed an early eluting peak of antithrombin antigen of more cathodal mobility than normal in crossed immunoelectrophoresis. The mobility of this peak was identical to the cathodal peak found in normal serum and in early eluting fractions from chromatography of normal serum. These results support the conclusion that factor IX concentrate-treated hemophilic plasma contained a non-functional, high molecular weight form of antithrombin, associated with the presence of neoantigen, which may represent complexed and/or modified antithrombin produced by the action of the concentrates in vivo.

Characterization of Raji cell binding IgG in patients with metastatic breast cancer.

The aim of the present study was to isolate and characterize the immune complexes detected by the Raji cell assay in metastatic breast cancer. However, the Raji cell binding material could not be separated from monomeric IgG by Sephacryl S-300 gel chromatography in any of the 16 sera investigated. The parallel elution profile of monomeric IgG and the Raji cell binding activity suggested that anti-Raji cell antibodies, rather than immune complexes of low molecular weight, were present in these sera. This was further substantiated by pepsin digestion of the Raji cell binding IgG fractions. The binding of F(ab')2 fragments was quantitatively comparable to the binding of undigested IgG, and the bound F(ab')2 fragments could be visualized by immunofluorescence at the cell membrane. The presence of antibodies against Raji cells was further confirmed by the complement-dependent cytotoxicity assay. These antibodies did not react with untransformed lymphocytes and there was no correlation with anti-Epstein-Barr virus antibodies. The incidence of cytotoxic anti-Raji cell antibodies in breast cancer was 12% compared to 20% in malignant lymphoma, to 0% in normals and patients with degenerative cardiovascular diseases, and to 5% in patients with auto-immune diseases.

Elastase activity in granulomatous inflammation in experimental murine leprosy

Proteolytic activity for [ 3H]elastin, pyro-Glu-Pro-Val-pNA(S-2484), and Suc-(Ala) 3-pNA(AAApNA) was demonstrated in the bound fraction extracted with 2 M KSCN + 0.1% Triton X-100 from hypersensitivity-type murine lepromas in C57BL/6N mice, while elastase-inhibitor activity was separately observed in the soluble fraction extracted with a Tris-saline buffer. Sephacryl S-200 gel chromatography showed a peak of elastolytic activity with approximately 20,000 in molecular weight. The following DEAE-Sepharose chromatography demonstrated three fractions of elastolytic activity (E-I, II, III). The inhibitory profile showed that E-I is a thiol proteinase, while E-II and E-III belong to serine proteinase-type elastases. Both E-II and E-III showed different properties with neutrophil elastase or elastase secreted from cultured macrophages, but identical characteristics to membrane bound-type elastase of monocytes. A lower level of elastolytic activity was detected in the bound fraction of nonhypersensitivity-type murine lepromas in CBA/N mice, suggesting a more involvement of membrane bound-type elastase from monocytes/macrophages during the tissue remodelings of hypersensitivity-type granulomas.

Suppression of human lymphocyte mitogen response by proteins of the type-D retrovirus PMFV.

A type-D retrovirus, derived from a human cell line and designated PMFV, which is related to, but distinct from the Mason-Pfizer monkey virus (MPMV) suppressed the in vitro mitogen response of human lymphocytes. PMFV was suppressive either as intact virus or after disruption by ether or detergents. In kinetic studies, the time course of the interaction between suppressive virus components and lymphocytes was characterized. Neither cytotoxicity nor time shifts in optimum 3H-thymidine incorporation of responding lymphocytes were observed. The suppressive activity of the disrupted virus was diminished by heating, freeze-thawing or treatment with pronase or trypsin, indicating temperature-sensitive proteins as suppressive components. By Sephacryl S-200 gel chromatography, molecular weights of approximately 70 and 15 kDa were determined for the suppressive components which were isolated from disrupted virus or virus-producing cells but not from non-infected cells.

乾せん表皮のＰｌａｓｍｉｎｏｇｅｎ Ａｃｔｉｖａｔｏｒ

Two molar potassium thiocyanate exract of psoriatic scales demonstrated fibrinolytic activity only in the pesence of plasminogen (0.54+0.51IU/mg protein). The extract also showed high level of activity on Ile-Pro-Arg-p-nitroanilide (pNA) (3.98±1.43nmol/min/mg protein). Sephacryl S-200 gel chromatography revealed that plasminogen activator (PA) activity positioned at approximately MW 60K. This PA activity was totally adsorbed to benzamidine-Sepharose column and dissociated with 2M KSCN. Enzymographic analysis demonstrated 62K, 43K and 33K PA in the adsorbed fraction. By the following high performance anion-exchange chromatography on Mono Q, three peaks of PA activities were separated. Among them, the major peak of PA activity was 62K. Futhermore, anti-urokinase (UK) IgG-Sepharose affinity chromatography of S-200 PA fraction showed that only 62K PA is adsorbed to this column and represents 70% of the total psoriatic PA activity. In order to demonstrate the localization of UK-type PA antigen in psoriatic epidermis, we performed an immunofluorescence study. UK-type PA was mainly found in upper layer of psoriatic epidermis, although diffuse fluorescence was seen in whole psoriatic epidermis.

Granulocyte-macrophage colony-stimulating factor in the sera of Schistosoma japonicum-infected mice

Infection of mice with Schistosoma japonicum engendered high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in serum. The rise in GM-CSF levels in serum was closely associated with the acute phase of the infection and seemed to be dependent on the dose of infection. GM-CSF activity was detected as a sharp single peak in DE-52 anion-exchange chromatography and Sephacryl S-200 and Sephadex G-200 gel chromatography and was almost entirely adsorbed to concanavalin A-Sepharose 4B affinity chromatography. The possible immunological and immunopathological importance of GM-CSF in S. japonicum infection is discussed.

Characterization of nontransformed and transformed androgen receptor from rat submandibular gland

Rat submandibular gland cytosol contained androgen receptor which had a single class of specific binding and an apparent dissociation constant of (1.1–1.2) · 10 −9 M. The process of transformation was investigated by a slightly modified minicolumn method in which the transformed receptor complexes were separated from the nontransformed receptor and meroreceptor. 10 mM ATP or pyrophosphate at 0°C induced transformation of androgen receptor as did heat or salt treatment. 20 mM of sodium molybdate completely inhibited transformation that resulted from ATP, heat or salt treatment. The nontransformed androgen receptor complexes sedimented at 8 S and eluted at 250–260 mM KCl from DEAE-Sephacel, and its molecular weight was found to be 220 000 on Sephacryl S300 gel chromatography. On the other hand, the transformed androgen receptor complexes sedimented at 4.1–4.3 S (ATP or KCl treatment) or 3.5–3.8 S (heat treatment) and eluted at 60–80 mM KCl from DEAE-Sephacel. The molecular weight of the transformed androgen receptor complexes was 80 000–85 000 (ATP or KCL treatment) or 70 000–80 000 (heat treatment). These results suggest that the transformation of androgen-receptor complexes from rat submandibular gland was induced by subunit dissociation and that salt bridges may be involved in the subunit interaction.

Interleukin 1 increases collagen type IV production by murine mammary epithelial cells.

The effects of human interleukin 1 (IL 1) on collagen type IV production by normal mouse mammary epithelial cells were examined. Human IL 1 was derived from the culture media of peripheral blood monocytes or placental cells that were stimulated with silica. Although crude culture media of silica-stimulated monocytes or placental cells had no enhancing activity for type IV collagen production, IL 1-containing fractions obtained by Sephacryl S-200 gel chromatography and isoelectrofocusing from such media possessed considerable activity. To confirm the effects of IL 1 on collagen production, human monocyte-derived IL 1 was highly purified by sequential isoelectrofocusing, anion-exchange (AX 300), high-performance liquid chromatography (HPLC), and HPLC gel filtration (TSK 3000). The same HPLC gel filtration fractions contained both an activity that stimulated collagen synthesis by mammary cells and thymocyte growth-promoting activity. These activities of IL 1 differed from a number of other factors, such as epidermal growth factor and another factor produced by placental cells that stimulated type IV collagen production but not thymocyte proliferation. In fact, IL 1 induced 100-fold less collagen type IV production by mammary epithelial cells than was needed to induce thymocyte proliferation. Our data suggest that IL 1-like molecules, which reportedly are produced by many tissue cell types, may therefore play a role in promoting a basement membrane formation at stromal-epithelial boundaries.

Isolation and reconstitution of the iron-sulfur protein in ubiquinol-cytochrome c oxidoreductase complex. Phospholipids are essential for the integration of the iron-sulfur protein in the complex.

An iron-sulfur protein has been purified from beef heart ubiquinol-cytochrome c oxidoreductase (Complex III) of the mitochondrial respiratory chain by phenyl-Sepharose column chromatography and Sephacryl S-200 gel chromatography. Depletion of most of the endogenous phospholipids in the complex was a prerequisite to the dissociation of the protein from the complex in the former chromatography. The iron-sulfur protein was nearly homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 76 ng atoms of nonheme iron and 66 nmol of acid-labile sulfide/mg of protein. When this preparation was incubated with an iron-sulfur protein-depleted complex in the presence of soybean phospholipids, the enzymic activity was restored up to 90% of that of the parent Complex III, whereas the recovery of the activity was marginal in the absence of the phospholipids. Thus it is clear that the iron-sulfur protein is integrated into the complex with the aid of phospholipids.