Sepharose 6B Gel Filtration Research Articles

Purification and Optimization of Extracellular Lipase from a Novel Strain Kocuria flava Y4.

The exogenous lipolytic activities of Kocuria sp. have been recognized earlier but the genus further contains many more unexplored strains. In this study, the extracellular lipase activity of Kocuria flava Y4 (GenBank accession no. MT773277), isolated from Dioscorea villosa during our previous study, was regulated by different physicochemical parameters, such as pH, temperature, shaking speed, and incubation time. For efficient immobilization of the extracellular lipase, 4% sodium alginate, 50 mL of 25 nM CaCl2.2H2O solution, and 15 min. Hardening time of gel beads in calcium chloride was used. For the first time, K. flava Y4 lipase was purified using ammonium sulphate precipitation followed by dialysis and DEAE-Sepharose anion exchange chromatography with Sepharose-6B gel filtration chromatography, yielding ∼15-fold purified lipase with a final yield of 96 U/mL. The SDS-PAGE of purified lipase displayed a single strong band, indicating a monomeric protein of 45 kDa. At a temperature of 35°C and pH 8, the purified lipase showed maximum hydrolytic activity. Using p-nitrophenyl acetate (p-NPA) as the hydrolysis substrate, the values of Km and Vmax derived from the Lineweaver–Burk plot were 4.625 mM and 125 mol/min−1mg−1, respectively.

Pharmacological Effects of Guava (Psidium guajava L.) Seed Polysaccharides: GSF3 Inhibits PC-3 Prostate Cancer Cell Growth through Immunotherapy In Vitro.

The inhibitory effects of purified fractions isolated from guava seed polysaccharides (GSPS) including guava seed polysaccharide fraction 1 (GSF1), GSF2, and GSF3 on prostate cancer cells remain unclear. To clarify the anti-prostate cancer potential, GSPS, GSF1, GSF2, and GSF3 were isolated using Sepharose 6B gel filtration chromatography to assay their inhibitory effects on prostate PC-3 cell growth with direct action or indirect immunotherapy using either splenocyte conditioned media (SCM) or macrophage conditioned media (MCM). Correlations between cytokine profiles in the conditioned media and pro-apoptotic gene expression levels in the corresponding treated PC-3 cells were analyzed. Results showed that GSPS, GSF1, GSF2, and GSF3, particularly GSF3, through either direct action or indirect treatments using SCM or MCM, significantly (p < 0.05) inhibited PC-3 cell growth. GSF3 direct treatments increased pro-apoptotic Bax/anti-apoptotic Bcl-2 mRNA expression ratios in corresponding treated PC-3 cells. Either SCM or MCM cultured with GSF3 increased Fas mRNA expression levels in corresponding treated PC-3 cells. Both Th2-polarized and anti-inflammatory cytokine IL-10 either secreted in SCM or MCM were positively correlated with Fas mRNA expression levels in corresponding treated PC-3 cells. Our results suggest that GSF3 is a potent biological response modifier to decrease PC-3 cell growth through inducing apoptosis.

Characterization of guava (Psidium guajava Linn) seed polysaccharides with an immunomodulatory activity

To clarify the property of a novel guava seed polysaccharide (GSPS), GSPS was subjected to purify using Sepharose 6B gel filtration chromatography and further characterize the property of each individual isolated fraction. GSPS further resolved into three purified fractions, guava seed polysaccharide fraction 1 (GSF1), GSF2 and GSF3. Isolated GSF1, GSF2 and GSF3 were respectively subjected to high performance size exclusion chromatography; molecular weights of three polysaccharide fractions were determined. GSPS, GSF1, GSF2 and GSF3 were suggested to be proteopolysaccharides or glycoproteins. GSPS, GSF1, GSF2 and GSF3, particularly GSF3, were found to have a Th2-inclination property and anti-inflammatory potential. Heated GSF3 did not significantly (P > .05) decreased its immunomodulatory activity, suggesting that GSF3 is a proteopolysaccharide. The deproteinated GSF3 markedly lost its immunomodulatory activity, suggesting that both protein and carbohydrate moiety in GSF3 are essential to its immunomodulatory function. Analyses of monosaccharides composition in GSF3 using a pre-column derivatization high performance liquid chromatography exhibited that GSF3 was composed of glucuronic acid (3.28%), galacturonic acid (28.13%), galactose (14.88%), mannose (3.96%), glucose (22.99%), arabinose (7.31%), ribose (1.55%), xylose (14.81%), fucose (1.68%) and rhamnose (1.43%). Overall, we evidence that GSF3 is a low molecular weight proteopolysaccharide with potent anti-inflammatory and immunomodulatory effects.

A lectin with anti-microbial and anti proliferative activities from Lantana camara, a medicinal plant

BackgroundLectins are known to possess interesting biological properties such as anti microbial, nematicidal, anti tumor and anti viral activities. Lantana camara from verbenaceae family is a medicinal plant known for possessing anti oxidant and anticancer activities. Since anticancer activity is reported in plant lectins, leaves of Lantana camara was used to check the presence of lectin. Methods and resultsHere we report the purification, characterization and biological properties of a lectin from Lantana camara (LCL) leaves. LCL was purified by ion exchange chromatography on CM-cellulose column followed by affinity chromatography on mucin coupled Sepharose 4B column and gel filtration chromatography on Superdex G75 column. LCL is a glycoprotein with 10% of the carbohydrate and is blood group non specific. SDS-PAGE analysis of affinity purified LCL showed two proteins with apparent molecular weight of 14.49 kDa and 17.4 kDa which were subsequently separated by Gel filtration chromatography on Superdex G75 column. Hapten inhibition studies of LCL revealed its highest affinity for Chitin, Milibiose, α-D-Methyl galactopyranoside and glycoproteins like mucin, asialomucin. LCL showed strong binding to human colon adenocarcinoma HT29 cells with MFI of 242 which was effectively blocked by 68.1 and 62.5% by both mucin and milibiose. LCL showed dose and time dependent growth inhibitory effects on HT29 cells with IC50 of 3.75 μg/ml at 48 h. LCL has potent antibacterial and anti fungal activity. ConclusionLCL can be explored for its clinical potential.

The Biological Activity of Some Extracted Proteins from Bacillus spp. Isolated from Soil Against Pathogenic Bacteria

Forty soil samples were collected from different regions in Baghdad city. Among 52 isolate, only 38 isolates (73.1 %) belong to Bacillus spp. All bacterial isolates have been submitted to the cultural, microscopical, biochemical examination and VITEK 2 System, Bacillus cereus was predominantly found 21 (55.3%) followed by Bacillus subtilis 11 (28.9%), then Bacillus amyloliquefaciens was 6 (15.8%). Bacillus cereus (B1) and Bacillus subtilis (B16) isolates were with the highest level with the antibacterial activity when selected. Extracellular proteins were extracted and purified with ammonium sulfate (80%) saturation, then submitted to Sepharose-6B gel filtration column. SDS-polyacrylamide gel electrophoresis was used to determine the molecular weights of proteins, and one band appeared to each of Bacillus cereus and Bacillus subtilis with molecular weight 68 and 50 KDa respectively. The results revealed that higher effect of antibacterial activity on Gram positive bacteria, and moderate on Gram negative bacteria except P.aeruginosa with no effect. The antibiofilm effect of the extract found higher effective against S.pyogens, moderate effect against each of (S.aureus, E.coli, S. marcescens, K. spp) and no effect against P.aeruginosa. The results of cytotoxicity showed that the purified extract was a higher cytotoxic effect on breast cancer MCF7 cells at 1000µg/ml concentration, while the crude extract had a higher cytotoxic effect on breast cancer MCF7 cells at 100µg/ml concentration.

Properties of arabinogalactan-proteins in European pear (Pyrus communis L.) fruits

Arabinogalactans (AGs) and arabinogalactan-proteins (AGPs) were partially purified from an extract of fruits of the European pear (Pyrus communis L.) by DEAE-cellulose ion-exchange and Sepharose 6B gel-filtration chromatography. Among 7 AG(P)-containing fractions, a neutral AGP (SE-1) was confirmed to be highly purified (Mr 67,000) and rich in L-Ara and Gal; this fraction included a small amount (2.6%, w/w) of protein and showed the highest reactivity forming precipitate with β-Glc Yariv reagent among the 7 fractions, the intensity of which was comparable to that of gum arabic, a standard AGP. Another accompanying minor low-Mr neutral AGP (SE-2; Mr approx. 7200) still contained other polysaccharide (starch fragments) and did not show Yariv reactivity. The carbohydrate moieties of SE-1 consisted of consecutive (1 → 3)-linked β-galactosyl backbone chains substituted with side chains of (1 → 6)-linked β-galactosyl residues at O-6, to which mainly single α-l-arabinofuranosyl residues were attached through O-3. This structural feature was also observed for SE-2. Successive digestion of SE-1 with α-l-arabinofuranosidase and exo-β-(1 → 3)-galactanase with the aid of endo-β-(1 → 3)-galactanase released most (more than 98%, w/w) of the carbohydrate moieties as low-Mr fragments. These consisted of free L-Ara and Gal, and a series of β-(1 → 6)-galactooligosaccharides with degree of polymerization (dp) up to at least 17, indicative of attachment of (1 → 6)-linked β-galactosyl side chains of varying length along the (1 → 3)-linked β-galactosyl backbone chains.

Structural elucidation and antioxidant activity of an arabinogalactan from the leaves of Moringa oleifera

A novel arabinogalactan (MOP-1) was isolated from leaves of Moringa oleifera and was purified by macro-porous resin, DEAE-52 Cellulose and Sepharose 6B gel filtration chromatography. High performance gel permeation chromatography (HPGPC) analysis showed that the molecular weight of MOP-1 was 7.65 × 107 Da. Monosaccharide composition analysis indicated that sugar composition is Rha: Ara: Gal in a molar ratio of 1:7.32:12.12. Structural analysis indicated that MOP-1 has a backbone of →1)-β-d-Galp-(3,4→ with highly branched chains at O-4 position. The branches were composed of →1)-β-d-Galp-(4→, →1)-α-d-Galp-(2→, Araf-(1→, Galp-(1→. Its antioxidant activities were evaluated by DPPH radical scavenging capacity, 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) radical cation scavenging capacity, and ferric-reducing antioxidant power (FRAP). The results indicated MOP-1 exhibited significant antioxidant activities.

Characterization of extracellular amylase produced by haloalkalophilic strain Kocuria sp. HJ014

The haloalkaliphilic bacterium Kocuria sp. (HJ014) has the ability to produce extracellular amylase. The aim of this study was to purify and characterize this protein. The amylase enzyme with a specific activity of 753,502 U/mg was purified 5.7– fold using Sepharose 4B and Sephacryl S-300 gel filtration columns. The molecular weight of the enzyme was 45,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amylase showed maximum activity at pH 9 and 50°C in the presence of 3.5 M NaCl. The Km was 3.0 mg/ml and Vmax 90.09 U/ml. It was found that extracellular amylase from Kocuria sp. has a high industrial potential.

Does hydrogen bonding contribute to lipoperoxidation-dependent membrane fluidity variation? An EPR-spin labeling study

This study is aimed at making clear the relationship between oxidative stress of the phospholipid bilayer and membrane fluidity. Di-(hydroperoxylinoleoyl)-phosphatidylcholine (diHpLPC) was used as a highly hydroperoxidized and unsaturated phospholipid species in order to investigate the issue. Hydrophylic Interaction Liquid Chromatography–ElectroSpray Ionization–Mass Spectrometry (HILIC-ESI-MS) and NMR spectroscopy were employed to define the structure of the peroxidized phospholipid as 1-(9-hydroperoxy-10c,12t)octadecadienoyl-2-(9t,11c-13-hydroperoxy)octadecadienoyl-sn-glycero-3-phosphorylcholine. This phospholipid's ability to form vesicular structures was confirmed by Sepharose 4B gel filtration and Dynamic Light Scattering (DLS) of its aqueous suspensions. Fatty acid misalignment and fluidity gradient were studied in the bilayer of both supported planar bilayers (SPB) and multilamellar vesicles (MLV) made of different DLPC/diHpLPC mixtures by means of spin labelling-EPR spectroscopy of either n-DSPC or 3-doxylcholestane spin labels embedded in the membranes. It was found that diHpLPC increases both fatty acid misalignment and rigidification with increasing molar ratio in spite of increasing unsaturation of the fatty acid core. Basing on our observations, the observed ability of pure diHpLPC to form rigid and disordered SPB and MLV bilayers is proposed to be dependent on the cross bridging of oxidized linoleoyl chains by mutual hydrogen bonding of hydroperoxyl groups. However, the contribution to the observed overall rigidification of the model membranes by trans double bonds in the peroxidized chains should not be neglected, as a second membrane fluidity effector also arising from lipid peroxidation.

Bidirectional Immunomodulatory Activities of Polysaccharides Purified From Pleurotus nebrodensis

A novel Pleurotus nebrodensis polysaccharide (PN50G) was purified, characterized, and cultured with RAW264.7 macrophages to evaluate its bidirectional immunostimulating characteristics. PN50G was purified by using Sepharose 4B gel filtration; the molecular weight of PN50G was distributed at 2,000 kDa. The total protein and carbohydrate constituent ratios in PN50G were 2.6 ± 0.9 % and 92.4 ± 6.1 % (w/w), respectively, which suggests that PN50G may be a major proteo-polysaccharide component. The phagocytosis of macrophages was improved significantly, and remarkable changes were observed in the morphology of PN50G-treated cells. Compared with the control group, the productions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and inducible nitric oxide synthase (iNOS) in the macrophages as well as the messenger RNA expressions were strongly induced by PN50G. Pro-/anti-inflammatory (IL-6/IL-10, TNF-α/IL-10, NO/IL-10) cytokine secretion ratios by lipopolysaccharide-stimulated RAW264.7 macrophages were significantly decreased by PN50G treatments in a dose-dependent manner under an excessive immune experimental model. This study suggests that purified PN50G can improve immunity and suppress immune overactivity in LPS-induced macrophages in a preventive manner to coordinate innate immunity and inflammatory responses.

겨우살이 Lectin이 흰쥐의 혈청지질 및 간조직에 미치는 영향

본 연구에서 국내산 겨우살이 lectin이 흰쥐 간의 지방형성에 미치는 영향을 알아보기 위해 혈청 내 지질 함량을 측정하였고, 이를 확인하고자 간조직의 지방형성 변화를 조사하였다. 렉틴의 분리는 sephadex G-150로 여과 및 sepharose 4B affinity chromatography 로 정제하였다. <TEX>$CCl_4$</TEX>를 복강 내에 투여시 72시간 경과 후 혈청 총cholesterol 및 triglyceride는 정상군 및 olive oil 투여군에 비해 유의하게 증가하였으며, lectin과 <TEX>$CCl_4$</TEX>를 동시 투여 하였을 때는 유의한 감소를 나타내였다. 흰쥐 간의 조직학적 관찰은 <TEX>$CCl_4$</TEX>투여군은 정상군 및 olive oil 투여군에 비해 지방변화 및 간세포 내에 많은 대소의 cytoplasmic vacuole가 보였으나, <TEX>$CCl_4$</TEX>와 lectin을 동시 투여시는 <TEX>$CCl_4$</TEX> 투여군에 비해 cytoplasmic vacuole은 현저히 감소하였고, 이에 비례하여 혈청 cholesterol 및 triglyceride 측정치가 현저히 감소하는 결과를 나타내었다. 이상의 결과를 볼때 흰쥐에서 lectin이 혈청 지질을 감소시키거나 또는 조직학적 관찰에서 간조직의 지질형성을 억제시키는 효과가 있는 것으로 확인되었다. This study was carried out to investigate effect of serum lipid and liver tissue in lectin from Viscum album on rats. The lectin was purified by sepharose 4B affinity chromatography and gel filtration using sephadex G-150 with plant material from Viscum album. After 72 h of <TEX>$CCl_4$</TEX> injection, there was a significant increase in serum total cholesterol and triglycerige levels relative to the control group. However, treatment of both Viscum album and purified lectin were significantly decreased lipid parameters against the <TEX>$CCl_4$</TEX>-induced. Histological observation of the liver section of rats challenged with <TEX>$CCl_4$</TEX> produced a marked increase of cytoplasmic vacuoles in number, while rats administered olive oil alone did not alter the normal hepatic architecture. Histological observation of the liver section in rats treated 72 h with either Viscum album purified lectin or <TEX>$CCl_4$</TEX>-induced liver lipogenesis showed decreased numbers of cytoplasmic vaculoes and necrotic cells. These results suggest that Viscum album lectin has a significantly decreased lipid in serum or histological observation of the liver section decreased in lipogenesis in rats.

The role of crude saliva and purified salivary mucins in the inhibition of the Human Immunodeficiency Virus type 1

BackgroundSub-Saharan Africa is the world’s worst HIV-AIDS affected region. More interventions to manage this pandemic are urgently required. Transmission of the virus through an exchange of saliva is rarely known to occur. This project sought to verify statistically previous findings in our laboratory, that crude saliva from uninfected individuals together with its purified mucin components inhibited HIV-1, whilst mucins from infected saliva did not show this inhibition, in an in vitro assay.MethodsSaliva was extracted in 4 M guanidinium hydrochloride and proteolytic inhibitors at pH 6.5, followed by the isolation of MUC5B and MUC7 by Sepharose 4B gel filtration and further purification of these mucins by density-gradient ultra-centrifugation in caesium chloride. Agarose gel electrophoresis, Western blotting and amino acid compositional analysis determined the size, purity and identity of the mucins. The inhibitory activity of crude saliva and purified MUC5B and MUC7, from HIV negative (n=20) and HIV positive (n=20) donors, was tested by their incubation with subtype C HIV-1 and subsequent infection of peripheral blood mononuclear cells (PBMCs). PCR was done on tandem repeat regions of MUC5B and MUC7 DNA to investigate whether any association existed between gene polymorphism and susceptibility to infection.ResultsThere was an inter-individual variation in the amounts of MUC5B and MUC7 in saliva. In contrast to previous studies, crude saliva and purified mucins from both HIV negative and HIV positive individuals inhibited the infection of HIV-1 in an in vitro assay. DNA analysis of the tandem repeat regions of MUC5B and MUC7 revealed no difference between groups.ConclusionsCrude saliva and its mucins, MUC5B and MUC7, from both uninfected controls and HIV positive individuals inhibited HIV-1 in an in vitro assay.

Purification, partial characterization and anti-inflammatory characteristics of lotus (Nelumbo nucifera Gaertn) plumule polysaccharides

A novel lotus plumule polysaccharide (LPPS) was purified, characterised and cultured with RAW264.7 macrophages to evaluate its anti-inflammatory characteristics. LPPS was purified using Sepharose 6B gel filtration and dissolved into two major components, fraction-1 (F1) and fraction-2 (F2). The molecular weights of native F1 and F2 were approximately distributed at >2,000 and 25.7kDa, respectively. The total protein and carbohydrate constituent ratios in LPPS, F1, and F2 were 30.0±0.9% vs. 70.0±0.9%, 30.1±2.6% vs. 69.9±2.6%, and 96.5±6.1% vs. 3.5±6.1% (w/w), respectively, suggesting that F1 may be a major proteo-polysaccharide component and F2 a glycoprotein constituent in LPPS. Pro-/anti-inflammatory (IL-6/IL-10) cytokine secretion ratios by lipopolysaccharide-stimulated RAW264.7 macrophages were significantly decreased by F1 and F2 treatments, particularly by F2, in a dose-dependent manner under a preventive experimental model. This study suggests that purified components, F1 and F2 from LPPS, have strong anti-inflammatory effects on LPS-induced inflamed macrophages in a preventive manner.

Purification and partial biochemical–genetic characterization of trehalose 6-phosphate synthase from muscles of adult femaleAscaris suum

Trehalose 6-phosphate (T6P) synthase (TPS; EC 2.4.1.15) was isolated from muscles of Ascaris suum by ammonium sulphate fractionation, ion-exchange DEAE SEPHACEL(TM) anion exchanger column chromatography and Sepharose 6B gel filtration. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), 265-fold purified TPS exhibited a molecular weight of 66 kDa. The optimum pH and temperature of the purified enzyme were 3.8-4.2 and 35°C, respectively. The isoelectric point (pI) of TPS was pH 5.4. The studied TPS was not absolutely substrate specific. Besides glucose 6-phosphate, the enzyme was able to use fructose 6-phosphate as an acceptor of glucose. TPS was activated by 10 mM MgCl2, 10 mM CaCl2 and 10 mM NaCl. In addition, it was inhibited by ethylenediaminetetra-acetic acid (EDTA), KCl, FeCl3 and ZnCl2. Two genes encoding TPS were isolated and sequenced from muscles of the parasite. Complete coding sequences for tps1 (JF412033.2) and tps2 (JF412034.2) were 3917 bp and 3976 bp, respectively. Translation products (AEX60788.1 and AEX60787.1) showed expression to the glucosyltransferase-GTB-type superfamily.

Structure elucidation of an immunoenhancing pectic polysaccharide isolated from aqueous extract of pods of green bean (Phaseolus vulgaris L.)

Abstract A water-soluble pectic polysaccharide (PS-I) isolated from the hot water extract of the pods of green bean (Phaseolus vulgaris L.) by successive purification through dialysis, centrifugation and Sepharose-6B gel filtration was found to consist of methyl ester of d -galacturonic acid, d -galactose, and l -arabinose in a molar ratio of nearly 2:2:1. This molecule showed splenocyte, thymocyte activation as well as antioxidant properties. Structural studies of PS-I was performed using acid hydrolysis, methylation analysis, periodate oxidation studies along with NMR analysis (1H, 13C, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC) and the repeating unit of PS-I was established as: Download : Download full-size image

Studies on cathepsin L in the surimi of southern blue whiting

According to the conventional method,actomyosin was extracted from the surimi of southern blue whiting (Micromesistius australias). After Sepharose-6B gel filtration of the actomyosin obtained,cathepsin L activity was monitored in each of the fractions collected. The results showed that the activity values in the actomyosin samples were much higher than that in the washing water,suggesting that cathepsin L could not be removed completely during conventional leaching and still remained in actomyosin sample. Further purification of actomyosin by dilution-precipitation could not remove cathepsin L effectively,indicating that cathepsin L might be dissolved together with actomyosin in the presence of high ion-strength solutions. The profile of the gel filtration showed hat the main peak of cathepsin L was obviously separated from that of actomyosin,suggesting that cathepsin L was non-binding with actomyosin. Fractions that showing the main peak of cathepsin L were pooled to be called L_ mix ,partially purified cathepsin L. L_ mix could strongly hydrolyze Z-Phe-Arg-MCA,a specific substrate for cathepsin L,rather than Z-Arg-Arg-MCA,spcefic substrate for cathepsin B,nor Arg-MCA,substrate for cathepsin H,as well as Boc-Gln-Ala-Arg-MCA,substrate for trypsin and trypsin-like proteases. The above results indicate that L_ mix was a crude cathepsin L. Cathepsin L specific inhibitors,such as E-64,specific inhibitor for cathepsins,and leupeptin,inhibitor for both cathepsins and trypsin-like proteases,could suppress the activity of L_ mix completely. Activators for cathepsin L,such as DTT,EDTA+DTT,could effectively improve the activity of L_ mix . As a result,the effect of activators and inhibitors confirmed the partially purified crude enzyme,L_ mix ,to be a thiol-type cysteine protease. The temperature dependence and pH dependence of the activity of L_ mix were also investigated. L_ mix had optimum temperature of 45 ℃ which was right in the temperature range of modori phenomena. L_ mix had optimum pH of 5.5,but kept high residue activity near neutral pH,and the pH range of surimi processing,suggesting its high potential ability in modori phenomena.

Expression, purification, and crystallization of a novel galactose mutarotase from Thermoanaerobacter tengcongensis

To purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction. The tte 1925 gene was subcloned into the prokaryotic expression vector pGEX-6P-1 and overexpression was obtained in the E.coli BL21 (DE3) through transformation of the right recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with glutathione S-transferase tag expressed highly by the induction of isopropyl beta-D-thiogalactoside and was purified in a three-step procedure, which included Glutathione Sepharose 4B affinity, ion chromatography (Resource Q 6 mL), and gel filtration chromatography (10/300 superdex 200). The purity of the purified protein was over 99% and a large amount of claval crystals whose X-ray diffraction reached 1.9 A were obtained. We successfully prepared TTE1925 with high purity and obtained crystals for X-ray diffraction. These work paved the way for the further research on the 3-D structure of TTE1925 and its biological mechanism.

Purification and characterization of lipase from Spirulina platensis

A lipase from photosynthetic cyanobacterium Spirulina platensis ( Arthrospira) was purified by sequential operation of ammonium sulphate precipitation, dialysis, DEAE-Sepharose anion exchange chromatography, and Sepharose-6B gel filtration chromatography for the first time. This purification procedure resulted in 375-fold purification of lipase with 29.35% final yield. The purified lipase showed a prominent single band on SDS-PAGE. It is a monomeric protein of 45 kDa molecular weight and its isoelectric point is 5.9. The purified lipase exhibited maximal hydrolytic activity at a temperature of 45 °C and pH of 6.5. The values of K m and V max calculated from the Lineweaver–Burk plot using p-nitrophenyl palmitate ( p-NPP) as hydrolysis substrate were 0.02 mM and 38.9 μmol min −1 mg −1, respectively. The catalytic efficiency ( k cat/ K m) of purified lipase was determined as 1.5 × 10 6 M −1 s −1. The remaining activity of the lipase was about 95% of its original activity at 25 °C for 24 h of preincubation. However, the remaining activity was about 26% of the original activity at 45 °C for 24 h. The purified lipase appears to be unique since it cleaved triolein at only 3-position releasing 1,2-diolein. Lipase activity was stimulated by Ca 2+, Mg 2+, Zn 2+, Triton X-100 and SDS, and inhibited by Li +, Fe 2+, Mn 2+, EDTA and PMSF.

ACTOMYOSIN NONBINDING CATHEPSIN L IN WALLEYE POLLOCK SURIMI

Cathepsin L remained with the actomyosin (AM) fraction which was extracted from walleye pollock surimi after three repeats of washing followed by one cycle of dilution–precipitation. The main peak of cathepsin L separated from the peak of AM by Sepharose 6B gel filtration chromatography. This result suggests that cathepsin L is not associated with AM in AM solution, and the enzyme might have interaction with AM in washed meat. In the presence of 0.3–1.0 M NaCl, the activity of cathepsin L was much higher in acidic condition (pH 5.5). In neutral condition (pH 7.0), the activity of cathepsin L still has about 85% that of at pH 5.5 in 0.6 M NaCl. This indicates that the enzyme contributes to the gel deterioration of surimi even at neutral conditions. The activity of cathepsin L in walleye pollock surimi was not affected by a serine-type protease inhibitor, but was inhibited by a cysteine-type protease inhibitor, suggesting that cathepsin L is not activated by trypsin-type serine protease. PRACTICAL APPLICATIONS Walleye pollock is one of the most popular material fish used in the surimi production. But the gel strength of the kamaboko made from this fish was much low because of the modori phenomena which was believed to be due to the hydrolysis of myofibrillar components by proteinases contained in the surimi. In this study, cathepsin L was confirmed to be still remained in actomyosin fractions after conventional treatments. The activity of cathepsin L at neutral condition in the presence of 0.3–1.0 M NaCl was much higher than that at acidic condition, indicating the potential hydrolytic ability of capthepsin L in walleye Pollock surimi.

A Case of Obstructive Jaundice with Severe Hypercholesterolemia Probably Due to Lipoprotein-Y

We analyzed the lipoproteins of a patient with pancreatic cancer causing obstructive jaundice, with marked hypercholesterolemia. The patient was a 49-year-old female. Serum total cholesterol, triglyceride, LDL-cholesterol and HDL-cholesterol levels were 1,170 mg/dL, 282 mg/dL, 1,070 mg/dL and 53 mg/dL, respectively. Cholesterol ester and lecithin:cholesterol acyltransferase decreased, and so-called remnant like particle-cholesterol increased remarkably. Fractionation of the patient's serum by polyacrylamide gel disc electrophoresis showed an abundant VLDL fraction, whereas agarose gel electrophoresis demonstrated a high level of beta-lipoprotein. Sepharose 6B gel filtration of the serum revealed that the levels of free cholesterol and apolipoprotein B were high in the VLDL- corresponding fraction. The results suggested that the high cholesterol level was due to the presence of abnormally large particles rich in free cholesterol and apoB, and this abnormal fraction may correspond to lipoprotein-Y that was increased in obstructive jaundice.