S-200 Gel Filtration Chromatography Research Articles

Isolation and Characterization of a Novel Sialoglycopeptide Promoting Osteogenesis from Gadus morhua Eggs.

Gadus morhua eggs contain several nutrients, including polyunsaturated fatty acids, lecithin and glycoproteins. A novel sialoglycopeptide from the eggs of G. morhua (Gm-SGPP) was extracted with 90% phenol and purified by Q Sepharose Fast Flow (QFF) ion exchange chromatography, followed by S-300 gel filtration chromatography. Gm-SGPP contained 63.7% carbohydrate, 16.2% protein and 18.6% N-acetylneuraminic acid. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that Gm-SGPP is a 7000-Da pure sialoglycopeptide. β-elimination reaction suggested that Gm-SGPP contained N-glycan units. Amino acid N-terminal sequence analysis indicated the presence of Ala-Ser-Asn-Gly-Thr-Gln-Ala-Pro amino acid sequence. Moreover, N-glycan was connected at the third Asn location of the peptide chain through GlcNAc. Gm-SGPP was composed of D-mannose, D-glucuronic acid and D-galactose. Fourier transform-infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance spectroscopy (1H-NMR) and methylation analysis were performed to reveal the structure profile of Gm-SGPP. In vitro results showed that the proliferation activity of MC3T3-E1 cells was significantly promoted by Gm-SGPP. In vivo data revealed that Gm-SGPP increased the calcium and phosphorus content of tibias and promoted longitudinal bone growth in adolescent rats.

Purification of chitosanase from Stenotrophomonas maltophilia KPU 2123 and Micromonospora sp. T5a1 for chitooligosacharide production

Stenotrophomonas maltophilia KPU 2123 and Micromonospora sp. T5a1 are bacterial strains isolated from shrimp waste capable of hydrolyzing chitosan to produce chitooligosaccharides (COSs). Their COS products showed antimicrobial activity. The aim of this study was to purify chitosanase from those bacteria and used for COSs production. Chitosanase from both strains were produced using 0.5% colloidal chitin as inducer. The chitosanase was then purified by ultrafiltration, DEAE Sepharose ion exchange and Separacyl S-300 gel filtration chromatography. The specific activity and the yield of chitosanase KPU 2123 increased 2.35-fold and 30.90% after three steps of purifications, respectively, whereas chitosanase T5a1 increased 3.32-fold and 22.79%, respectively. The molecular weight of both chitosanases KPU 2123 and T5a1 were about 76 and 20 kDa, respectively. The COS products generated by chitosanase KPU 2123 contained N-acetyl-D-glucosamine, Di-N-acetyl-D-glucosamine, Tri-N-acetyl-D-glucosamine, Tetra-acetyl-D-glucosamine and Penta-N-acetyl-D-glucosamine while by chitosanase T5a1 contained N-acetyl-D-glucosamine, Di-N-acetyl-D-glucosamine. Based on their COS products, chitosanase KPU2123 can be categorized as endo-type chitosanase. Further study is needed to analysis the bioactivity of COSs obtained from the pure of chitosanases KPU 2123 and T5a1.

Interaction of tubulin and protein kinase CK2 in Trypanosoma equiperdum.

A polypeptide band with an apparent molecular weight of 55,000 was phosphorylated in vitro in whole-cell lysates of Trypanosoma equiperdum. This band corresponds to tubulin as demonstrated by immunoprecipitation of the phosphorylated polypeptide from T. equiperdum extracts when anti-α and anti-β tubulin monoclonal antibodies were employed. A parasite protein kinase CK2 was in charge of modifying tubulin given that common mammalian CK2 inhibitors such as emodin and GTP, hindered the phosphorylation of tubulin and exogenously added casein. Interestingly, a divalent cation-dependent translocation of the T. equiperdum tubulin and the CK2 responsible for its phosphorylation was noticed, suggesting a direct interaction between these two proteins. Additionally, this fraction of tubulin and its kinase coeluted using separations based on parameters as different as charge (DEAE-Sepharose anion-exchange chromatography) and size (Sephacryl S-300 gel filtration chromatography). Analyses by non-denaturing polyacrylamide gel electrophoresis and immunoblot of the purified and radioactively labeled fraction containing both tubulin and the CK2 enzyme, established the phosphorylation of a single band that was recognized by anti-CK2 α-subunit and anti-tubulin antibodies. All these findings revealed a physical association between a pool of tubulin and a CK2 in T. equiperdum.

Purifi cation and Characterization of Protease From Bacillus sp. TBRSN- 1

Potato Cyst Nematode (PCN), Globodera rostochiensis, is one of the important potato’s pests and causedeconomic looses up to 70% in the several centrals of potato plantations in Indonesia. PCN’s shell componentof egg shell containing chitin (inner layer) and viteline/ protein (outer layer). The purpose of this researchwas to purify of protease Bacillus sp. TBRSN-1, isolate from tomato’s rhizosfer in Yogyakarta province. Thepurifi ed protease could be used for cutting the life cycle of PCN. Results showed that Bacillus sp. TBRSN-1could produce extracellular protease and purifi cation using DEAE-cellulose ion-exchange chromatographyand Sephacryl S-300 gel fi ltration chromatography resulted in specifi c activity 4.31 fold and 1.68% recovery.Analysing using SDS-PAGE 12.5% and molecular weight 48.1 kDa. Km and Vmax values of the protease forcasein substrate were 7.83 mg/ml and 4.03 μg/h, respectively. The optimum activity at the temperature30oC and pH 7.0. Keywords : protease, purifi cation, indigenous Bacillus sp. TBRSN-1

BACE2의 대량발현 및 리폴딩

BACE2 is a membrane-bound aspartic protease that is highly homologous with BACE1. While BACE1 processes the amyloid precursor protein (APP) at a key step in generating <TEX>${\beta}$</TEX>-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be involved in APP processing directly, and its physiological functions are unknown. To determine its function and to develop inhibitors from marine sources, we constructed an overexpression vector for producing BACE2. The gene encoding human BACE2 protease was amplified using the polymerase chain reaction and cloned into the pET11a expression vector, resulting in pET11a/BACE2. Recombinant BACE2 protease was overexpressed successfully in E. coli as inclusion bodies, refolded using the rapid-dilution method, and purified via two-step fast protein liquid chromatography using Sephacryl S-300 gel filtration and Resource-Q column chromatography. The BACE2 protease produced was an active form. This study provides an efficient method not only for studying the basic properties of BACE2, but also for developing inhibitors from natural marine sources.

Purification and Characterization of an Endo-d-arabinase Produced by Cellulomonas

An extracellular endo-D: -arabinase enzyme produced by the bacterial strain of Cellulomonas was purified 77.1-fold with 0.20% recovery for protein by DEAE Sepharose anion exchange, Sephacryl S-300 gel filtration and blue Sepharose affinity chromatography, and designated as CEDAase. The apparent molecular mass of CEDAase was 45kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CEDAase is an endoenzyme for arabinogalactan with the main and specific product of hexa-arabinofuranoside. It reacts optimally with its substrate, arabinogalactan, at approximately pH 8.0 and at 40°C. CEDAase shows stability in the pH range of 6.0-9.0 and at the temperature below 50°C. The K(m) measured for the CEDAase was 55.6μM, with an apparent V(max) of 0.083μmol/min. To our knowledge, for the first time, the current work obtains an extracellular Cellulomonas endo-D: -arabinase enzyme that might be potentially served as a tool enzyme for hydrolyzing specific cell wall such as Mycobacterium cell. It is purified as an important potential initial material basis for mass spectrometric sequencing and chemical gene synthesis. It may make it possible to clone and express this valuable endo-D: -arabinase and make it available to the mycobacteria scientific community.

Monoclonal antibodies recognizing mucus immunoglobulin and surface immunoglobulin-positive cells of flounder (Paralichthys olivaceus)

Mucus immunoglobulin (Ig) of flounder (Paralichthys olivaceus) was purified by the combination of salting-out, Sephacryl S-300 gel filtration chromatography and DEAE Sepharose chromatography. According to the SDS-PAGE and native-PAGE, the purified mucus Ig showed apparent molecular weights of 72kDa (heavy chain) and 26kDa (light chain), and a total molecular weight of 798kDa, which indicated mucus IgM was in tetrameric form. Purified mucus Ig was used to immunize the Balb/C mice, nineteen hybridomas secreting monoclonal antibodies (mAbs) against flounder mucus Ig were obtained by indirect enzyme-linked immunosorbent assay, and three of them designated as 1A-M2, 1C-M10 and 3F-M9 were cloned by limiting dilution. In Western blotting, the three mAbs specifically reacted to the heavy (H) chain of mucus Ig, but not reacted with serum Ig of flounder, whereas mAb 2D8 against serum Ig previously produced could react with the H chain of both mucus and serum Ig, indicating the composition of the mucus and serum Ig H chains was different. Meanwhile, surface Ig positive (sIg+) lymphocytes in the peripheral blood, spleen, skin and gills of healthy flounder, were analyzed by flow cytometry using mAb 1A-M2 and mAb 2D8, and the results revealed that both mAbs were reactive with the sIg+ lymphocytes. The positive reactivity rates for mAb 1A-M2 were 38.64% in the peripheral blood, 23.6% in the spleen, 16.56% in the skin and 6.26% in the gills, while the positive reactivity rates for mAb 2D8 were 48.89%, 33.7%, 15% and 6.02%, respectively, suggesting mucus Ig was similar, but not identical, to serum Ig. These results generated important mucosal immunological information and gave a valuable insight into understanding the mucosal immunity in flounder.

Purification and partial characterization of a thermostable alkaline protease from Bacillus licheniformis LHSB-05 isolated from hot spring

An alkaline protease produced from Bacillus licheniformis LHSB-05 isolated from Nigerian hot spring was purified in a 2-step procedure by ammonium sulphate precipitation and Sephacryl S-300 gel filtration chromatography. The enzyme was purified 13.4-fold with a yield of 6.6%. The molecular weight of the protease was 16 kDa on SDS-PAGE. The purified protease had optimum temperature of 50°C. The enzyme retained 90% of its maximum activity at 60°C and had 45% relative activity at 70°C. This enzyme exhibited high thermostability with 100% stability at 50°C and almost 80% stability at 60°C after 60 min of incubation. The protease had optimum pH of 9.0 and showed relative activity between 81 and 71% in the pH range of 10.0 to 12.0. The enzyme was stable over a broad pH range (6.0 to 12.0). Ca2+ and Mg2+ increased protease activity with 27 and 9%, whereas Hg2+ strongly inhibited its activity by 83%. The enzyme was relatively stable in the presence of Ca2+, Mg2+, Al3+, Zn2+ and ethylene diamine tetra acetic acid (EDTA). These characteristics suggest the suitability of protease from B. licheniformis LHSB-05 for use in detergent industries. Key words: Alkaline protease, Bacillus licheniformis LHSB-05, characterization, purification, thermostability.

PURIFICATION, CHARACTERIZATION AND IN VITRO ANTIOXIDANT ACTIVITIES OF POLYSACCHARIDE FRACTIONS ISOLATED FROM THE FRUITS OF PHYSALIS ALKEKENGI L.

The water-soluble crude polysaccharide PAPS, isolated from the fruits of Physalis alkekengi L. by hot water extraction and ethanol precipitation, was fractionated by diethylaminoethyl-cellulose ion exchange chromatography and Sephacryl S-300 gel filtration chromatography on ATKA explore 100 purifier, giving four polysaccharide fractions coded as PAPSA-a, PAPSA-b, PAPSB-c and PAPSB-d. Their chemical and physical characteristics were determined by chemical methods, gas chromatography and high performance size exclusion chromatography. The in vitro antioxidant activities of polysaccharides were studied by various antioxidant assays. Experiment results showed that all fractions exhibited antioxidant activity in a concentration-dependent manner, and PAPSB-c and PAPSB-d had stronger antioxidant ability than PAPSA-a and PAPSA-b in all the tests. The polysaccharide fractions can be developed as new antioxidant agents. PRACTICAL APPLICATIONS More and more researches are focused on natural products including vegetables and wild fruits sources. Physalis alkekengi is a kind of wild fruit in north of China, famous for its good taste and high nutritional quality. But, at present, the fruits of P. alkekengi are not fully utilized. This work reported on isolation and characterization of polysaccharides from P. alkekengi, and the in vitro antioxidant activities were studied by various antioxidant assays. The objective of this research work was to extend the potential use of P. alkekengi fruits in functional food or antioxidant biomedicine.

Purification and mass spectrometric analysis of the κ opioid receptor

A clonal human embryonic kidney (HEK) 293 cell line was established that stably expressed the rat κ-opioid receptor (rKOR) with a FLAG epitope at the amino terminus. The K d for [ 3H]diprenorphine was 1.1 ± 0.2 nM, and the B max was 2.6 ± 0.4 pmol/mg. Dynorphin A (1–13), U69,593 and naloxone competitively inhibited [ 3H]diprenorphine binding with K i values of 2.0, 18 and 18 nM, respectively, in good agreement with previously reported affinities for the unmodified receptor. U69,593 stimulated [ 35S]GTPγS binding in a concentration-dependent manner and caused phosphorylation of mitogen-activated protein (MAP) kinase, indicating that the activated epitope-tagged receptor triggered appropriate signaling pathways. Immunoblot analysis demonstrated that two immunoreactive receptor species with apparent molecular masses of 42 and 52 kDa were expressed. Previous studies indicated that the 42 kDa protein was localized intracellularly and was a precursor of the 52 kDa receptor, which was present at the cell surface. rKOR was extracted from transfected HEK 293 cell membranes with n-dodecyl-β- d-maltopyranoside. Sequential use of wheat germ agglutinin chromatography, Sephacryl S300 gel filtration chromatography, anti-FLAG immunoaffinity chromatography and SDS/PAGE permitted purification of the 52 kDa receptor. MALDI-TOF mass spectrometry was used to identify peptides derived from rKOR following sequential in-gel digestion with trypsin and cyanogen bromide. Eighteen rKOR peptides were detected, corresponding to 27.1% coverage of the receptor. Precursor-selective MS/MS confirmed the identity of most of these peptides. In addition, we have identified heat shock protein 70 (HSP70) as a rKOR-interacting protein.

Comparative Structural, Emulsifying, and Biological Properties of 2 Major Canola Proteins, Cruciferin and Napin

Canola is an economically important farm-gate crop in Canada. To further explore the potential of canola protein as value-added food and nutraceutical ingredients, a better understanding of fundamental properties of 2 major canola proteins is necessary. Two major protein components, cruciferin and napin, were isolated from defatted canola meal by Sephacryl S-300 gel filtration chromatography. SDS-PAGE showed that cruciferin consists of more than 10 polypeptides, and noncovalent links are more important than disulphide bonds in stabilizing the structural conformation. Napin consists of 2 polypeptides and is stabilized primarily by disulphide bonds. Purified cruciferin showed 1 major endothermic peak at 91 degrees C compared with that of 110 degrees C for napin. Emulsion prepared by cruciferin showed significant higher specific surface area and lower particle size than that of napin. The study indicated that the presence of napin could detrimentally affect the emulsion stability of canola protein isolates. Hydrolysates from cruciferin and napin showed potent angiotensin I-converting enzyme inhibitory activity (IC(50): 0.035 and 0.029 mg/mL, respectively), but weaker than that of canola protein isolate hydrolysate (IC(50): 0.015 mg/mL).

Structure and Function of Both Domains of ArnA, a Dual Function Decarboxylase and a Formyltransferase, Involved in 4-Amino-4-deoxy-L-arabinose Biosynthesis

Modification of the lipid A moiety of lipopolysaccharide by the addition of the sugar 4-amino-4-deoxy-L-arabinose (L-Ara4N) is a strategy adopted by pathogenic Gram-negative bacteria to evade cationic antimicrobial peptides produced by the innate immune system. L-Ara4N biosynthesis is therefore a potential anti-infective target, because inhibiting its synthesis would render certain pathogens more sensitive to the immune system. The bifunctional enzyme ArnA, which is required for L-Ara4N biosynthesis, catalyzes the NAD(+)-dependent oxidative decarboxylation of UDP-glucuronic acid to generate a UDP-4'-keto-pentose sugar and also catalyzes transfer of a formyl group from N-10-formyltetrahydrofolate to the 4'-amine of UDP-L-Ara4N. We now report the crystal structure of the N-terminal formyltransferase domain in a complex with uridine monophosphate and N-5-formyltetrahydrofolate. Using this structure, we identify the active site of formyltransfer in ArnA, including the key catalytic residues Asn(102), His(104), and Asp(140). Additionally, we have shown that residues Ser(433) and Glu(434) of the decarboxylase domain are required for the oxidative decarboxylation of UDP-GlcUA. An E434Q mutant is inactive, suggesting that chemical rather than steric properties of this residue are crucial in the decarboxylation reaction. Our data suggest that the decarboxylase domain catalyzes both hydride abstraction (oxidation) from the C-4' position and the subsequent decarboxylation.

Purification and mass spectrometric analysis of the δ opioid receptor

A mouse δ opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B max of 10.5 pmol/mg protein. [ 3H]Bremazocine exhibited high affinity binding to the epitope-tagged δ opioid receptor with a K D of 1.4 nM. The agonists DADL, morphine, and DAMGO competitively inhibited bremazocine binding to the tagged δ receptor with K I's of 0.9, 370, and 620 nM, respectively. Chronic treatment of cells expressing the epitope-tagged δ receptor with DADL resulted in downregulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The δ receptor was solubilized from HEK 293 cell membranes with n-dodecyl-β- d-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of Sephacryl S300 gel filtration chromatography, wheat germ agglutinin (WGA)-agarose chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and SDS/PAGE permitted purification of the receptor. The purified δ opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 65 kDa. MALDI-TOF mass spectrometry was used to identify and characterize peptides derived from the δ opioid receptor following in-gel digestion with trypsin, and precursor-derived ms/ms confirmed the identity of peptides derived from enzymatic digestion of the δ opioid receptor.

Characterization of a novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86 was purified to homogeneity by ammonium sulfate precipitation and Sephacryl S-300 gel filtration chromatography. The purified xylanosome appeared as a single protein band on the non-denaturing (native) polyacrylamide gel electrophoresis (PAGE) gel with a molecular mass of approximately 1200 kDa. The optimal temperature and pH for xylanase activity was 60 °C and pH 6.0, respectively. The xylanase activity was stable within pH 4.1–10.3. It was stable up to 60 °C at pH 6.0. The xylanosome was highly specific towards oat-spelt xylan, and showed low activity towards corncob powder, but exhibited very low activity towards lichenan, CMC and p-nitrophenyl derivatives. Apparent K m values of the xylansosome for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 2.5, 3.6, 1.7 and 4.9 mg ml −1, respectively. The main hydrolysis products of birchwood xylan were xylotriose, xylobiose and xylose. Analysis of the products from wheat arabinoxylan degradation by xylanosome confirmed that the enzyme had endoxylanase and debranching activities, with xylotriose, xylobiose, xylose and arabinose as the main degradation products. These unique properties of the purified xylanosome from Streptomyces olivaceoviridis E-86 make this enzymatic complex attractive for biotechnological applications.

Purification and mass spectrometric analysis of the μ opioid receptor

A mouse μ opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl-terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B max of 10 pmol/mg protein. 3[H]Bremazocine exhibited high affinity binding to the epitope-tagged μ opioid receptor with a K D of 1.0 nM. The agonists [ d-Ala 2, N-Me-Phe 4,Gly 5-ol]enkephalin (DAMGO), morphine and [ d-Ala 2, d-Leu 5]enkephalin (DADL) competitively inhibited bremazocine binding to the tagged μ receptor with K I's of 3.5, 17 and 70 nM, respectively. Chronic treatment of cells expressing the epitope-tagged μ receptor with DAMGO resulted in down-regulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The μ receptor was solubilized from HEK 293 cell membranes with n-dodecyl-β- d-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of wheat germ agglutinin (WGA)–agarose chromatography, Sephacryl S300 gel filtration chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) permitted purification of the receptor. The purified μ opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 80 kDa. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify and characterize peptides derived from the μ opioid receptor following in-gel digestion with trypsin or chymotrypsin, and precursor-derived tandem mass spectrometry (ms/ms) confirmed the identity of several peptides derived from enzymatic digestion of the μ opioid receptor.

Tumor Necrosis Factor-α Induces Bax-Bak Interaction and Apoptosis, Which Is Inhibited by Adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.

Purification and Preliminary Characterisation of a Thiamine-binding Protein from Maize Seeds

Summary A thiamine-binding protein was isolated from seeds of maize ( Zea mays L.) using a sequence of precipitation techniques, DEAE-cellulose batch adsorption, Sephacryl S-300 gel filtration and EAH-Sepharose chromatography. It was a 130 kDa globulin composed of some combination of 54 kDa and 39 kDa subunits not linked by disulphide bridges. Its isoelectric point was estimated to be 6.8. The protein preparation contained a high percentage of glutamine/glutamic acid, arginine and glycine. Thiamine was bound to a capacity of 13.5 nmoles per mg protein, indicating the 2:1 stoichiometry of thiamine-protein interaction. In optimal 0.05 mol/L sodium phosphate buffer, pH 8.0, the dissociation constant of the thiamineprotein complex was 0.68 μmol/L. This protein also bound several thiamine analogues though with affinity always lower than that to thiamine, discriminating thiamine phosphates by 2–3 orders of affinity constant magnitude.

Detection and characterization of a type IIA secretory phospholipase A2 inhibitory protein in human amniotic fluid.

Two types of phospholipase A2 (PLA2) inhibitory protein (PLIP-I, PLIP-II) were detected and isolated from human amniotic fluid by Sephacryl S300 gel filtration chromatography. The lower molecular weight-fraction (PLIP-II) was further purified by Sephadex G75 gel filtration and analyzed by SDS-PAGE. Its molecular weight was estimated to be approximately 18 kDa, and it was sensitive to heat treatment. Inhibition of phospholipase A2 (sPLA2 type IIA) by PLIP-II occurred in a dose-dependent manner (IC50 about 0.82/microm), and the effect was stronger on sPLA2 IIA than on pancreatic sPLA2 (IC50 about 3.11 microL). The ratio of the inhibitions of the sPLA2 IIA by PLIP-II remained consistent over an entire range of substrate concentrations. Furthermore, addition of excess Ca2+ at concentrations of up to 10 mm did not antagonize the inhibitory activity of PLIP-II.

Purification and characterization of liver ferritins from different animal species.

The ferritins were purified from liver homogenates of buffalo, camel, cattle, sheep and shark by thermal denaturation, ammonium sulphate fractionation, Sephacryl S-300 gel filtration and DEAE-blue gel affinity chromatography. The yield and iron content of affinity-purified liver ferritins ranged from 0.008 to 0.052 mg/g and 3.17% to 11.4% respectively. As they are glycoproteins, the ferritins contained variable amounts of neutral carbohydrates. Except for shark ferritin, the ferritins all exhibited immunological cross-reactivity with anti-buffalo liver ferritin and anti-horse spleen ferritin by immunodiffusion and immunoelectrophoresis. Gel electrophoresis, gel filtration and ultracentrifugal analysis indicated the presence of a monomeric ferritin in all cases. SDS-gel electrophoresis of shark ferritin gave a protein band of 18 kDa. Ovine, buffalo and bovine ferritin comprised two protein subunits, the H (20 and 21 kDa) and the L types (18 and 19 kDa). Oligomeric ferritin subunits with molecular weights of 27, 37 and 55 kDa were also found for bovine and buffalo ferritin. SDS-PAGE of camel ferritin revealed a complex pattern with four prominent bands of 61, 51, 44 and 39 kDa. Two fast-migrating components of 15 and 16 kDa were also found in the purified liver ferritins, including reference preparations. The PO4(3-)/Fe ratios of purified shark (0.10) and bovine ferritin (0.12) were similar to that of standard equine spleen ferritin (0.11). However, the ratio was higher in ovine (0.17), camel (0.22) and bovine (0.26) ferritins. The amino acid compositions, molecular weights and sedimentation coefficients of the different liver ferritins were similar.

Characterization of small GTP-Binding proteins in plant cell

To identify and characterize small GTP-binding proteins in plant cells, GTP-binding studies were performed with electroblotted plant proteins following SDS-polyacrylamide gel electrophoresis using [α-32P]GTP. Three species of small GTP-binding protein (21, 23, and 27 kD) which have a specific GTP-binding property were identified in the membrane and cytosolic fractions of both monocotyledons (Zea mays) and dicotyledons (Glycine max). Moreover, these three species of small GTP-binding protein were gradually decreased when membranes were treated with hydroxylamine. This result indicates that these small GTP-binding proteins in plant cells are fatty acylated to the membrane lipids. The 27 kDa component was partially purified from hypocotyl membranes of Glycinemax, following S-300 gel filtration, phenylsepharose CL-4B, hydroxyapatite, and Q-sepharose column chromatography. This 27 kD protein was found to have both GTP-binding and GTPase activities.