A crustacean-specific neurotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus

A method of the isolation of a crustacea-specific neurotoxin from the venom of the Latrodectus mactans tredecimguttatus spider by means of ion exchange chromatography on Mono Q and Mono S columns and hydrophobic chromatography on Phenyl-Superose column has been developed. LD50 of the toxin has been elucidated.     

Simultaneous isolation of protein C activator,fibrin clot promoting enzyme (fiprozyme) and phospholipase A2 from the venom of the southern copperhead snake

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal–chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A₂.     

Chitinase-like proteins with antifungal activity from emperor banana fruits

Two 30-kDa proteins with N-terminal sequence homology to chitinases have been isolated from fruits of the emperor banana by using a protocol that involved (NH(4))(2)SO(4) precipitation, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S and gel filtration by FPLC on Superdex 75. The proteins were adsorbed on Affi-gel blue gel and Mono S. They both inhibited mycelial growth in Fusarium oxysporum but not in Mycosphaerella arachidicola. The chitinase-like protein more strongly bound on Mono S was obtained with a slightly lower yield and exhibited a higher antifungal potency toward F. oxysporum when compared with the less strongly bound chitinase-like protein.     

Purification of hormone-sensitive lipase by high-performance ion exchange chromatography

We have developed an improved method for purification of hormone-sensitive lipase from adipose tissue. The method employs two preparative high-performance ion-exchange chromatography steps on Mono Q and Mono S after detergent solubilization and partial fractionation of the enzyme by gradient sievorptive chromatography on QAE-Sephadex. About 0.2 mg of greater than 70% pure enzyme is prepared at 10% yield within 6-7 days from adipose tissue of 500 rats. This protocol is a major improvement over the previously established procedure in terms of accessibility, rapidity, enzyme purity, and yield.     

First isolation of an antifungal lipid transfer peptide from seeds of a Brassica species

An antifungal peptide with a molecular mass of 9412 and an N-terminal sequence exhibiting notable homology to those of lipid transfer proteins was isolated from seeds of the vegetable Brassica campestris. The purification protocol entailed ion exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on a Superdex peptide column. The antifungal peptide was adsorbed on Affi-gel blue gel and Mono S. It inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola with an IC(50) value of 8.3 microM and 4.5 microM, respectively. It exhibited dose-dependent binding to lyso-alpha-lauroyl phosphatidylcholine. The present findings constitute the first report on a non-specific lipid transfer protein from the seeds of a Brassica species.     

Isolation and structural characterization of twenty-one sialyloligosaccharides from galactosialidosis urine. An intact N,N'-diacetylchitobiose unit at the reducing end of a diantennary structure

Galactosialidosis urine was fractionated by gel-permeation chromatography on Bio-Gel P-6. The obtained sialic acid-containing carbohydrate fractions were purified by reversed-phase chromatography and separated according to charge by medium-pressure anion-exchange chromatography on Mono Q. The Mono Q fractions, being mixtures of sialyloligosaccharides differing mainly in sialic acid-linkage type (alpha 2-3/alpha 2-6), were subfractionated by high-performance liquid chromatography on Lichrosorb-NH2. The purified compounds were analysed by 500-MHz 1H-NMR spectroscopy. Twenty-one fully and partially sialylated N-acetyllactosamine-type compounds include mono-, di-, tri- and tetra-antennary structures. All structures have the sequence Man beta 1-4Glc-NAc at the reducing terminus in common, except one diantennary structure bearing an intact N,N'-diacetylchitobiose unit at the reducing end, which is a new feature in human glycoproteinosis urine.     

Exo- and endo-glucanolytic activity of cellulases purified from Trichoderma reesei

Four cellulases, produced by Trichoderma reesei, have been purified by preparative isoelectric focusing (Rotofor), size exclusion (Sephacryl 100 HR), anionic (Mono Q) and cationic (Mono S) chromatography and chromatofocusing (Mono P). Enzymatic activity with a large number of substrates allowed the proteins to be classified as: cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II. The exo- or endo-glucanase character of these enzymes was analysed by using a technique based on the measurement of the Avicel insoluble fibres reducing power. © Rapid Science Ltd. 1998     

A one-step preparative method for separating SER 6-phosphorylated HMG 14 from unphosphorylated HMG 14 and in vitro phosphorylation reaction components

While clear evidence exists for the regulation of the phosphorylation of the very basic high mobility group (HMG) and histone chromatin proteins, the physiological role of their phosphorylation remains poorly understood. Elucidation of these roles has been difficult, in part, because of the inability to obtain sufficient quantities of purified phosphorylated derivatives. We have used Mono S cation-exchange chromatography to prepare milligram quantities of pure Ser 6-phosphorylated HMG 14 (Ser 6-PO4-HMG) from unphosphorylated Mono S-purified calf thymus HMG 14 following in vitro phosphorylation with cAMP-dependent protein kinase (A-kinase). In one step, this technique separates the phosphorylated derivative from A-kinase, ATP, unphosphorylated HMG 14, and a minor phosphorylated by-product which evidence suggests may be the previously reported Ser 6, 24-diphospho-HMG 14. Mono S chromatography also enhances the purity of calf thymus HMG 14 prepared by perchloric acid extraction, acetone and ethanol precipitations, and CM-Sephadex chromatography. In addition, it permits the detection of apparent microheterogenous forms of both unphosphorylated and Ser 6-PO4-HMG 14. The significant reductions in binding affinity resulting from the incorporation of phosphate groups into HMG 14 suggest that Mono S chromatography could have more general application in the isolation of phosphorylated derivatives of other basic proteins, including other chromatin-associated DNA-binding proteins which are known to undergo specific phosphorylation. It would especially be useful when the proteins and their phosphorylated derivatives bind more tightly to Mono S than the kinases used for their phosphorylation.     

Purification, partial amino acid sequence and structure of the product of raucaffricine-O-beta-D-glucosidase from plant cell cultures of Rauwolfia serpentina

Plant cell suspension cultures of Rauwolfia produce within 1 week approximately 250 nkat/l of raucaffricine-O-beta-D-glucosidase. A five step procedure using anion exchange chromatography, chromatography on hydroxylapatite, gel filtration and FPLC-chromatography on Mono Q and Mono P delivered in a yield of 0.9% approximately 1200-fold enriched glucosidase. A short protocol employing DEAE sepharose, TSK 55 S gel chromatography and purification on Mono Q gave a 5% recovery of glucosidase which was 340-fold enriched. SDS-PAGE showed a Mr for the enzyme of 61 kDa. The enzyme is not glycosylated. Structural investigation of the enzyme product, vomilenine, demonstrated that the alkaloid exists in aqueous solutions in an equilibrium of 21(R)- and 21(S)-vomilenine in a ratio of 3.4:1. Proteolysis of the pure enzyme with endoproteinase Lys C revealed six peptide fragments with 6-24 amino acids which were sequenced. The two largest fragments showed sequences, of which the motif Val-Thr-Glu-Asn-Gly is typical for beta-glucosidases. Sequence alignment of these fragments demonstrated high homologies to linamarase from Manihot esculenta (81% identity) or to beta-glucosidase from Prunus avium (79% identity). Raucaffricine-O-beta-D-glucosidase seems to be a new member of the family 1 of glycosyl hydrolases.     

Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets

The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.     

Macrophage differentiation inducing factor from human monocytic cells is equivalent to murine leukemia inhibitory factor

Human macrophage differentiation inducing factor (DIF) can induce differentiation of human myeloid leukemic cells into macrophage-like cells in vitro. A procedure is described for purification of DIF from serum-free human monocytic leukemia THP-1 cell-conditioned medium. The procedure included concentration of a conditioned medium by ultrafiltration, lentil lectin-Sepharose affinity chromatography, and fast protein liquid chromatography using Mono S and Mono Q. DIF-A of pI 9.0 and DIF-B of pI 8.8 were obtained after a final purification with a Mono Q column, and both DIF gave a single peak with a molecular weight of approximately 51,000 determined by gel chromatography. NH2-terminal amino acid analysis of DIF-A showed a noticeable homology with murine leukemia inhibitory factor. Human DIF-A was found to induce maturation of human and murine leukemic cells into both macrophage-like cells with nitro blue tetrazolium reducing activity and phagocytic cells, but was found to suppress proliferation of these leukemic cells.     

Analytical cation-exchange chromatography to assess the identity,purity, and N-terminal integrity of human lactoferrin

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the innate host defense. The positively charged N-terminal domain of hLF mediates several of its activities by interacting with ligands such as bacterial lipopolysaccharide (LPS), specific receptors, and other proteins. This cationic domain is highly susceptible to limited proteolysis, which impacts on the affinity of hLF for the ligand. An analytical method, employing cation-exchange chromatography on Mono S, was developed to assess the N-terminal integrity of hLF preparations. The method, which separates N-terminally intact hLF from hLF species lacking two (Gly(1)-Arg(2)) or three (Gly(1)-Arg(2)-Arg(3)) residues, showed that 5-58% of total hLF in commercially obtained preparations was N-terminally degraded. The elution profile of hLF on Mono S unequivocally differed from lactoferrins from other species as well as homologous and other whey proteins. Analysis of fresh human whey samples revealed two variants of N-terminally intact hLF, but not limitedly proteolyzed hLF. Mono S chromatography of 2 out of 26 individual human whey samples showed a rare polymorphic hLF variant with three N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Ser(5)-) instead of the usual variant with four N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Arg(5)-Ser(6)-). In conclusion, Mono S cation-exchange chromatography appeared a robust method to assess the identity, purity, N-terminal integrity, and the presence of polymorphic and intact hLF variants.     

A single-column chromatographic system for the analysis and preparation of high mobility group proteins 1 and 2 and other chromosomal proteins using nondenaturing solvents

One-step chromatography on a Mono S column allows the purification of high mobility group (HMG) proteins 1 and 2 under nondenaturing conditions. Chromatography of HMG1 and -2 on Mono S can be achieved with three of the most widely employed extraction techniques for chromosomal proteins, 0.35 M sodium chloride, 0.74 M perchloric acid, and 0.4 N sulfuric acid. In each case HMG1 and -2 are purified away from the other chromosomal proteins, histone H1, and core histones, and are resolved into both their reduced and oxidized forms. Additionally histone H1 and the core histones are fractionated on Mono S, thus the entire complement of chromosomal proteins can be analyzed in a single rapid chromatographic step.     

The carbohydrate moiety of human platelet glycocalicin: the structures of the major Asn-linked sugar chains

Glycocalicin, a predominant glycoprotein on the human platelet surface, has been purified from a platelet suspension by means of sonication, ammonium sulfate precipitation and acid treatment followed by chromatography on columns of wheat germ agglutinin-Sepharose and Mono Q. Asparagine-linked (N-linked) oligosaccharides were released by hydrazinolysis, and then N-acetylated and reduced with NaBH4 or NaB3H4. The released carbohydrate chains were found to be of the complex-type from their interaction with immobilized lectin columns. The structures of the two major oligosaccharide-alditols separated by ion-exchange chromatography on a Mono Q column were investigated by means of methylation analysis, glycosidase digestion, and Smith periodate degradation, and they were assigned as typical di- and trisialylated complex-type oligosaccharide-alditols with two and three peripheral chains consisting of Gal-GlcNAc sequences, respectively.     

A galactose-specific lectin from the hemolymph of the pearl oyster, Pinctada fucata martensii

1. A lectin in the serum of Pinctada fucata martensii was purified by a combination of affinity chromatography on Sepharose 4B coupled with bovine submaxillary gland mucine, anion exchange chromatography on Mono Q and gel filtration on Superose 6. 2. The purified lectin was indicated to be homogeneous by polyacrylamide electrophoresis and rechromatography on Mono Q. 3. The purified lectin was approximately 440,000 in molecular weight and was composed of identical subunits with a molecular weight of approximately 20,000. 4. D-galactose and N-acetylgalactosamine gave a 50% inhibition of agglutination of horse erythrocytes by the lectin at 0.3 and 1.2 mM, respectively. 5. The antibody obtained from rabbit immunized with the purified lectin was monospecific to the lectin judged from the hemagglutination blocking test, immunoelectrophoresis and immunoblotting.