Isolation of a ribosome-inactivating and abortifacient protein from seeds of Luff a acutangula

A glycoprotein with a molecular weight of 28 000 as estimated by SDS-polyacrylamide gel electrophoresis was isolated from seeds of Luffa acutangula using a procedure that involved acetone precipitation, ion exchange chromatography on CM Sepharose CL-6B and gel filtration on Sephadex G-50. In immunodiffusion studies it was found to be immunologically distinct from abortifacient proteins isolated from other members of the Cucurbitaceae family including Momordica charantia, Momordica cochinchinensis, Trichosanthes kirilowii and Trichosanthes cucumeroides. There were some differences in amino acid composition among the proteins although there was a gross similarity. The protein from L. acutangula was capable of inducing mid-term abortion in mice and inhibiting protein synthesis in a cell-free system.     

Purification and characterization of charantin,a napin‐like ribosome‐inactivating peptide from bitter gourd (Momordica charantia) seeds

Abstract: A peptide designated charantin, with a molecular mass of 9.7 kDa, was isolated from bitter gourd seeds. The procedure comprised affinity chromatography on Affi‐gel blue gel, ion‐exchange chromatography on Mono S and gel filtration on Superdex 75. The N‐terminal sequence of charantin exhibited marked similarity to that of the 7.8‐kDa napin‐like peptide previously isolated from bitter gourd seeds. Charantin inhibited cell‐free translation in a rabbit reticulocyte lysate system with an IC₅₀ of 400 nm , a potency lower than that of the previously reported small ribosome‐inactivating protein γ‐momorcharin (IC₅₀ = 55 nm ) which also exhibited an abundance of arginine and glutamate/glutamine residues. Charantin reacted positively in the N‐glycosidase assay, yielding a band similar to that formed by the small ribosome‐inactivating proteins γ‐momorcharin and luffin S.     

β-Trichosanthin: a new abortifacient protein from the Chinese drug,Wangua, Trichosanthes cucumeroides

β-Trichosanthin was a new abortifacient protein purified from the Chinese drug, Wangua, root tubers of Trichosanthes cucumeroides (Cucurbitaceae). The purification procedure involved acetone fractionation, ammonium sulfate precipitation, ion-exchange chromatography on CM-Sepharose and preparative agarose electrophoresis. Homogeneity of β-trichosanthin was demonstrated in immunoelectrophoresis, agarose electrophoresis and SDS-polyacrylamide gel electrophoresis. It had a molecular weight of 28 000 and no cysteine in its molecule. It differed from trichosanthin, a known abortifacient protein isolated from a related Chinese drug, Tianhuafen, root tubers of Trichosanthes kirilowii (Cucurbitaceae), in molecular weight, carbohydrate content, charge and amino acid composition. β-Trichosanthin was, however, immunochemically identical to trichosanthin and was about twice as potent as trichosanthin in inducing mid-term abortion in mice.     

Complete amino acid sequence of a subunit from rapeseed high molecular weight protein

A subunit (M, 15 600) from the high molecular weight protein from rapeseed was separated and isolated; its purity and homogeneity were ascertained. The subunit was cleaved with cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease. The fragments were separated and isolated by polyacrylamide gel electrophoresis, gel filtration, column chromatography on Dowex 1 × 2, and paper electrophoresis. The amino acid compositions of the intact subunit and different fragments obtained from enzymatic and chemical cleavages were determined. The subunit and its fragments were sequenced by manual Edman method. The phenylthiohydantoin amino acids obtained after each step were identified by thin-layer chromatography and ultraviolet spectroscopy. The complete amino acid sequence of the subunit consisting of 125 amino acid residues has been established by the overlapping method.     

Equinatoxin, a lethal protein from Actinia equina--I. Purification and characterization

A lethal protein from Actinia equina named equinatoxin has been purified by fractional precipitation with acetone and by gel chromatography. The purified protein is electrophoretically homogenous. It is highly basic with an isoelectric point of 12·5. It contains many aspartic acid residues, probably as the amide, no cysteine and little methionine. The total number of amino acid residues per molecule is estimated to be from 49 to 53, and the molecular weight is about 20,000. It is thermolabile, is hydrolyzed by pepsin and trypsin and can act as an antigen. Following intravenous injection into rats the ld₅₀ is 33·3 μg per kg.     

Erinacin, an antihaemorrhagic factor from the european hedgehog, Erinaceus europaeus

An antihaemorrhagic factor named erinacin was purified from the skeletal muscle extract of the European hedgehog, Erinaceus europaeus, by ammonium sulfate precipitation followed by various steps of ion-exchange (DEAE-cellulose), absorption chromatography (hydroxylapatite), and gel filtration (cellofine gel). A 625-fold purification was achieved with an overall yield of 19% antihaemorrhagic activity. The protein effectively inhibited the activity of Bothrops jararaca venom haemorrhagin and did not inhibit the enzymatic activity of trypsin and chymotrypsin. Erinacin is a large molecule (about 1,000,000 mol. wt). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of two subunits: one with an apparent mol. wt of 35,000 forming a larger subunit (350,000) by cross-linking with disulfide bridges, and a second with a mol. wt of 39,000 without disulfides. Dissociation of erinacin into its subunits resulted in complete loss of its antihaemorrhagic activity.     

ISOLATION OF A TRYPSIN-LIKE ENZYME FROM. STREPTOMYCES PAROMOMYCINUS (PAROMOTRYPSIN) BY AFFINITY ADSORPTION THROUGH KUNITZ INHIBITOR-SEPHAROSE

A trypsin-like enzyme has been isolated from the filtrate of a Streptomyces rimosus forma paromomycinus culture. Purification involves acetone fractionated precipitation, ultrafiltration on a Diaflo UM 10 membrane and affinity adsorption on to Kunitz pancreatic trypsin inhibitor linked to Sepharose. The trypsin-like enzyme (paromotrypsin) appears homogeneous by zone electrophoresis on gelatinized cellulose acetate. Specific activity toward Tos-Arg-OMe, calculated from amino acid analysis, is about 220 u mg^-1. The overall yield in activity is about 30%. The molecular weight of the trypsin-like enzyme, determined by gel filtration, is around 22,000–25,000 daltons. Electrophoretic migration on cellulose acetate strips indicates an isoelectric point around 8. Amino acid composition has been determined; the protein comprises about 210 residues on the basis of a single histidine residue per molecule. Paromotrypsin is unstable in acidic medium and is not stabilized by calcium ions. Enzymic activity towards Bz-Arg-OEt is not increased by the addition of calcium ion in contrast to the activating effect observed on bovine trypsin. Paromotrypsin is inhibited by TLCK and NPGB; it interacts with naturally occurring bovine trypsin inhibitors such as soya bean and Kunitz pancreatic inhibitors, but not with chicken ovomucoid. Proteolytic specificity, examined by hydrolysis of oxidized Kunitz pancreatic inhibitor and characterization of resulting peptides, seems similar to that of bovine trypsin.     

DETERMINATION OF THE COMPLETE AMINO ACID SEQUENCE OF BOVINE NEUROPHYSIN II

Bovine neurophysin II (BNP-II), a major neurohypophyseal hormone-binding protein in the cow, was isolated from acetone-dried posterior pituitary powder by gel filtration, ion exchange chromatography and preparative disc electrophoresis. The single-chain protein is composed of 97 amino acids, possesses a molecular weight of 10,029, and contains seven disulfide bonds. The fully S-alkylated protein, S-carboxamido-[C]- methylcysteine BNP-II ([C]BNP-II), was subjected to 80 automated cycles of the Edman degradation with positive identification of every amino acid residue through 65 cycles and many identifications through 80 residues. [C]BNP-II was digested with chymotrypsin and the peptides were isolated by gel filtration and purified by ion exchange chromatography. A 55-amino acid C-terminal chymotryptic peptide was sequenced through 46 residues by automated Edman degradations producing a 38-amino acid overlap with the sequence of [C]BNP-II. This established the sequences of the first 88 residues of the protein. A chymotryptic nonapeptide was sequenced from NH₂- to COOH-terminus by manual Edman degradations which established the sequence through residue 94. COOH-terminal analysis of aminoethylated BNP-II elucidated the tetrapeptide sequence and produced an overlap with the sequenced chymotryptic nonapeptide, completing the proposed amino acid sequence, which is supported by amino acid compositions of three other chymotryptic and two tryptic peptides.     

Characterization of a basic phospholipase A2-homologue myotoxin isolated from the venom of the snake Bothrops neuwiedii (yarará chica) from Argentina

A basic protein was isolated by CM-Sephadex C-25 chromatography from the venom of Bothrops neuwiedii from Argentina, and named B. neuwiedii myotoxin I. This protein exerted local myotoxic and edema-forming effects in mice, with potencies comparable to other myotoxins isolated from Bothrops spp. venoms. When injected by i.v. route at doses up to 4.7 mg/kg of body weight, the toxin was not lethal. In vitro, the toxin had no detectable phospholipase A₂ activity on egg yolk phospholipids. B. neuwiedii myotoxin I appeared as a homodimer in sodium dodecylsulphate–polyacrylamide gel electrophoresis, with a subunit molecular weight of 15 kD. Gel immunodiffusion revealed a pattern of partial antigenic identity between the newly isolated myotoxin and myotoxin II from Bothrops asper venom. The sequence of B. neuwiedii myotoxin I was determined for the first 40 amino acid residues, showing high homology to several class II phospholipase A₂ myotoxins of the Lys-49 family from crotalids. Altogether, results suggest that this toxin is a new member of the Lys-49 phospholipase A₂-homologues with myotoxic, cytolytic, and edema-inducing activities.     

Biochemical characterization of a toxin from Indian cobra (Naja naja naja) venom

A major toxic component was isolated from the venom of Indian cobra (Naja naja naja) by ammonium sulfate fractional precipitation followed by carboxymethyl cellulose column chromatography and Sephadex gel filtration. This component constituted 2% of the venom and produced a block of neuromuscular transmission in nerve muscle preparations. Three other toxic fractions comprising 3% of the venom were also detected. The major toxic component was homogeneous on starch and polyacrylamide gel electrophoresis and on rechromatography on CM-cellulose. Its molecular weight was approximately 6300. This toxin contained 61 amino acid residues including 8 half-cystine residues whereas alanine, methionine and phenylalanine were totally absent. Its ld₅₀, as determined by i.p. injection in mice, was 0·2 mg per kg body weight. The fraction did not possess any enzymatic, hemolytic or hemagglutinin activities of crude venom but showed a close resemblance to the major neurotoxin of Formosan cobra venom.     

A napin‐like polypeptide with translation‐inhibitory,trypsin‐inhibitory,antiproliferative and antibacterial activities from kale seeds

Abstract: A heterodimeric napin‐like polypeptide with translation‐inhibiting and antibacterial activities has been isolated from kale seeds. The purification procedure entailed ion‐exchange chromatography on dielthylaminoethyl (DEAE)‐cellulose, 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 napin‐like polypeptide was unadsorbed on DEAE‐cellulose but adsorbed on Affi‐gel blue gel and Mono S. Its 7‐kDa large subunit differs in N‐terminal amino acid sequence from the 4‐kDa small subunit. The polypeptide inhibited translation in the rabbit reticulocyte lysate system with an IC₅₀ of 37.5 nm . This activity was preserved between pH 5 and pH 11, and between 10 and 40 °C. It fell to a low level at pH 3 and pH 13 and at 70 °C. Antibacterial activity against Bacillus, Megabacterium, and Pseudomonas species and antiproliferative activity against leukemia L1210 cells were observed. However, the polypeptide did not exert antifungal, ribonuclease, or protease activity.     

Isolation,characterization and primary structure of two γ- variants from ostrich pituitary glands

Two γ-LPH variants have been isolated from ostrich pituitary glands using acid acetone extraction, salt fractionation, ion exchange and gel permeation chromatography and HPLC. The two fractions appeared homogeneous on PAG-IEF (pI = 4.7) and both displayed an alanine N-terminal residue. Amino acid composition and fragmentation data for these two peptides are in agreement with that expected for the N-terminal 44 and 46 amino acid residues in ostrich β-LPH, with corresponding molecular masses of 4911 and 5154 respectively. The molecular mass of the smaller variant was confirmed by means of sedimentation equilibrium centrifugation to be 4717. The two variants displayed lower lipolytic potencies than the corresponding peptides from other species.     

Reconstitution of rapeseed high molecular weight protein from isolated subunits

The high molecular weight protein was isolated from rapeseed and characterised. Six subunits were isolated in SDS (0.01%) solution on polyacrylamide gel electrophoresis and by gel filtration on Sephadex G-100. Reassociation by removing SDS by co-dialysis, against 10mM sodium phosphate buffer (pH 7.9) was done and the yield was about 90%. The reconstituted protein was indistinguishable from the intact protein in all respects. The subunits isolated from the native protein and the reconstituted protein were found to have identical molecular weights and N-terminal amino acids. No disulphide bonds were observed in the subunit association. Amino acid analysis of the proteins and the six subunits was performed and the number of each amino acid residue calculated.     

Solid phase synthesis and biological activity of mast cell degranulating (MCD) peptide: a component of bee venom

Mast cell degranulating (MCD) peptide, a 22 amino acid residue basic peptide from bee venom, was synthesized by stepwise solid phase synthesis on a benzhydrylamine resin support. N^α-t-butyloxycarbonyl and benzyl type side chain protection was used. The two disulfide bridges were formed selectively by using S-acetamidomethyl protection for the cysteine residues in positions 5 and 19 and S-methylbenzyl protection for the cysteine residues in positions 3 and 15. Crude synthetic MCD peptide was obtained following deprotection and cleavage from the resin by the low/high HF method. The peptide was isolated in pure form by ion exchange chromatography and gel filtration. The final product has physical, chemical, and biological properties identical with those reported for the natural product. The synthetic strategy utilized for MCD peptide will facilitate the availability of structurally similar analogs for evaluating antihistaminic and anti-inflammatory activities.     

Isolation and characterization of a toxic protein from Canavalia ensiformis (jack bean) seeds,distinct from concanavalin A

A toxic protein present in the crude extract of Canavalia ensiformis seeds induces within 24 hr dyspnoea, ataxia, hypothermia, coma, tonic convulsions and death in mice injected i.p. with 100–200 mg of protein per kg. The toxin was separated from concanavalin A by affinity adsorption of the latter on Sephadex G-100, and further purified by sequential fractionation by (a) removal of polysaccharides with 30% v/v ethanol; (b) ammonium sulfate precipitation at 0.35–0.55 saturation and (c) DEAE-cellulose chromatography. The purified toxin, with an ld₅₀ of 2–5 mg of protein/kg mouse, is unstable. Gel-filtration of the purified toxin on Bio-Gel P-200 showed a single protein peak with a molecular weight corresponding to 88,000 daltons. Polyacrylamide gel electrophoresis of this material indicated the presence of two small contaminants. A central convulsive effect of the toxin can be proposed since no effects were found on isolated pharmacological preparations. The name CANATOXIN is suggested for this new toxic protein.     

Complete amino acid sequence of arachin subunit of molecular weight 21000

A subunit of molecular weight 21 000 from arachin, the major peanut protein, was isolated in pure form and primary structure was determined. The subunit was fragmented with CNBr, trypsin, and NBS; the fragments were separated and isolated by PAGE, gel filtration, Dowex treatment, and paper electrophoresis, and Edman degradation on each fragment, including the intact subunit, was performed. The PTH-amino acids thus obtained were identified by UV spectroscopy and TLC. The complete sequence of 176 residues was established by overlapping technique.     

Processed forms of neuroendocrine proteins 7B2 and secretogranin II are found in porcine pituitary extracts

The complete structure of the novel polypeptide 7B2 recently deduced from cDNA clones has been reported to be highly conserved in a variety of species. The deduced amino acid sequence of the mature protein is predicted to be 185 or 186 amino acids long. While its biological role is still unknown, its occurrence in neuroendocrine secretory granules has been largely documented. This report shows: (i) that the protein, isolated from a large quantity of porcine pituitary glands, does not correspond to the full predicted cDNA structure but, on the contrary, to a truncated form; (ii) that the latter could arise from proteolytic cleavage at position ISO following pairs of basic residues; (iii) that it contains an extra residue at position 100 which is absent in the cDNA sequence; and, finally, (iv) that it displays a higher than expected molecular weight on SDS-polyacrylamide gel electrophoresis. In addition, a copurifying peptide was identified as an NH₂-terminal related fragment of the secretogranin II molecule. Protein sequencing of the latter demonstrates (i) that the correct amino terminus of mature porcine secretogranin II is an Ala residue and not the previously proposed Gin residue and (ii) that this fragment could also arise from proteolytic cleavage at a pair of basic residues located within the secretogranin II sequence.     

Purification and properties of a toxin from the South African sea anemone, Pseudactinia varia

A comparison was made of the hemolytic potency of aqueous extracts prepared from five species of intertidal sea anemones from the coast of South Africa. The active agent in an extract of Pseudactinia varia was purified by ammonium sulfate precipitation, gel permeation chromatography and isoelectric focusing. The hemolytic toxin, termed variolysin, is a protein having a molecular weight of 19,500 and an isoelectric pH of 9.8. It retained appreciable activity after heating to 70 degrees for 40 min. Amino acid analysis revealed that it lacked methionine and cysteine. Its hemolytic activity was inhibited by sphingomyelin. The properties of variolysin show that it is broadly similar to cytolytic toxins isolated from a number of other anthozoans.     

Some chemical properties of the venom of the rattlesnake,Crotalus viridis helleri

The venom of the Southern Pacific Rattlesnake, Crotalus viridis helleri, was separated into three lethal and several non-lethal peaks by gel filtration. Peak I was a protein having a mol. wt of ca. 100,000 and an intravenous ld₅₀ of 0.58 mg/kg. Peak II had a mol. wt of ca. 30,000 and a ld₅₀ of 1·7 mg/kg. Peak III, the peptide, had a mol. wt of ca. 6000 and a ld₅₀ of 1·96 mg/kg and moved as a cation on strip and polyacrylamide gel electrophoresis. On ion exchange chromatography the peptide was resolved into three lethal fractions. The major fraction, C, was a basic polypeptide containing 43 amino acid residues with six half cystine residues. Its mol. wt was 4990, as calculated from its sequence, 7600 as estimated from Sephadex G-50 gel filtration and 5200 by SDS- disc gel electrophoresis. The differences are being studied. Analysis showed the peptide contained almost 20% lysine. On sequencing, the most basic amino acid residues were distributed in the N-terminal and C-terminal parts. The middle part was rather hydrophobic.