Purification and characterization of the endonuclease present in Physalia physalis venom

• 1.1. Physalia physalis nematocyst venom contains a DNase which has a non-specific endonucleolytic action.
• 2.2. This enzyme has an approximate molecular weight of 75,000 daltons.
• 3.3. The enzyme can cleave DNA over a wide pH range with an optimum near neutrality.
• 4.4. The enzyme is thermolabile and its activity can be stimulated by 80 nM NaCl or 10 mM MgCl₂.

     

Isolation, identification and characterization of a novel antioxidant protein from the nematocyst of the jellyfish Stomolophus meleagris

Reactive oxygen species (ROS) can damage the lipids, proteins and DNA when produced excessively in cells. Here, we describe the isolation and identification of a novel antioxidant protein named SmP90 from the nematocyst of jellyfish Stomolophus meleagris by 50% ammonium sulfate precipitation and gel filtration chromatography, superdex75. HPLC and SDS-PAGE analysis revealed >95% purity of SmP90 with apparent molecular weight of 90 kDa, approximately. The identification of SmP90 was confirmed by both N-terminal amino acids sequencing, with the sequences of NLDTPYCFYSGDYGG, and peptide mass fingerprint (PMF) analysis by MALDI-TOF-MS. However, no known protein had been completely matched in the database, which indicated that SmP90 might be a novel protein. The antioxidant assay result showed that it had strong superoxide anion radical-scavenging activity with the half-scavenging concentration (EC(50)) of about 16 μg/mL. Therefore, the present study is the first time to demonstrate a high efficient method of isolating a novel antioxidant protein from the nematocyst of jellyfish S. meleagris.     

Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Isolation and characterization of a protein C activator from the moccasin snake venom

The protein C activator detectable in the venom of the Southern Copperhead snake (Agkistrodon contortrix contortrix) was isolated by a combination of chromatofocusing on PBE-94 in the range pH9-7 and gel-filtration on Sephadex G-100 column. The peak protein C activator from Sephadex G-100 column appeared as double diffuse bands with apparent molecular weight of 37,700 and 31,400 after electrophoresis in the presence of sodium dodecylsulfate and 2-mercaptoethanol. The isolated enzyme does not clot human fibrinogen and when mixed with normal plasma generates activity of Protein C. It can be used for the measurement of protein C functional activity.     

Isolation and characterization of a convulxin-like protein from Crotalus durissus collilineatus venom

A convulxin (Cvx)-like protein was isolated from Crotalus durissus collilineatus venom by a combination of molecular exclusion and reversed-phase HPLC chromatographies. The molecular mass of the Cvx-like protein in the absence and presence of DTT was 78 kDa and 12-13 kDa, respectively. The Cvx-like protein consisted of two nonidentical polypeptide chains ( and ). The N-terminal amino-acid sequences of the and subunits were GLHCPSDWYAYDGHCYKIFNEEMNWED and GFCCPSHWSSYSRYCYKFFSQEMNWEDAEK, respectively, with both subunits having a high content of Glu, Ser, Cys, and Asp. The Cvx-like protein showed high homology with other venom C-type lectins, but had low hemagglutinating activity on intact and trypsinized erythrocytes. The Cvx-like protein stimulated insulin receptor phosphorylation and potentiated insulin secretion from isolated islets in the presence of sub- (2.8 mM) or supra-physiological (16.7 mM) glucose concentrations. These results suggest that the increase in insulin secretion induced by Cvx-like protein may be mediated by a protein tyrosine kinase-dependent pathway and may involve other membrane receptors, such as GP VI or Scr family proteins.     

Possible involvement of red cell membrane proteins in the hemolytic action of Portuguese Man-of-War toxin.

Glycophorin and the fragments isolated from trypsinizing intact rat, dog, sheep and human red blood cells (rbc's) neutralize the hemolytic action of the Portuguese Man-of-War venom. This action can be blocked by rabbit antisheep hemolysin and phytohemagglutinin, a lectin which preferentially binds to glycophorin. Concanavalin A, which binds to band-3 protein of rbc membranes, does not block the neutralizing action of rbc tryptic fragments or glycophorin. The concentrations of rat, dog, human and sheep glycophorin which half neutralize venom induced hemolysis are inversely and linearly proportional to the hemolytic sensitivities of these rbc's to the venom. These data implicate glycophorin as a possible binding site for the hemolytic component of the Portuguese Man-of-War venom.     

Isolation and partial characterization of a carbohydrate-binding protein from a nematode-trapping fungus

A developmentally regulated carbohydrate-binding protein from the capture organs of Arthrobotrys oligospora, not present on hyphae, was isolated and partially characterized. Surface structures of A. oligospora were radiolabeled with [125I]iodosulfanilic acid. The fungus was homogenized, and the homogenate was passed over an affinity column containing N-acetyl-D-galactosamine immobilized to Sepharose 6B. The bound radiolabeled protein was eluted from the affinity column with a glycine-hydrochloride buffer (pH 3.0), concentrated, and chromatographed on a metal chelate affinity gel containing Ca2+. EDTA was used as an eluant for the radiolabeled protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with autoradiography revealed a molecular weight for the carbohydrate- and cation-binding polypeptide of ca. 20,000.     

Isolation and partial characterization of a glial hyaluronate-binding protein

A glial hyaluronate-binding protein (GHAP) with an isoelectric point of 4.3-4.4 was isolated from human brain white matter. The 60-kDa glycoprotein appeared to be quite resistant to proteolysis, and comparison with GHAP from a viable glioma removed at surgery showed that the protein isolated from autopsy material was not a degradation product resulting from postmortem autolysis. The protein was localized immunohistochemically with mouse monoclonal and rabbit polyclonal antibodies in cerebral white matter. Only small amounts could be found in the gray matter. After enzymatic deglycosylation, an immunoreactive 47-kDa polypeptide was obtained. Two amino acid sequences of GHAP showed a striking similarity (up to 89%) with a highly conserved region of cartilage proteins (bovine nasal cartilage proteoglycan and rat and chicken link protein). However, the amino acid composition and other amino acid sequences suggested that there are also differences between brain-specific GHAP and cartilage proteins.     

Isolation and partial characterization of a cystine-rich basic heparin-binding protein from bovine platelets

We report the isolation and partial characterization of a so far unidentified basic platelet protein. Delipidated bovine platelets were extracted at pH 2.1. The extract was subjected to differential precipitation at pH 5.4-5.5 and by ammonium sulfate, then it was further purified by ion exchange chromatography on DEAE and CM cellulose columns in an urea containing medium. The major protein peak eluted from the CM cellulose column by NaCl gradient contained a protein in electrophoretically homogeneous form. It consists of a single polypeptide chain with an Mr of 28,000 as estimated by SDS PAGE. It was shown to be extremely rich in lysine and cystine and possessed a highly basic character (pI 9.8-10.1). On this basis the term cystine-rich basic protein (CRBP) was proposed for the new protein. Unlike some other low Mr basic proteins it did not bind calmodulin and troponin C, however, it showed significant heparin neutralizing activity.     

Isolation and partial characterization of a protein kinase NII from wheat germ chromatin

A protein kinase, type NII, has been purified from wheat germ chromatin. The enzyme, which uses both ATP and GTP as phosphoryl donors, catalyzes the phosphorylation of casein, phosvitin and E. coli RNA polymerase, but not of histone proteins. Polypeptide bands at 46 kDa, 37 kDa and 25 kDa were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autophosphorylation of the 25 kDa subunit was observed following incubation of the purified kinase with (-³²P)ATP and (-³²P)GTP.     

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 μg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.     

Isolation, purification and partial characterization of a 30-kDa chlorophyll-a/b-binding protein from spinach

A 30-kDa chlorophyll-a/b-binding protein was purified from photosystem II membrane fragments using Ca(2+)-chelating Sepharose 6B chromatography. The protein binds approximately four chlorophyll a molecules, one chlorophyll b molecule and carotenoids. Its 77-K fluorescence-emission spectrum exhibits a maximum at 680 +/- 1 nm. The protein has a high tendency to form a dimer in the presence of Ca2+.Ca2+ binding affects the low-temperature fluorescence-emission maximum, leading to a decrease in its intensity and a blue shift of 1 nm. Similar spectral changes were obtained in the presence of Mg2+, possibly indicating a common binding domain for both cations. We interpret these observations as cation-induced conformational changes of the protein, which were reversible upon subsequent incubation in EDTA. Evidence is presented for the involvement of carboxyl groups in the coordination sphere of the bivalent cations. The possible structural and functional role of the protein is discussed.     

Isolation and partial characterization of a cadmium-binding protein from Lumbriculus variegatus (Oligochaeta,annelida)

1. A low molecular weight, cadmium-binding protein has been isolated from Lumbriculus variegatus.2. The protein has an apparent molecular weight of 19 kD as estimated by Sephadex G-75 chromatography and is thought to be a dimer. It shows high absorbance at 254 nm and low absorbance at 280 nm.3. Amino acid analysis revealed a high content of cysteine (16.5%), and elevated amounts of aspartate (10.9%), serine (10.6%), glutamate (9.3%), glycine (11.7%), leucine (10%) and lysine (10%). The content of aromatic amino acids was low.4. After SDS-polyacrylamid gel electrophoresis three distinct bands with molecular weights of 19, 11.5 and 10.2 kDa, respectively, were found. The three bands are assumed to represent the dimer, the monomer and a partially carboxymethylated monomer.5. The protein is suggested to play an important role in the detoxification of cadmium.     

Isolation, stabilization, and characterization of a toxin from timber rattlesnake venom.

A rapid and convenient method for the purification of a toxin from timber rattlesnake, Crotalus horridus horridus, venom using carboxymethyl cellulose ion-exchange chromatography has been devised. The toxicity of this venom component is labile, but it is stabilized by the addition of 20+ V/V glycerol to the buffer solution. This toxin has a molecular weight of 15,000 +/- 700 as determined by SDS gel electrophoresis. It is both heat and protease resistant. Treatment of this venom component with 2-mercaptoethanol followed by G-50 Sephadex chromatography causes no loss of toxicity although incubation of the toxin with 1% SDS and 1% 2-mercaptoethanol prior to electrophoresis does result in a faster migrating species. The toxin does not affect neuromuscular junctions but does appear to act on the nervous system. It causes no local responses in mice.