Purification and amino-acid sequence of a nerve growth factor from the venom of Vipera russelli russelli.

Nerve growth factor (NGF) was purified from the venom of Vipera russelli russelli by Sephadex G-50 gel filtration, S-Sepharose column chromatography and Blue-Sepharose CL-6B column chromatography. The purified NGF was found to be a glycoprotein, whose apparent molecular mass was estimated to be about 17.5 kDa by SDS-PAGE. The amino-acid sequence was determined by a combination of conventional methods. The V. r. russelli NGF was composed of 117 amino-acid residues with one residue, Asn-21, being N-linked glycosylated and the molecular mass of its protein portion was calculated to be 13,280 Da.     

Isolation and characterization of a novel postsynaptic/cytotoxic neurotoxin from Daboia russelli russelli venom.

A postsynaptic neurotoxin was purified from Daboia russelli russelli venom using gel filtration, ion-exchange chromatography and reverse-phase high-performance liquid chromatography. The N-terminal sequence, molecular mass and pharmacological activities of the neurotoxin/cytotoxin indicate that it is a short-chain neurotoxin like that found in Elapid venom. This is the first report on the presence of such a postsynaptic neurotoxin from D. r. russelli venom.     

Analysis of Vipera russelli venom using polyclonal antibodies prepared against its purified toxic phospholipase A2 VRV PL-V.

Vipera russelli venom induces predominantly neurotoxic, myotoxic necrotic and hemorrhagic symptoms in experimental animals and has several hydrolytic enzyme activities. In this study, V. russelli venom is characterized both as a PLA2 and as a toxin. Anti PL-V Ig (antibodies to a toxic phospholipase A2 VRV PL-V of V. russelli venom) nullifies the toxicity of whole V. russelli venom to a great extent. The neurotoxic symptoms vanish completely in the presence of anti PL-V Ig. The cross reacting components of whole V. russelli venom were removed by precipitating them from whole venom by the addition of anti PL-V Ig. The non-cross reacting components present in the supernatant were checked for toxicity. There was a significant reduction in toxicity. The LD50 value of the supernatant had increased from 4.1 mg/kg body weight to 11.7 mg/kg body weight and it showed about 34% of the total venom phospholipase A2 activity. It had edema forming, hemorrhagic and hemolytic activity but failed to induce neurotoxic, anticoagulant and myotoxic effects.     

Purification and characterization of an organ specific haemorrhagic toxin from Vipera russelli russelli (Russell's viper) venom

A haemorrhagic toxin (VRR-12) from Vipera russelli russelli (Russell's viper) venom has been purified by ion-exchange chromatography on CM-Sephadex C-50 followed by size-exclusion HPLC to electrophoretically homogeneous state. It is a 12 kDa single polypeptide having 1 mole of Zn+2 ion. This toxin induces intense intestinal haemorrhage and to a lesser extent skeletal muscle haemorrhage in mice. It does not show detectable proteolytic and esterolytic activity with selected substrates under specified conditions, haemolytic and phospholipase activity. When VRR-12, preincubated with bivalent antiserum against Saw-scaled and Russell's viper venom or EDTA was injected, haemorrhagic activity was not reduced, on the other hand preincubation with phenylmethyl sulphonyl fluoride reduced the activity markedly. Biodistribution studies with 125I VRR-12 show that haemorrhagic manifestation by this toxin is not a direct function of the fraction of the totally administered toxin distributed to that tissue.     

Purification of the muscarinic acetylcholine receptor from porcine brain

The muscarinic acetylcholine receptor of porcine cerebrum has been purified to apparent homogeneity by affinity chromatography, with conjugated 3-(2'-aminobenzhydryloxy)tropane (ABT) as described previously (Haga, K., and Haga, T. (1983) J. Biol. Chem. 258, 13575-13579). In a single step purification using 900 ml of digitonin/cholate-solubilized preparations and 300 ml of the ABT-agarose gel, we obtained, in a yield of 10-15%, more than 250 pmol of muscarinic receptors which bind [3H]N-methylscopolamine with a specific activity of 1,000-5,000 pmol/mg of protein (1,000-5,000-fold purification). The muscarinic receptors eluted from the ABT-agarose gel with 0.1 mM atropine were adsorbed to hydroxylapatite and then recovered as a concentrated solution. Muscarinic receptors were further purified by rechromatography with the same gel or by gel permeation high pressure liquid chromatography. The amino acid composition of the purified receptor was determined, and the specific activity of the purified preparation was estimated to be 13,100 pmol/mg of protein on the basis of amino acid composition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptors with or without radioiodination revealed a single, major band with an apparent Mr of 70,000 either by silver staining or radioautogram. The major band corresponded to the band which specifically bound [3H]propylbenzylcholine mustard (irreversible muscarinic ligand). The purified receptor showed essentially the same specificity for muscarinic ligands as unpurified receptors.     

Purification and characterization of a low molecular weight transforming growth factor from the urine of melanoma patients

A low Mr human transforming growth factor (TGF) present in melanoma patients' urine has been purified approximately 200,000-fold to apparent homogeneity. Initial purification of an acid-soluble fraction of urine was achieved by Bio-Gel P-30 gel filtration chromatography in 1 M acetic acid. TGF activities were demonstrated in the Mr ranges of 30,000 and 6,000-10,000. These competed with epidermal growth factor (EGF) for binding to A431 membrane receptors and induced anchorage-independent growth of untransformed fibroblasts. The low Mr TGF activity obtained from P-30 chromatography was purified to apparent homogeneity by two sequential reverse-phase high performance liquid chromatography steps with a mu Bondapak C18 column first using a linear gradient of acetonitrile going from 0-60% in 120 min and then by rechromatography of the activity over the same column using a shallower gradient of acetonitrile going from 20-40% in 160 min. The isoelectric point of the melanoma patient-derived urinary TGF was determined to be 6.2, which is distinct from that for human EGF. Amino acid composition analysis of the purified urinary TGF (uTGF) revealed that it is composed of at least 42 amino acid residues with a minimum estimated Mr of 4,545. Compositional analysis further revealed distinct similarities and differences between the uTGF, human EGF and TGFs secreted by various transformed human and rodent cell lines.     

Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native alpha 2 beta 2 insulin receptor subunit complex

The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing (0.1% SDS) or nondenaturing (0.1% Triton X-100) conditions. Pretreatment of 32P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the alpha 2 beta 2 insulin receptor complex (Mr 400,000) into the monomeric 95,000 beta subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the alpha 2 beta 2 heterotetrameric complex with essentially no alpha beta heterodimeric or free monomeric beta subunit species present. This suggests that the insulin receptor can reoxidize into the Mr 400,000 complex after the removal of DTT by gel filtration chromatography. Surprisingly, these apparently reoxidized insulin receptors were also observed to be functional with respect to insulin binding, albeit with a 50% decrease in affinity for insulin and insulin stimulation of the beta subunit autophosphorylation. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT followed by incubation with excess N-ethylmaleimide prior to gel filtration chromatography in 0.1% Triton X-100. Under these conditions the insulin receptors migrated as the Mr 400,000 alpha 2 beta 2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vipoxin. A protein from Russell's viper venom with high affinity for biogenic amine receptors

We have purified a small, basic protein with high affinity and selectivity for biogenic amine receptors to apparent homogeneity from the venom of Russell's viper (Vipera russelli). This protein, which we designate "vipoxin," has Mr = 13,000, and appears to exist in solution as a single polypeptide chain. It may contain 2 atypical amino acids. Vipoxin inhibits in a dose-dependent manner the binding of 3H-ligands to biogenic amine receptors, with apparent Ki values of 3 nM at alpha 1-adrenergic receptors, 5 nM at alpha 2-adrenergic receptors, 15 nM at dopamine receptors, and 32 nM at serotonin receptors. At concentrations up to 1 microM, vipoxin is inactive at beta-adrenergic, histamine, nicotinic cholinergic, muscarinic cholinergic, adenosine, gamma-aminobutyric acid, benzodiazepine, or opiate receptor binding sites. The effect of vipoxin is essentially irreversible over 20 h at alpha 1- and alpha 2-adrenergic receptors and serotonin receptors and is only slightly reversible at dopamine receptors. Norepinephrine protects alpha-adrenergic receptors from inhibition by vipoxin, while dopamine does not. Vipoxin has no protease activity but does have phospholipase A2 activity, which cannot account for its action on receptors, since receptor binding is assayed in the presence of 1 mM CoSO4 which completely and selectively inhibits the phospholipase activity. Other phospholipases A2 in the same venom lack vipoxin's action on receptors. In physiologic experiments, vipoxin behaves as an agonist at alpha 2-adrenergic receptors in the rat vas deferens and is over an order of magnitude more potent than norepinephrine itself. At alpha 1-adrenergic receptors, it is neither a simple agonist nor an antagonist, but selectively potentiates norepinephrine. Vipoxin may be a useful tool for biogenic amine receptor characterization.     

Two L-amino acid oxidase isoenzymes from Russell's viper (Daboia russelli russelli) venom with different mechanisms of inhibition by substrate analogs

Two isoforms, L(1) and L(2), of L-amino acid oxidase have been isolated from Russell's viper venom by Sephadex G-100 gel filtration followed by CM-Sephadex C-50 ion exchange chromatography. The enzymes, with different isoelectric points, are monomers of 60-63 kDa as observed from size exclusion HPLC and SDS/PAGE. Partial N-terminal amino acid sequencing of L(1) and L(2) showed significant homology with other snake venom L-amino acid oxidases. Both the enzymes exhibit marked substrate preference for hydrophobic amino acids, maximum catalytic efficiency being observed with L-Phe. Inhibition of L(1) and L(2) by the substrate analogs N-acetyltryptophan and N-acetyl-L-tryptophan amide has been followed. The initial uncompetitive inhibition of L(1) followed by mixed inhibition at higher concentrations suggested the existence of two different inhibitor-binding sites distinct from the substrate-binding site. In the case of L(2), initial linear competitive inhibition followed by mixed inhibition suggested the existence of two nonoverlapping inhibitor-binding sites, one of which is the substrate-binding site. An inhibition kinetic study with O-aminobenzoic acid, a mimicking substrate with amino, carboxylate and hydrophobic parts, indicated the presence of three and two binding sites in L(1) and L(2), respectively, including one at the substrate-binding site. An inhibitor cross-competition kinetic study indicated mutually excluding binding between N-acetyltryptophan, N-acetyl-L-tryptophan amide and O-aminobenzoic acid in both the isoforms, except at the substrate-binding site of L(1). Binding of substrate analogs with different electrostatic and hydrophobic properties provides useful insights into the environment of the catalytic sites. Furthermore, it predicts the minimum structural requirement for a ligand to enter and anchor at the respective functional sites of LAAO that may facilitate the design of suicidal inhibitors.     

Identification, isolation and purification of neurotoxic phospholipases A2 from Vipera russelli venom using polyclonal antibodies.

All the neurotoxic phospholipases A2 present in whole Vipera russelli venom were precipitated selectively from other non-neurotoxic phospholipases A2 and non-phospholipases A2 fractions using antibodies (anti PL-V Ig) raised against one of the purified neurotoxic phospholipases A2 (VRV PL-V). These neurotoxins were identified and isolated in their homogeneous form by chromatographic and electrophoretic methods. The present report of selective isolation and purification of all the neurotoxic phospholipases A2 of V. russelli venom is first of its kind.     

Isolation and characterization of biologically active and inactive cholecystokinin-octapeptides from human brain

Cholecystokinin-like immunoreactivity (CCK-LI) in 0.9 kg human brain was extracted by 2% trifluoroacetic acid at 4 degrees C. Sephadex G50 gel filtration of crude extract revealed one main molecular form of CCK, detected by a carboxy-terminal antibody (5135), that eluted in the position of CCK8. When the CCK-LI in the extract was purified by affinity chromatography using another carboxyl-terminal CCK antibody followed by several steps of reverse phase high pressure liquid chromatography (HPLC), a component was isolated that was found by sequence analysis to be identical to the carboxyl-terminal CCK-octapeptide of porcine CCK33, isolated from intestinal mucosa, and to CCK-octapeptide, isolated from sheep brain. This component possessed comparable biological potencies to synthetic sulfated CCK8 in eliciting amylase release and in competitively displacing radioiodinated CCK33 from isolated mouse pancreatic acini. Furthermore, it exhibited a similar binding characteristic to CCK8 in binding to specific receptors on mouse brain cortical particulate preparations. On high pressure liquid chromatography another minor, earlier eluting immunoreactive peak was observed, which had the same amino acid composition and sequence as CCK8. These findings suggested that this material was oxidized CCK8. This earlier eluting component, exhibiting CCK8-like immunoreactivity, did not induce amylase release from acini and had no or minimal effect in inhibiting tracer CCK33 binding to receptors on isolated acini or on mouse brain cortical particulate preparations at the concentrations tested.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.     

Purification and characterization of fatty acid-binding protein from rat kidney

We detected the presence of a fatty acid-binding protein (FABP) in rat kidney cytosols. This protein was eluted and purified 9.3-fold by sequential gel filtration and anion-exchange chromatography. Homogeneity was shown by a single band on polyacrylamide gel with a molecular weight of about 15,500. It had an optimum binding pH of 7.4. The binding of palmitate to the protein was saturable. Examination of fatty acid binding revealed the presence of a single class of fatty acid-binding sites. The apparent dissociation constant was 1.0 microM and the maximal binding capacity was 48 nmol/mg of protein. This protein showed similar binding characteristics for palmitate, oleate, and arachidonate. Rabbit antibody to this cytosolic FABP gave a single precipitin line with the antigen and selectively inhibited [14C]palmitate binding to the protein.     

Neuropharmacological studies on the venom of Vipera russelli.

Neuropharmacological studies have been conducted on the venom of V. russelli on experimental animals. The venom was found to produce alteration in general behaviour pattern, reduction in spontaneous motility, hypothermia, potentiation of pentobarbitone hypnosis, analgesia, reduction in exploratory behaviour pattern, muscle relaxant action, and suppression of aggressive behaviour. The venom caused a significant increase in brain GABA content in mice. The observations are suggestive of a potent CNS-depressant action of V. russelli venom.     

Isolation and partial characterization of an amphiphilic 56-kDa fatty acid binding protein from rat renal basolateral membrane

We have identified a 56-kDa fatty acid binding protein in rat renal basolateral membrane and purified it by extraction in nonionic detergent (Triton X-100), followed by gel filtration, DEAE-cellulose chromatography, and affinity chromatography. The purified protein was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration, polyacrylamide gel electrophoresis, and oleate-Sepharose 4B chromatography. Its molecular mass was estimated to be 56 kDa by SDS-polyacrylamide gel electrophoresis. The protein showed optimal binding activity at pH 7.5 and 37 degrees C. The apparent Kd for palmitic acid was 0.79 microM. It was immunologically clearly distinct from renal cytosolic fatty acid binding protein.     

Purification and characterization of a low molecular weight zinc binding protein from human placenta

A low molecular weight, native zinc binding, cytosolic protein (LMZP) has been isolated, purified and characterized from human normal term placenta. Gel filtration of heat treated placental cytosol after sequential acetone precipitation (80% ppt) revealed a major zinc binding protein in the range of low molecular weight. This partially purified zinc binding fraction was further fractionated on DEAE-Sephadex A-25. The zinc was eluted in one of the three peak fractions. Further, the purity of zinc binding protein was confirmed on fast protein liquid chromatography (FPLC). The purified placental LMZP was homogenous on SDS-polyacrylamide gel electrophoresis with a single band. Ultraviolet (UV) spectrum of LMZP showed an absorption maximum at 257 nm which disappeared at pH 2. Molecular weight of LMZP as determined by gel chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis was 6 kDa. It was calculated that 1 g atom of zinc was bound to 1 mole of the LMZP. Unlike in classical metallothionein, the amino acid composition of placental LMZP revealed the presence of aromatic amino acids, lower content of cysteine and higher content of histidine, glutamic acid and aspartic acid (10, 9 and 5 residues/mole, respectively).     

几种药品抗圆斑蝰蛇毒作用的比较

目的探讨几种药品抗圆斑蝰蛇毒的作用。方法将蝰蛇毒造模后的小鼠随机分组,给予美兰、葛根素、葛根总黄酮和生理盐水于小鼠皮下注射,观察24 h内各组动物各个时间段内的局部症状以及呼吸、食欲、活动等全身症状,以动物的死亡数累计各组的实验结果。结果美兰在规定的4 h内对小鼠的保护率高于葛根总黄酮与葛根素。结论美兰有较强的抗蝰蛇毒作用。     

Purification and characterization of prolactin receptors from rat ovary

Prolactin receptors were purified to homogeneity by two affinity chromatography steps using concanavalin A-Sepharose and human growth hormone (hGH)-Sepharose. The purified receptors showed specificity and high affinity for lactogenic hormones and a binding capacity of 20 nmol/mg of protein. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis revealed that purified receptors were composed of two major protein bands of Mr = 41,000 and 88,000, which were identified as radioactive bands by binding of 125I-hGH to blotted renatured receptors and by autoradiogram of free and 125I-radiolabeled purified receptors. Autoradiographic analysis of SDS-polyacrylamide gel electrophoresis of cross-linked 125I-hGH-receptor or hGH-125I-iodinated receptor complexes showed two radioactive bands of Mr = 63,000 and 106,000. Analysis of the free receptors by high performance liquid chromatography using Superose 12 revealed two peaks of binding activity for 125I-hGH eluting in the positions of Mr approximately 150,000 and 250,000. After cross-linking with 125I-hGH, SDS-polyacrylamide gel electrophoresis analysis revealed that both peak fractions contained two binding species with Mr = 63,000 and 106,000. Chromatography of 125I-hGH-receptor complexes showed two radioactive fractions with approximate Mr approximately 180,000 and 300,000. The treatment of 125I-iodinated receptors with SDS and reductant resulted in the dissociation of the higher Mr form into the lower Mr form upon gel filtration. Chromatofocusing of free receptors showed three isoforms with pI 4.0, 5.0, and 5.3. These results indicate that detergent-solubilized prolactin receptors appear to be aggregated forms of holoreceptor containing two binding species of Mr = 41,000 +/- 2,000 and 88,000 +/- 3,000.     

Solubilization of the Neuropeptide Y Receptor from Rat Brain Membranes

The neuropeptide Y (NPY) receptor was solubilized from rat brain membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The binding of 125I-NPY to CHAPS extracts was protein, time, and temperature dependent. Unlabeled NPY and the related peptides peptide YY (PYY) and pancreatic polypeptide inhibited 125I-NPY binding to solubilized receptors with relative potencies similar to those seen with membrane-bound receptors: NPY greater than PYY much greater than pancreatic polypeptide. Scatchard analysis of equilibrium binding data showed the CHAPS extracts to contain a single population of binding sites with a KD of 3.6 +/- 0.4 nM (mean +/- SEM) and a Bmax of 5.0 +/- 0.2 pmol/mg of protein. In addition the 125I-NPY binding to the soluble receptor was not inhibited by guanosine-5'-O-(3-thiotriphosphate), in contrast to the GTP sensitivity displayed by the membrane-bound receptor. Gel filtration chromatography using Sepharose 6B revealed a single peak of binding activity corresponding to a Mr of approximately 67,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after chemical cross-linking revealed a single band at Mr 62,000. After solubilization and gel chromatography a 50- to 100-fold purification of the NPY receptor was obtained.     

Role of Riboflavin and Certain Amino Acids in the Excystment of Schizopyrenus russelli*

SYNOPSIS. A simple medium, consisting of riboflavin and a mixture of L-glutamic acid, L-tryptophan, L-isoleucine, L-serine, and L-proline has been shown to induce rapid and mass scale excystment in Schizopyrenus russelli. Whereas percentage excystment was found to depend on the concentration of riboflavin and amino acids, more than 80% of cysts were found to excyst within 4 hr, when adequate amounts of these were supplied. Several individual amino acids, particularly L-glutamic acid, L-tryptophan, and L-proline, also supported considerable excystment, but riboflavin was always indispensable.