Phospholipase isoenzymes from Naja naja venom. I. Purification and partial characterization

Six phospholipase A-containing fractions were isolated from cobra (Naja naja) venom by ion-exchange and gel filtration chromatography. Each fraction was homogeneous by immunological criteria and comprised more than 1 phospholipase isoenzyme, their total number amounting to 14. The molecular weights of the isoenzymes ranged from 11000 to 24000. None of them contained free SH groups. The six fractions exhibited similar specific activities.     

Phosphatase of regenerating liver: a novel target for cancer therapy

Introduction: Phosphatases of regenerating livers (PRLs) are novel oncogenes that interact with many well-established cell signaling pathways that are misregulated in cancer, and are known to drive cancer metastasis when overexpressed.

Areas covered: This review covers basic information of the discovery and characteristics of the PRL family. We also report findings on the role of PRL in cancer, cell functions and cell signaling. Furthermore, PRL's suitability as a novel drug target is discussed along with current methods being developed to facilitate PRL inhibition.

Expert opinion: PRLs show great potential as novel drug targets for anticancer therapeutics. Studies indicate that PRL can perturb major cancer pathways such as Src/ERK1/2 and PTEN/PI3K/Akt. Upregulation of PRLs has also been shown to drive cancer metastasis. However, in order to fully realize its therapeutic potential, a deeper understanding of the function of PRL in normal tissue and in cancer must be obtained. Novel and integrated biochemical, chemical, biological, and genetic approaches will be needed to identify PRL substrate(s) and to provide proof-of-concept data on the druggability of the PRL phosphatases.     

Phospholipase isoenzymes from Naja naja venom —I. Purification and partial characterization

Six phospholipase A-containing fractions were isolated from cobra (Naja naja) venom by ion-exchange and gel filtration chromatography. Each fraction was homogeneous by immunological criteria and comprised more than 1 phospholipase isoenzyme, their total number amounting to 14. The molecular weights of the isoenzymes ranged from 11000 to 24000. None of them contained free SH groups. The six fractions exhibited similar specific activities.     

灵芝多糖的提纯、组成及活性研究

目的探讨灵芝多糖的分离纯化、单糖组成以及抗肿瘤活性。方法采用热水提取,Sevag法去蛋白质,乙醇分级沉淀,DEAE-32离子交换色谱法从灵芝子实体粉中分离得到灵芝多糖。用SephadexG-100凝胶柱色谱法判断其纯度及相对分子质量,用薄层色谱法及气相色谱法鉴定其单糖组成,溴化四唑蓝法测定其体外抗肿瘤活性。结果从灵芝子实体粉中分离得到两种粗多糖GLPⅠ、GLPⅡ。GLPⅠ进一步分离得到两种均一多糖GLPH1、GLPH2。GLPH1糖苷键为β型,相对分子质量为45000。GLPH1由葡萄糖、半乳糖和痕量甘露糖、岩藻糖组成。GLPH1对KB细胞、BGC细胞、B16细胞的增殖具有一定的抑制作用。结论灵芝多糖GLPH1可望开发成抗肿瘤药或辅助抗肿瘤药。     

Isolation of a soluble cadmium-binding protein from pulmonary macrophages

A soluble cadmium-binding protein, with properties similar to metallothionein, has been isolated from rabbit alveolar macrophages. The macrophages were cultured in Medium 199 with Earle's salts for 24 hr in the presence of 10 μm CdCl₂ and carrier-free ¹⁰⁹Cd as a tracer. The isolation procedure began with application of a 100,000 g cell supernatant to a column of Sephadex G-75 Fine. The fraction containing the greatest amount of cadmium was eluted at a relative elution volume, $\text{V}{}_{\mathrm{<mtext>e</mtext>}}\text{V}{}_{\mathrm{<mtext>o</mtext>}}$, of 1.87. A molecular weight determination performed following Sephadex chromatography indicated that the apparent molecular weight of the impure protein was approximately 11,000. The fractions containing cadmium were pooled and purification procedures were applied, including acetone fractionation, DEAE-cellulose chromatography, and polyacrylamide gel electrophoresis. DEAE-Cellulose chromatography following acetone fractionation indicated the presence of two forms of metalloprotein as has been demonstrated previously in the isolation of cadmium-thionein from liver and kidney. The two forms of metalloprotein were subjected to polyacrylamide gel electrophoresis and, although separation was incomplete, bands obtained corresponded to those typically observed in rat liver.     

Isolation of two functionally different kininogens from human plasma--separation from proteinase inhibitors and interaction with plasma kallikrein

A high (HMW) and a low (LMW) molecular weight kininogen were isolated in highly purified form from human plasma, using QAE-Sephadex chromatography, followed by ammonium sulfate precipitation, gel filtration through Sephadex G-200, re-precipitation with ammonium sulfate, CM-Sephadex and SP-Sephadex chromatography. The initial preparative step was done at room temperature and the remaining procedures at 4°. In aqueous media, the apparent molecular size of the HMW-kininogen was about four times the size of the LMW-kininogen (200,000 vs 50,000). During the process of purification, proteinase inhibitors were separated from the two kininogens: α₁-antitrypsin and α₂-macroglobulin from the LMW-kininogen preparations: Cl-inactivator and inter-α-trypsin inhibitor from the HMW-kininogen preparations. There was a well defined functional difference between the two kininogens with respect to kinin generation by plasma kallikrein. This enzyme released kinin at a much faster rate from the HMW-kininogen than from the LMW-kininogen. When equipotent preparations of kininogens were incubated for 10 min with kallikrein, 60 times more enzyme was required to release the same amount of kinin from the LMW-kininogen as from the HMW-kininogen.     

Purification and characterization of antithrombotics from Syzygium aromaticum (L.) MErr. & PERRY

Two antithrombotic polysaccharides with relatively high molecular weight (HMW) and low molecular weight (LMW) were isolated from the flower buds of Syzygium aromaticum (L.) MERR. & PERRY (clove) by anion-exchange chromatography, hydrophobic interaction column chromatography and size exclusion chromatography (LMW: EC-2B-IIIa-2, M.W. ca. 34000; HMW: EC-2C-Ia-2, M.W. ca. 103000). The LMW polysaccharide was mainly composed of Rha, Gal, GalA and Ara (molar %: 24.1, 18.9, 18.0 and 17.9, respectively) with 10.8% of sulfate and 18.2% of protein. The HMW fraction consisted of Ara, Gal, Glc and Rha (molar %: 26.0, 23.7, 17.5 and 12.4, respectively) with 15.4% of sulfate and 8.0% of protein. Both polysaccharides had the backbone of type I rhamnogalacturonan and the side chain of arabinan. Also, most of the sulfates were attached at the position 6 of 3-linked galactosyl residues. Compared to the antithrombotic activity of the HMW fraction (plasma clotting time of 145 s in APTT assay), the LMW fraction displayed a slightly low activity (90 s). However, animal studies indicated that crude LMW polysaccharide did not show acute toxicity, while the acute LD50 of the HMW fraction was approximately 2-fold lower than that of heparin.     

Purification and characterization of trypsins from the digestive tract of Locusta migratoria

Two trypsin-like enzymes were isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides. Primary purification was carried out on a DEAE-cellulose column, from which the two trypsins emerged in the anionic fraction. Further purification was achieved by affinity chromatography on a p-aminobenzamidine (PABA)-Sepharose column, which also separated the two trypsins (TLE_Aff.1.. and TLE_Aff.2.), or by HPLC on an anion exchange column. The purity and homogeneity of the trypsins were demonstrated by electrophoresis on cellulose acetate strips and in polyacrylamide gels, with and without SDS. The molecular weights of TLE_Aff.1 and TLE_Aff.2, as determined by SDS-PAGE, were 17000 and 24000 respectively. The amino acid compositions of the locust trypsins were similar to those of trypsins from the digestive systems of other insects, which are characterized by the lack or low content of half cystines. The isoelectric points were 3.2 for TLE_Aff.1 and 3.5 for TLE_Aff.2. Since most of the locust trypsin comprised TLE_Aff.2, the latter served as the main object of this study. TLE_Aff.2 was unstable at low pH, differing in this respect from mammalian trypsins. The optimum activity was at pH 8.5-9.0. The Km and k_cat, values were similar to those for bovine trypsin. Activation by substrate, a phenomenon in bovine trypsin, was also observed for TLE_Aff.2. The locust trypsin was fully inhibited by the proteinaceous trypsin inhibitors Bowman-Birk (BBI) and Kunitz from soybeans, CI from chickpeas, chicken ovomucoid (COM), and turkey ovomucoid (TOM). It was inactivated by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone (TLCK), indicating the involvement of serine and histidine in the active site.     

Merbarone induces activation of caspase-activated DNase and excision of chromosomal DNA loops from the nuclear matrix

Studies were carried out to address possible cellular mechanisms by which merbarone, a catalytic inhibitor of DNA topoisomerase II, can block tumor cell growth without inducing extensive DNA cleavage. Merbarone induced the release of high molecular weight DNA fragments from the nuclear matrix of HL-60 leukemia cells, which preceded the internucleosomalsize DNA fragmentation characteristic of late-stage apoptosis. The chromatin fragments were enriched in a matrix attachment region (MAR) sequence compared with a non-MAR sequence and were similar in size to DNA loops extracted from nuclear matrices. However, merbarone did not directly induce the excision of high molecular weight DNA fragments from the nuclear matrix by promoting topoisomerase II-catalyzed DNA cleavage, because the drug inhibited topoisomerase II-mediated cleavage in isolated nuclear matrix preparations. Instead, merbarone induced rapid activation of the mitochondrial apoptosis pathway, which included the following temporal sequence of events: dissipation of the mitochondrial transmembrane potential within 30 min, release of mitochondrial cytochrome c, and activation of caspase-activated DNase (CAD) by its inhibitor ICAD. The excision of high molecular weight DNA was inhibited at least 80% in merbarone-treated cells preincubated with the pan-caspase inhibitor z-VAD-fmk [Z-Val-Ala-Asp(OMe)-fluoromethyl ketone] and in caspase-resistant Jurkat cells (ICAD/double-mutated) that express a mutant form of ICAD. These results provide evidence that merbarone can induce rapid disorganization of DNA in tumor cells that have a functional mitochondrial apoptosis pathway without inducing extensive DNA cleavage.     

Partial characterization of the low molecular weight fractions of the extract of the venom apparatus of the brown recluse spider and of its hemolymph.

Fractions containing low molecular weight components have been isolated from the venom apparatus extract and the hemolymph of the Loxosceles reclusa spider by Sephadex G-25 column chromatography. The presence of inosine in these low molecular weight fractions was positively identified by Dowex ion exchange chromatography, paper chromatography and u.v. absorption spectrometry. Nucleotides containing guanine-like moieties were also present. Studies with mice indicated that the low molecular weight fraction was not important to the lethality of the toxic fraction (the high molecular weight fraction) of the venom apparatus extract.     

Pharmaceutics: Comparative Measurement of the Molecular Weight of an Antineoplastic Glucan from BCG Vaccine

Bacillus Calmette-Guérin (BCG) vaccine, developed originally for the prophylaxis of tuberculosis, is a potent immunostimulant used to treat superficial bladder carcinoma in man. The aim of this study was to compare the molecular weight and self-association properties of an antineoplastic glucan (PS1A1) extracted from BCG vaccine as determined by different techniques including diffusion, light-scattering and chromatographic methods. In the diffusion experiments, a semi-empirical relationship was derived between the effective diffusion coefficients, D_p, and the weight-average molecular weights, M_w, of several dextrans used as standards, according to the equation D_p = 2.233 times 10^?6 x M_w^?0.66. On the basis of this relationship, the molecular weight of PS1A1 was found to be 57.4 kDa, although, unexpectedly, membrane association was high, most probably because of molecular branching. In the light-scattering experiment it was observed that, unlike dextran, PS1A1 undergoes concentration-dependent multimerization in water. However, the molecular weight of PS1A1 in 0.1 M sodium chloride ranged from 60 to 68 kDa, with a mean of 65 kDa, over the same concentration range. This value was in agreement with the molecular weight determined for PS1A1 by gel-filtration chromatography in previous studies, suggesting that 65 kDa represents the approximate monomeric size of the unassociated molecule. Thus, it was evident that the aggregation was suppressed by electrolyte. Elemental analysis by X-ray fluorescence showed that PS1A1 contained carbon, oxygen, hydrogen and phosphorus, indicating that hitherto unobserved ionized phosphate groups might promote electrostatic interactions.     

RAT BETA-LPH,GAMMA-LPH AND BETA-ENDORPHIN BIOSYNTHESIZED BY ISOLATED CELLS OF PARS INTERMEDIA AND PARS DISTALIS

Rat pars intermedia cells were incubated for 3 h with the following amino acids: (a) ³⁵S-methionine and ³H-phenylalanine; (b) ³H-valine; and (c) ³H-valine and ³H-lysine. Radioactive gamma-lipotropin, beta-lipotropin and beta-endorphin were purified on carboxymethyl-cellulose and characterized by polyacrylamide disc gel electrophoresis at pH 4.5, molecular weight estimation and micro-sequencing. Rat gamma-lipotropin was shown to differ slightly from ovine gamma-lipotropin in its NH₂-terminal amino acid sequence, in containing no methionine and having phenylalanine at position 6, valine at positions 13 and 27, and lysine at position 20. The same variations were observed in the sequence of rat beta-lipotropin, while rat beta-endorphin was shown to be identical to the ovine beta-endorphin. Following a 3-h pulse of rat pars distalis, the cells were extracted with care to avoid beta-lipotropin degradation by proteolytic enzymes. A peptide was purified and identified to be rat beta-endorphin, thus demonstrating that beta-endorphin is biosynthesized in pars distalis and is not an extraction artifact.     

Identification of insect-selective and mammal-selective toxins from Parabuthus leiosoma venom.

Venoms were collected from two scorpion species: Parabuthus leiosoma and Parabuthus pallidus from Kenya. Subcutaneous injection and oral toxicity tests of crude and pure fractions of scorpion venoms were done in Mus musculus (mice), Chilo partellus and Busseola fusca. The highest activity against C. partellus was found in P. leiosoma venom (LC(50) 0.689 mg/50mg body weight). Bioassay-guided purification by a combination of cation-exchange (CE) and reverse-phase high-performance liquid chromatography (RP-HPLC) led to the isolation of three toxic peptides. A lepidopteran-selective toxin (P. leiosoma insect toxin, Plit) was isolated, and the partial N-terminal amino acid sequence (-KDGYPVDNANCKYE-) plus the molecular weight (6688.5 Da) determined. A peptide with significant insect toxicity coupled with mild effects on mice (P. leiosoma toxin, Plt) was isolated, and the partial N-terminal amino acid sequence (-LCEKFKVQRLVELNCVD-) plus the molecular weight (6742.5 Da) was determined. Another toxin with anti-mammalian activity (P. leisoma mammal-selective toxin, Plmt), and N-terminal partial amino acid sequence of ADVPGNYPLDKNGNRYY- plus a molecular weight of 7145.5 Da was also isolated. Comparison of the partial N-terminal amino acid sequences with other toxins revealed that Plit shows high homology to other known insect toxins. Similarly, Plmt shows high homology with several birtoxin-like anti-mammalian toxins. Plt does not exhibit homology with any known scorpion toxin with combined anti-insect and anti-mammalian activity.     

Antimicrobial peptide from the skin secretion of the frog Leptodactylus syphax.

Antimicrobial peptides are considered part of the innate immune system of the majority of living organisms. Most of these molecules are small, cationic and show amphiphilic nature. The skin secretions of Leptodactylus syphax were extracted by mild electrical stimulation and its semipreparative reverse-phase chromatography was resolved in more than 40 fractions. Among these fractions, an antimicrobial peptide was isolated and its amino acid sequence determined by de novo sequencing. Six other truncated forms were characterized in skin secretion. The longest one (25 amino acid residues), named syphaxin (SPX), is amidated at the C-terminal, and shares strong sequence similarity with antimicrobial peptides found in the skin secretion of leptodactylid frogs. Two of the truncated peptides (SPX(1-22) and SPX(1-16)) were tested against Escherichia coli and Staphylococcus aureus, showing low minimal inhibitory concentration (MIC) and no significant toxicity towards blood cells, including both leukocytes and erythrocytes, based on their direct incubation in whole blood at the highest MIC concentration (64 microg/mL).     

Purification, characterization, and cDNA cloning of acidic platelet aggregation inhibiting phospholipases A2 from the snake venom of Vipera lebetina (Levantine viper)

Two novel acidic phospholipase A₂s (PLA₂) were isolated by size exclusion chromatography and reversed-phase chromatography from the crude Vipera lebetina venom. The molecular masses of VLPLA₂-1 (13,704 Da) and VLPLA₂-2 (13,683 Da) and their internal tryptic peptides were determined by MALDI-TOF mass-spectrometry. When tested in human platelet-rich plasma, both enzymes showed a potent inhibitory effect on aggregation induced by ADP and collagen. Chemical modification with p-bromophenacylbromide abolished the enzymatic activity of PLA₂; its anti-platelet activity was fully inhibited in case of collagen as inducer and partially inhibited in case of ADP as inducer. The complete cDNAs encoding PLA₂ were cloned from a single venom gland cDNA library. Complete amino acid sequences of the VLPLA₂ were deduced from the cDNA sequences. The full-length cDNA sequences of the VLPLA₂ possess 615 bp and encode an open reading frame of 138 amino acids that include signal peptide (16 amino acids) and mature enzyme (122 amino acids). The VLPLA₂s have significant sequence similarity to many other phospholipase A₂s from snake venoms. The phylogenetic analysis on the basis of the amino acid sequence homology demonstrates that VLPLA₂s grouped with other Asp49 PLA₂s and they appear to share a close evolutionary relationship with the European vipers.     

人参细胞培养物中水溶性多糖的研究

人参细胞培养物中水溶性多糖经提取后,用果胶酶和Sevag法除去其中蛋白,用Sephadex G-100柱进行分离、纯化得到DPSCG1和DPSCG2两个洗脱峰。经醋酸纤维素薄膜电泳和琼脂糖凝胶电泳,证明DPSCG1和DPSCG2均为均一性多糖,两者经水解和薄层层析结果均由鼠李糖、木糖、阿拉伯糖、半乳糖和半乳糖醛酸等组成,经Sephadex G-200柱层析并与标准分子量对比,DPSCG1和DPSCG2的分子量分别为1.22×10⁵D和2.79×10⁴D。     

Characterization and amino acid sequences of two lethal peptides isolated from venom of Wagler's pit viper, Trimeresurus wagleri

Two lethal toxins were isolated from Trimeresurus wagleri venom by fast protein liquid chromatography (molecular sieve) and high performance liquid chromatography (reverse phase). The toxins (termed peptide I and II) had mol. wt of 2504 and 2530, respectively, pIs of 9.6-9.9 and lacked phospholipase A, proteolytic, and hemolytic activity. Lethal peptide I had a murine i.p. LD50 of 0.369 mg/kg, while lethal II had a murine i.p. LD50 of 0.583 mg/kg. Peptide I retained full toxicity after autoclaving at 121 degrees C for 40 min. The lethal activity was found to represent less than 1% of the total venom protein, which was only 62-65% of crude venom. The amino acid sequence of peptide I revealed a proline-rich (over 30% of total sequence) sequence unique among snake venom toxins. Lethal peptide II showed the same sequence except for a second tyrosine in the position of histidine (residue No. 10) in peptide I. The toxin lacked antigenic identity with a number of representative neurotoxins and myotoxins. The crude venom shared at least one antigen with Crotalus scutulatus scutulatus venom. This antigen was not Mojave toxin. The toxin appears symptomatologically suggestive of a vasoactive peptide or neurotoxin.     

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.     

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.     

Isolation and structure elucidation of bovine pineal arginine vasopressin: arginine vasotocin not identified

A large number of reports have demonstrated the presence of neurohypophysial hormone-like peptides in mammalian pineal glands and an antigonadotropic function has been ascribed to pineal arginine vasotocin (AVT). We have undertaken large scale purification of bovine pineal neurohypophysial hormone-like substances which demonstrate mouse mammary milk-ejection activity (ME-activity) in vitro. Peptides with ME-activity were extracted from more than 5 kg of bovine pineal glands. ME-activity containing peptides were found in both high (M_r～ 10 000–15 000) and low (M_r～ 500–1000) M_r species from Sephadex G-25 chromatography of 0.2 n acetic acid extracts. After ultrafiltration in 5% formic acid, the neurohypophysial hormone-like peptides were localized to an ultrafiltration M_r 500–1000 retentate. A homogeneous peptide, which shared an identical retention time (RT) and amino acid sequence with synthetic 8-arginine vasopressin (AVP), was isolated by serial semipreparative high performance liquid chromatography. On the other hand, the non-mammalian nonapeptide AVT was not identified.