Purification and characterization of alkaline phosphatase from human seminomas.

Despite the apparent link between the presence of alkaline phosphatase (ALP) and various cancers, it has so far been difficult to determine distinct differences between seminoma-derived ALP and placental ALP (PLAP). In order to determine specificity, we purified ALP from a seminoma type of human testicular cancer tissue and compared its biochemical and immunological properties with those of PLAP. The purified ALP had a specific activity of 66 units per mg of protein, and it was possible to obtain 169 microg of purified preparation from 60 g of tissue. The molecular weight of the purified seminoma enzyme was approximately 500 kDa. We found that a novel type of ALP from human testicular cancer tissue exists, with a high molecular weight and differing in degree, from the seminoma ALP previously reported.     

Biochemical characterization of fibrinogenolytic serine proteinases from Vipera lebetina snake venom.

Two glycosylated serine fibrinogenases isolated from Vipera lebetina venom have homologous N-terminal sequences and antigenic determinants but can be clearly differentiated according to substrate specificity, glycosylation levels, molecular mass and fibrinogen degradation. alpha-Fibrinogenase has no homolog among known serine proteinases. It has N-terminal similarity with snake venom arginine esterases but does not hydrolyze the esters of arginine, lysine and tyrosine. The enzyme has strong proteolytic activity and degrades alpha-chain of fibrinogen altering its clottability by thrombin. beta-Fibrinogenase is a typical arginine esterase which hydrolyzes esters and amides of arginine and attacks the beta-chain of fibrinogen.     

毒蛇的毒器和蛇咬的生物学

本文描述毒蛇的毒器,包括毒腺、毒牙、导管和有关肌群,并讨论毒蛇咬伤的生物学。     

A study on the venom yield of venomous snake species from Argentina

A study on the venom yield of snakes from Argentina over a three year period was carried out on adult specimens of Bothrops alternatus (n=74); Bothrops neuwiedii (n=127); Bothrops ammodytoides (n=30); Bothrops moojeni (n=14); Bothrops jararaca (n=14); B. jararacussu (n=6); Crotalus durissus terrificus (n=120) and Micrurus spp. (n=6) as well as with 12 specimens of newborn C. d. terrificus kept in captivity. While for each species there was a positive correlation between venom yield and number of snakes milked, the correlation with the snake's body weights after individual milkings was even better, suggesting that the size of the snakes is more important in determining the venom yield than the number of snakes milked or the specimen's sex. Individual milkings indicated that, in addition to the snake size, when the amount of venom is normalized per 100 g body weight there is a species specific difference in venom yield. It follows the order B. jararacussu>B. moojeniB. jararacaB. alternatus>B. neuwiedii>Micrurus sppB. ammodytoides>C. d. terrificus. Although the venom yield per 100 g body weight of newborn C. d. terrificus specimens is 2-fold higher than that of adults, no correlation was observed between venom yield and body weight.     

Synopsis of recent developments in venomous snake systematics, No. 3.

We present recent findings in the systematics of venomous snakes, with emphasis on those which affect the nomenclature and our understanding of species limits in these animals. Changes in systematics reviewed here include particularly the genera Acanthophis, Elapsoidea, Bitis, Lachesis, Porthidium, Trimeresurus/Tropidolaemus and Vipera. Other new publications of more general interest to toxinologists are also presented.     

Biochemical characterization of soluble phospholipase A2 from rheumatoid synovial fluid

Radiolabeled E. coli, Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC), were used to characterize the phospholipase A2 (PLA2) activity in synovial fluid (SF) from rheumatoid arthritis (RA) patients. Cell-free fractions of SF contain a PLA2 enzyme that preferentially releases [14C]oleic acid from E. coli, requires calcium and is optimally active at neutral pH. Purified PE, but not PC is also readily degraded by the soluble enzyme. A cell-associated PLA2 present in sonicates of SF mononuclear cells and neutrophils preferentially releases [3H]AA from E. coli. These studies suggest the presence of at least two different enzymes with activity of PLA2 in rheumatoid SF.     

舟山眼镜蛇毒蕈碱样多肽MP的部分理化性质测定

目的对舟山眼镜蛇毒蕈碱样多肽MP成分进行纯化及部分理化性质测定。方法采用色谱技术纯化蛇毒MP成分,质谱仪测定其相对分子质量(Mr);Edman降解法分析部分氨基酸序列,与已知成分比对;以卵磷脂为底物测定MP组分的磷脂酶A2活性;采用Bliss法测定MP对小鼠的急性毒性。结果 MP的Mr为13 260,其N端16位氨基酸序列为NLYQFKNMIQCTVPSR,与已知蛇毒磷脂酶A2基本一致,并具有较强的磷脂酶A2活性;腹腔注射MP对小鼠的LD50值为14.3 mg/kg。结论 MP为一种眼镜蛇毒磷脂酶A2。     

Biochemical and morphological characterization of subcellular fractions isolated from rabbit colon muscle.

From a homogenate of rabbit colon muscle subcellular fractions were isolated by differential centrifugation. The crude microsomal fraction could be separated into subfractions, a fraction of vesicular microsomes at 35% sucrose, a fraction containing sarcolemma, mitochondrial fragments and microsomal vesicles at 35--45% sucrose and a small protein fraction at 45--55% sucrose. Their biochemical properties and their morphological characterization were investigated. The cholesterol and the phospholipid content was equally distributed between the microsomal fractions 35% and 35--45% while the RNA was localized to the mitochondria and the microsomal fraction 35%. The enzyme cytochrome c oxidase was found to be concentrated in the mitochondria while a high contamination was found in the microsomal fractions 35--45%. The NADH-oxidase activity was highest in the 35% fraction and the 5'-nucleotidase activity in the 40,000 X g supernatant. The microsomal subfractions contained the enzymes ATPase, adenylate cyclase and phosphodiesterase. In the 35% fraction Ca stimulated the hydrolysis of ATP. The binding of [3H]-ouabain and the incorporation of [3H]-leucine was most pronounced in the 35% fraction. In a K+-free Krebs Ringer medium the binding of the glucoside was stimulated in all the fractions. From these results we concluded that the fraction 35% sucrose may be mainly derived from the endoplasmic reticulum and the plasma membrane while the 35--45% originates from the plasma membrane, mitochondria and to a lesser extent the endoplasmic reticulum.     

Pharmacological characterization of mikatoxin, an alpha-neurotoxin isolated from the venom of the New-Guinean small-eyed snake Micropechis ikaheka.

Symptoms of envenomation by the New-Guinean small-eyed snake Micropechis ikaheka (Elapidae) include peripheral neurotoxicity and myotoxicity. We have now purified to homogeneity a long-chain neurotoxin, mikatoxin, from M. ikaheka venom by successive gel filtration and reverse-phase chromatography. Electrospray ionization mass spectrometry showed mikatoxin to be a homogenous peptide of MW 7775.6. Mikatoxin was devoid of any phospholipase A(2) activity associated with the crude venom and did not exhibit any intrinsic anticholinesterase activity. In the chick biventer cervicis muscle, it produced an irreversible, concentration-dependent block of responses to exogenously applied acetylcholine and carbachol as well as twitches evoked by nerve, but not by direct muscle stimulation. Moreover, mikatoxin, like alpha-bungarotoxin and erabutoxin-b, did not show significant fade response to train-of-four stimulation of the mouse phrenic nerve-hemi diaphragm muscle. It also failed to block ganglionic transmission in the guinea pig ileum and muscarinic responses in the rat anococcygeus muscle. Our study provides strong evidence for the presence of a neurotoxin (mikatoxin) in M. ikaheka venom that produces neuromuscular blockade in skeletal muscle attributable to selective and irreversible antagonism of postsynaptic nicotinic acetylcholine receptors of the neuromuscular junction and likely contributes to the peripheral neurotoxicity observed in M. ikaheka envenomation.     

Purification and functional characterization of bothrojaractivase, a prothrombin-activating metalloproteinase isolated from Bothrops jararaca snake venom.

Bleeding at the site of bite and/or systemic hemorrhage are symptoms frequently observed in envenomation by Bothrops jararaca snakes. In this study, we purified and characterized a prothrombin activator from B. jararaca that is probably involved in these clinical manifestations. The enzyme was isolated by a combination of gel filtration and ion exchange chromatographies and named bothrojaractivase. It has a single polypeptide chain with a molecular weight of 22,829 Da as measured by mass spectroscopy. Bothrojaractivase generates active thrombin from prothrombin, independently of cofactors. SDS-PAGE analysis of the prothrombin activation products shows that bothrojaractivase converts prothrombin into meizothrombin producing similar fragments to those generated by group A prothrombin's activators. In addition, bothrojaractivase degraded fibrinogen and fibrin. Chelating agents completely inhibited the enzymatic activity, whereas inhibitors of serine and cysteine proteinases had no effect. Amino acid sequence of four peptides demonstrated high similarity of bothrojaractivase with P-I class of snake venom metalloproteinases. Thus, our results indicate that bothrojaractivase is a new metalloproteinase that acts on different protein factors of the clotting cascade especially displaying a key and most relevant functional action in the generation of thrombin through prothrombin activation in a similar mode of action as that of group A activators.     

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M_r = 34,000 under reducing conditions and pI  6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (−70 to 37 °C), pH values (3–9) or in the presence of divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺ and Mn²⁺). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom.     

Biochemical characterization of a thrombin-like enzyme and a fibrinolytic serine protease from snake (Agkistrodon saxatilis) venom.

A thrombin-like enzyme and a fibrinolytic serine protease were purified to homogeneity from the venom of a Korean snake Agkistrodon saxatilis emelianov. Both the purified enzymes migrated as a single protein band corresponding to 39 kDa in SDS-PAGE. However, the molecular mass was reduced to 28 kDa by enzymatic removal of the N-linked carbohydrates in those two different enzyme species. Although the thrombin-like enzyme and the fibrinolytic protease show homologous features in their molecular sizes and N-terminal amino acid sequences, yet they can be clearly distinguished from each other in terms of substrate specificity, susceptibility to inhibitors and fibrinogen degradation. It is postulated that these two enzymes are capable of functioning in a cooperative manner to effectively remove fibrinogen and consequently to reduce the blood viscosity.     

Streptozotocin-induced diabetes: significant changes in the kinetic properties of the soluble form of rat bone alkaline phosphatase.

A soluble form of an alkaline phosphatase, obtained from the osseous plate of streptozotocin-induced diabetic rats, was purified 90-fold with a yield of 26%. The calculated Mr of the purified enzyme was 80,000 by denaturing polyacrylamide gel electrophoresis and 160,000 by gel filtration on Sephacryl S-300, suggesting a dimeric structure for its native form. In the absence of metal ions, the p-nitrophenylphosphatase activity of the purified enzyme was 4223.1 U/mg. Magnesium or calcium ion concentrations up to 2 mM increased the specific activity of the enzyme to 9896.5 and 10,796.2 U/mg, respectively. The enzyme was stimulated to a lesser extent by MnCl2 (5390.1 U/mg) and CoCl2 (5088.2 U/mg). The purified soluble alkaline phosphatase showed a broad substrate specificity, and among the less hydrolyzed substrates were pyrophosphate (1517.6 U/mg) and bis-p-nitrophenylphosphate (499.6 U/mg). The enzyme was relatively stable at 45 degrees for periods as long as 180 min, but was denatured rapidly above 50 degrees, following first order kinetics with T1/2 values ranging from 3.5 to 57.7 min. The results reported herein suggested that the soluble form of alkaline phosphatase from streptozotocin-induced diabetic rats had its kinetic properties altered, apparently as a consequence of changes in metal-binding properties.     

骨碱性磷酸酶与碱性磷酸酶在佝偻病诊断中的比较

目的 了解定量骨碱性磷酸酶( BAP-E)、定性骨碱性磷酸酶(BAP-CIT)、血清碱性磷酸酶(AP)三个指标在佝偻病诊断中的意义。方法 检测55例佝偻病患儿、26例对照婴幼儿血清AP、BAP-E、BAP-CIT,BAP-E采用ELISA法,BAP-CIT用全血干化学和免疫浓缩技术。结果 佝偻病患儿的AP和BAP-E较...     

血液透析滤过治疗毒蛇咬伤并急性肾功能衰竭的分析

目的探讨毒蛇咬伤并急性肾功能衰竭的血液净化治疗。方法回顾性分析2007—2008年收治的43例毒蛇咬伤并急性肾功能衰竭病例,分为血液透析组（HD组）,血液透析滤过组（HDF组）。结果两组死亡存活率无明显区别,但平均进入多尿期时间血液透析滤过组优于血液透析组。结论毒蛇咬伤并急性肾功能衰竭应早期血液净化治疗,血液透析滤过更有效。     

Acid and alkaline phosphatase activities of a fraction isolated from Parawixia bistriata spider venom

This present study describes the isolation of a high molecular weight fraction (F1) from the venom of the social spider Parawixia bistriata, by gel filtration and also its subfractions by further purification with affinity chromatography on a Concanavalin A-Sepharose column. Acid and an alkaline phosphatase activities were found in fractions. The effects of pH, temperature and metallic ions on these activities were evaluated. Optimal temperature for both enzymes was 55 °C and optimal pH was 5.0 and 8.5 for the acid and alkaline phosphatase activities, respectively. As ZnCl₂ inhibited enzymatic activities and the chelating agent ethylenediaminetetracetic acid (EDTA) raised the basal phosphatase activities, it was speculated that the venom itself could contain Zn⁺⁺; this was confirmed with the use of an atomic absorption flame spectrometer. In conclusion, the high molecular weight components of the spider venom of P. bistriata have acid and alkaline phosphatase activities, which may reflect the presence of at least two different enzymes.     

Purification and characterization of a phospholipase A2 isoenzyme isolated from Lachesis muta snake venom

A new phospholipase A2 (PLA2) isoenzyme was isolated from Lachesis muta crude venom, and was named LM-PLA2-II. This enzyme was purified by gel filtration on a Sephacryl S-200 HR column followed by reverse-phase chromatography on a C2/C18 column. LM-PLA2-II consists of a single polypeptide chain with an apparent molecular mass of 18 kDa and an isoelectric point at pH 5.4. The amino terminal sequence of the enzyme revealed a high degree of homology with other PLA2s from several sources. LM-PLA2-II has a high indirect hemolytic activity and a potent inhibitory effect on platelet aggregation induced by ADP and collagen. It also produces a significant paw edema reaction in rats. The edematous response in rats was abolished by pretreatment with either indomethacin or dexamethasone, suggesting the involvement of cyclo-oxygenase. Pretreatment of LM-PLA2-II with p-bromophenacyl bromide abolished all of these actions, clearly indicating that the biological activities, including the edematogenic effect, are dependent entirely on its enzymatic activity.     

Effects of several snake venoms on serum and tissue transaminases,alkaline phosphatase and lactate dehydrogenase

The effect of lethal doses of five venoms (Bitis arietans, Bitis gabonica, Dendroaspis polylepis, Naja nigricollis and Naja haje) on the activities of transaminases, alkaline phosphatase and lactate dehydrogenase were studied. Samples from the serum, liver, heart and kidney were collected 4 hr following lethal venom injections in albino rats. The activity of these enzymes were markedly increased in serum and variably decreased in the liver, heart and kidney after envenomation.     

Isolation and chemical characterization of a toxin isolated from the venom of the sea snake, Hydrophis torquatus aagardi

Sea snakes (family: Hydrophiidae) are serpents found in the coastal areas of the Indian and Pacific Oceans. There are two subfamilies in Hydrophiidae: Hydrophiinae and Laticaudinae. A toxin, aagardi toxin, was isolated from the venom of the Hydrophiinae snake, Hydrophis torquatus aagardi and its chemical properties such as molecular weight, isoelectric point, importance of disulfide bonds, lack of enzymatic activity and amino acid sequence were determined. The amino acid sequence indicated a close relationship to the primary structure of other Hydrophiinae toxins and a significant difference from Laticaudinae toxins, confirming that primary toxin structure is closely related to sea snake phylogenecity.