Identification of an unusual AT(D)Pase-like activity in multifunctional NAD glycohydrolase from the venom of Agkistrodon acutus

NAD-glycohydrolases (NADases) are ubiquitous enzymes that possess NAD glycohydrolase, ADPR cyclase or cADPR hydrolase activity. All these activities are attributed to the NADase-catalyzed cleavage of C-N glycosyl bond. AA-NADase purified from the venom of Agkistrodon acutus is different from the known NADases, for it consists of two chains linked with disulfide-bond(s) and contains one Cu(2+) ion. Here, we show that AA-NADase is not only able to cleave the C-N glycosyl bond of NAD to produce ADPR and nicotinamide, but also able to cleave the phosphoanhydride linkages of ATP, ADP and AMP-PNP to yield AMP. AA-NADase selectively cleaves the P-O-P bond of ATP, ADP and AMP-PNP without the cleavage of P-O-P bond of NAD. The hydrolysis reactions of NAD, ATP and ADP catalyzed by AA-NADase are mutually competitive. ATP is the excellent substrate for AA-NADase with the highest specificity constant k(cat)/K(m) of 293+/-7mM(-1)s(-1). AA-NADase catalyzes the hydrolysis of ATP to produce AMP with an intermediate ADP. AA-NADase binds with one AMP with high affinity determined by isothermal titration calorimetry (ITC). AMP is an efficient inhibitor against NAD. AA-NADase has so far been identified as the first unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities.     

Purification and properties of the soluble NAD glycohydrolase from Bungarus fasciatus venom

The NAD glycohydrolase (NADase) (EC 3.2.2.5) from Bungarus fasciatus (banded krait) venom was purified (1000-fold) to electrophoretic homogeneity through a 3-step purification procedure, the last step being affinity chromatography on Cibacron blue agarose. The purified NADase is a glycoprotein containing two subunits of Mr = 62,000 each. Nicotinamide and adenosine diphosphoribose were produced in a 1:1 stoichiometry and were the only products formed when the purified NADase was incubated with NAD. These results were confirmed by high performance liquid chromatography. The enzyme exhibited a brod pH profile with optimum pH for hydrolysis at 7.5 with very little change in Km from pH 6.0 to pH 8.5. The NADase is only slightly affected by changes in ionic strength. The enzyme studied titrimetrically at pH 7.5 and 38 degrees C exhibited a Km of 14 microM and a Vmax of 1380 mumol of NAD cleaved/min/mg of protein. The activation energy for the enzyme-catalyzed hydrolysis of NAD was 15.7 kcal/mol. In addition to NAD and NADP, a number of NAD analogs were shown to function as substrates for the enzyme. Product inhibition studies demonstrated nicotinamide to be a noncompetitive inhibitor with a KI of 1.5 mM and adenosine diphosphoribose a competitive inhibitor with a KI of 0.36 mM. Procion blue HB (Cibacron blue F3GA) was shown to be a competitive inhibitor with a KI of 33 nmol. The purified NADase catalyzed the pyridine base exchange reaction between 3-acetylpyridine and the nicotinamide moiety of NAD.     

Characterization of clotting factors from Agkistrodon acutus venom

1. Four clotting factors, Cf-1(C), Cf-2(C), Cf-1(T) and Cf-2(T) were isolated from Agkistrodon acutus (collected on mainland China and Taiwan) venom by Komori et al. (1987). It was reported that all factors possessed coagulant activity in the conversion of fibrinogen to fibrin, although they showed different chemical properties and antigenicities. 2. Their role in the clot formation system was clarified and compared with that of thrombin. Clotting factors from A. acutus venom released only fibrinopeptide A from the A alpha chain of fibrinogen, while thrombin released fibrinopeptide A and B from the A alpha and B beta chains. 3. Cf-1(C) and Cf-2(T), like thrombin, rapidly activated factor XIII in the presence of calcium ions, whereas Cf-2(C) and Cf-1(T) had little effect on factor XIII. These effects are shown by Cf-1(C) and Cf-2(T) forming a clot that remained insoluble in 8 M urea or 0.44 M monochloroacetic acid, whereas Cf-2(C) and Cf-1(T) formed a soluble clot in these agents.     

Purification and characterization of the fibrinolytic principle of Agkistrodon acutus venom.

By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon acutus venom was separated into twelve fractions. The fibrinolytic activity was concentrated in Fraction 9. This fraction was rechromatographed on Sephadex G-75 three times and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 2.44 S was obtained by ultracentrifugation, the molecular weight of which was estimated to be 24 100, and the isoelectric point 3.8. The specific activity was four times higher than that of crude venom. The optimal pH value on fibrinolysis was 7.4. In addition to fibrinolytic activity, the purified principle also had fibrinogenolytic and caseinolytic activities. The purified fibrinolytic principle had a specific action on the a(A) chain subunit of fibrinogen, leaving the beta(B) chain and the gamma chain unaffected.     

尖吻蝮蛇毒中精氨酸脂酶的分离纯化与性质研究

目的：从尖吻蝮蛇蛇毒中纯化出精氨酸脂酶并进行性质研究。方法：利用亲和层析、DEAE—Sepharose FF、Superdex 75柱对尖吻蝮蛇毒进行分离纯化。结果：分离纯化出的2个精氨酸脂酶活性组份分子量分别为36000、30000,它们的HPLC纯度也都达到97％以上。结论：为其临床应用打下了基础。     

Purification, characterization, and cDNA cloning of a new fibrinogenlytic venom protein, Agkisacutacin, from Agkistrodon acutus venom

Agkisacutacin is a new fibrinogenlytic protein from Agkistrodon acutus venom. It consists of two heterologous subunits linked by an intersubunit disulfide bond. The cDNAs encoding the two chains of Agkisacutacin were cloned from a lambdagt11 cDNA library of the snake venom gland and sequenced, including the leader peptides (23/23 amino acid residues) and mature subunits (129/123 amino acid residues). It is structurally related to the family of IX/X-binding protein (IX/X-bp)-like proteins and shows high similarity (alpha-70%/beta-64%) to habu IX/X-bp from Trimeresurus flavoridis, but displays distinct biological activity with direct action on fibrinogen.     

Purification and functional characterization of AAV1, a novel P-III metalloproteinase, from Formosan Agkistrodon acutus venom

AAV1, an alkaline glycoprotein (GP), was purified from Agkistrodon acutus venom by two chromatographic steps on successive DEAE-Sephadex A-50 and Superdex 75 FPLC columns. AAV1 on SDS-PAGE under non-reducing conditions migrated as a monomeric and a polymeric forms with apparent molecular mass of 57 and 180 kDa, respectively. Upon reduction, it appeared as a single broad band with a mass of 50.3 kDa corresponding to the size of a typical P-III metalloproteinase acurhagin. The N-terminal sequence of an autoproteolytical 30 kDa-fragment of AAV1 showed a high homology to that of venom proteins with Metalloproteinase, Disintegrin-like, and Cysteine-rich (MDC) domains. Although it was devoid of cleaving activity toward gelatin, fibronectin and prothrombin, AAV1 preferentially digested the Aalpha chain of fibrinogen and followed by the Bbeta chain, leading to the inhibition of fibrinogen-induced platelet aggregation in elastase-treated human platelets. However, the proteolytic activity of AAV1 was completely inactivated by the chelating agent but not serine proteinase inhibitor. Furthermore, AAV1 could concentration-dependently inhibit platelet aggregation and suppress tyrosine phosphorylation of intracellular proteins in collagen- and convulxin-stimulated platelets, respectively. The interaction of MDC domains in AAV1 molecule with platelet GPVI was responsible for the inhibitory effect of AAV1 on collagen- and convulxin-induced platelet aggregation. Taken together, these pieces of evidence suggest that AAV1 from Formosan viper venom belongs to a new member of high-molecular mass metalloproteinase family and functions as a GPVI antagonist.     

Presence of zinc in proteolytic hemorrhagic toxin isolated from Agkistrodon acutus venom

1. Hemorrhagic toxin (Ac1-proteinase) was isolated from the venom of Agkistrodon acutus (Formosa) and the zinc content was determined (1.15 mol/mol protein). The results we extensively compared with hemorrhagic toxin e of Crotalus atrox venom (U.S.A.). Comparable results were obtained for zinc content, hemorrhagic and proteolytic activities for native hemorrhagic toxins from two different venoms. It is of interest that the zinc content of hemorrhagic toxins is identical even though the venoms are obtained from snakes inhabiting totally different continents. 2. Zinc content, hemorrhagic and proteolytic activities were compared before and after the removal of zinc. It was found that both hemorrhagic and proteolytic activities disappeared upon removal of the zinc. 3. Zinc content of all hemorrhagic toxins with proteolytic activity reported so far were also compared and it is concluded that regardless of their geographic origin, zinc is present in venom hemorrhagic toxins on a unimolar basis. 4. When zinc is removed there is a drastic change in the low-frequency region of the Raman spectrum suggesting the presence of a zinc ligand co-ordination. The decrease of alpha-helical content and increase of random coil are reflected in the amide I and III bands of Raman spectroscopy after the removal of zinc. Increase of random coil and the loss of zinc are probably responsible for the loss of hemorrhagic and proteolytic activities.     

Properties and applications of immobilized snake venom NAD glycohydrolase

The NAD glycohydrolase (NADase) from Bungarus fasciatus snake venom was adsorbed on concanavalin A-Sepharose, and demonstrated to retain both hydrolase and transglycosidase activities in the bound form. The matrix-bound enzyme was stable to repeated washing with buffer and storage at 4°C. The bound enzyme exhibited the same K_m value for hydrolysis of nicotinamide-1,N⁶-ethenoadenine dinucleotide as previously measured with the soluble, purified form of the enzyme. The bound NADase was used repeatedly for a preparative-scale synthesis of 3-acetylpyridine adenine dinucleotide. It was further demonstrated that the immobilized enzyme could be prepared directly from crude snake venom, thus avoiding the time required for purification. The application of the immobilized snake venom NADase for the preparation of pyridine nucleotide coenzyme analogs has many advantages over procedures used previously for analog synthesis.     

Recombinant production of fibrinogenase IV from Agkistrodon acutus venom and its preliminary evaluation

A novel metalloproteinase, recombinant fibrinogenase IV (rFIV_a), was expressed and purified from Agkistrodon acutus venom. It is a single-chain protein with an apparent molecular weight of 27 kDa. Western blot showed that it had a good immunological reaction against anti-FIV_a rabbit serum. The kinetic parameters K_m and K_cat of rFIV_a on the substrate T6140 were 7.471×10⁻⁴ mol/l and 5.103×10⁻⁵ s⁻¹. RFIV_a cleaved preferentially the α-chain, and the β- and γ-chains of fibrinogen were also cleaved when the incubation time was prolonged. The administration of rFIV_a (1.8 and 5.4 mg/kg) to animals with acute blood-stasis model produced a decrease in fibrinogen to control values. To our knowledge, this is the first report of the expression, purification, and evaluation of recombinant fibrinogenase IV, which belongs to class P-I metalloproteinase from A. acutus venom.     

Primary structure and antiplatelet mechanism of a snake venom metalloproteinase, acurhagin, from Agkistrodon acutus venom

Acurhagin has been characterized as a P-III hemorrhagic metalloproteinase. We herein report the complete sequence of acurhagin by molecular cloning. Analysis of the cDNA-predicted amino acid sequence encoding acurhagin precursor revealed that this mosaic Asn-linked glycoprotein possesses a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domains (189/205/102/114 residues), with an overall 87% identity to that of jararhagin, an integrin alpha2beta1-cleaving metalloproteinase. Acurhagin has a Ser-Glu-Cys-Asp sequence in the disintegrin-like domain instead of the typical Arg-Gly-Asp motif. In contrast to inhibiting fibrinogen-integrin alphaIIbbeta3 interaction by disintegrins, acurhagin selectively showed a dose-dependent inhibition on platelet aggregation induced by collagen, and suppression on tyrosine phosphorylation of several signaling proteins in convulxin-stimulated platelets. Although the immobilized acurhagin was shown to bind platelet GPVI and collagen in a primary structure- and steric conformation-dependent manner, respectively, the mechanism of acurhagin under short incubation is mainly through its binding to GPVI and collagen, instead of binding to alpha2beta1, or cleaving platelet membrane glycoproteins. Moreover, the molecular conformation maintained by divalent cations is required for the proteolytic activity of acurhagin toward extracellular matrix fibronectin. Taken together, these results suggest that all the three domains in mature acurhagin may cooperatively contribute to its biological function.     

蕲蛇蛇毒中一个新的类凝血酶的分离纯化与表征

使用DEAE－Sephadex A-50、POROS 50HS及POROS 20PI等色谱分离技术,从蕲蛇粗毒中分离提纯得到1个具有凝血活力的组分,经SDS－PAGE和PAGE检测均为一条带,非还原条件下相对分子质量24.3kDa,还原条件下相对分子质量33.0kDa;其凝血活力为91.0NIH u/mg。该组分不具有激活因子ⅩⅢ的活性,肝素不影响该组分的凝血活性,EDTA部分抑制其活性,苯甲基磺酰氟（PMSF）则会产生不可逆抑制作用,该酶N－末端氨基酸序列为VIGGNGXDINEHRFLVAFF,经分析表明该酶是一种新的类凝血酶。     

五步蛇毒降纤酶的快速分离与纯化

目的：建立快速分离纯化降纤酶工艺方法适合于规模化生产。方法以五步蛇毒为原料。采用ＥＤＴＡ络合－硫氰酸钾沉淀初步分离与快速离子交换和凝胶层析化相结合。分离纯化出符合新部颁标准的降纤酶成分。结果经初步分离五步蛇粗毒可使出血毒去除９２％,非类凝血酶（ＴＬＥ）成分去除６５．１７％,ＴＬＥ活性回收保持９２．６８％以上。层析组份中至少含有４种ＴＬＥ,其中１种为降酶成分。初步化学分离,。离子交换柱层析和凝胶过滤     

五步蛇蛇毒磷脂酶A_2的纯化及部分性质

经Sephadex G-75和QAE-Sephadex A-50离子交换层析等方法,从湖南产五步蛇(Agkistrodon acutus)蛇毒中纯化一种均一的酸性磷脂酶A_2。SDS-PAGE测得分子量为15.8kD,按氨基酸残基计算其分子量为14.352kD,IEF-PAGE测得等电点为5.32。氨基酸组份分析表明磷脂酶A_2分子由128个氨基酸残基组成,富含Asp和Glu,不含中性糖。PLA_2酶活性的最适温度为45℃,最适pH为8.5左右,没有抗胰蛋白酶的活性,具一定的热稳定性。K~+、Ca~(++)和Na~+离子激活,而Cd~(++)、Sn~(++)、Cu~(++)、Li~+、Hg(++)、Zn~(++)、Fe~(++)和Co~(++)离子可抑制或完全丧失酶活力。手工微量顺序分析测得PLA_2分子N-末端氨基酸为Leu。此酶对小白鼠的LD_(50)至少大于10mg/kg(ip)。     

尖吻蝮蛇毒活性肽K组分对血小板聚集和超微结构的影响

目的应用电镜研究尖吻蝮蛇毒活性肽(K组分)对血小板聚集和超微结构的影响,以探讨其抗血小板聚集的机制。方法取2周内未服任何药物健康志愿者静脉血,观察K组分对二磷酸腺苷(ADP)和胶原(collagen)诱导的血小板聚集作用的影响。将已调好的PRP分为5组,A组为空白对照组,B、D组为加入等体积的生理盐水组,C、E组为加K组分组。B、C组分别加血小板聚集诱导剂ADP,D、E组分别加血小板聚集诱导剂胶原,各组分别制成超薄切片样品进行电镜观察、摄片,测出其聚集抑制率。结果K组分能抑制由ADP和胶原诱导的血小板聚集,剂量与抑制率成量效关系,IC50经直线回归分别为0.067和0.088μmol/L。ADP和胶原组血小板形态不规则,突起明显增多。K组分组血小板形态基本规则,膜表面清晰光滑,颗粒较ADP与胶原组明显增加,胞浆空泡化现象减轻。结论K组分明显抑制ADP和胶原诱导的血小板聚集反应及其超微结构的改变。     

Metal ion-induced stabilization and refolding of anticoagulation factor II from the venom of Agkistrodon acutus

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding/refolding of apo-ACF II, holo-ACF II, and Tb(3+)-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solutions was studied by following the fluorescence and circular dichroism (CD). Metal ions were found to increase the structural stability of ACF II against GdnHCl and irreversible thermal denaturation and, furthermore, influence its unfolding/refolding behavior. The GdnHCl-induced unfolding/refolding of both apo-ACF II and Tb(3+)-ACF II is a two-state process with no detectable intermediate state, while the GdnHCl-induced unfolding/refolding of holo-ACF II in the presence of 1 mM Ca(2+) follows a three-state transition with an intermediate state. Ca(2+) ions play an important role in the stabilization of both native and I states of holo-ACF II. The decalcification of holo-ACF II shifts the ending zone of unfolding/refolding curve toward lower GdnHCl concentration, while the reconstitution of apo-ACF II with Tb(3+) ions shifts the initial zone of the denaturation curve toward higher GdnHCl concentration. Therefore, it is possible to find a denaturant concentration (2.1 M GdnHCl) at which refolding from the fully denatured state of apo-ACF II to the I state of holo-ACF II or to the native state of Tb(3+)-ACF II can be initiated merely by adding the 1 mM Ca(2+) ions or 10 microM Tb(3+) ions to the unfolded state of apo-ACF II, respectively, without changing the concentration of the denaturant. Using Tb(3+) as a fluorescence probe of Ca(2+), the kinetic results of metal ion-induced refolding provide evidence for the fact that the first phase of Tb(3+)-induced refolding should involve the formation of the compact metal-binding site regions, and subsequently, the protein undergoes further conformational rearrangements to form the native structure.     

Characterization of Ac3-proteinase from the venom of Agkistrodon acutus (hundred-pace snake)

Ac3-Proteinase from the venom of Agkistrodon acutus was isolated in a homogeneous form by a previously published method. Ac3-Proteinase possessed lethal, hemorrhagic, caseinolytic, azocaseinolytic, dimethylcaseinolytic and hide powder azure hydrolytic activities. These activities were inhibited when Ac3-Proteinase was incubated with the metal chelators ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), tetraethylenepentamine (TEP), 1,10-phenanthroline, phosphoramidon or beta-mercaptoethanol. The toxin also hydrolyzed the oxidized A and B chains of both insulin and fibrinogen. The cleavage sites in the oxidized B chain of insulin were identified as His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25). The A alpha chain of fibrinogen was digested first followed by hydrolysis of the B beta chain. Toxicological and biochemical properties of Ac3-Proteinase were investigated further and are reported in this paper.     

Purification and properties of a mitochondrial NAD+ glycohydrolase

A 60- to 70-fold purification of an NAD⁺ glycohydrolase from the inner membrane of rat liver mitochondria to apparent homogeneity on sodium dodecyl sulfate (SDS)-polyacrylamide slab gel is described. The minimum molecular weight of the enzyme on polyacrylamide gels in the presence of SDS is around 62,000. The enzyme splits NAD⁺ to ADP-ribose and, presumably, nicotinamide. No phosphatase or phosphodiesterase activity is detected in the purified enzyme preparation. The enzyme shows high activity with NAD⁺ and moderate activity with NADP⁺ as substrates NAD(P)Hs are poor substrates. ATP and nicotinamide inhibit the enzyme. A possible participation of the enzyme in the mechanism of calcium release from rat liver mitochondria is discussed.