Biochemical,Pharmacological and Structural Characterization of Two PLA<Subscript>2</Subscript> Isoforms Cdr-12 and Cdr-13 from <Emphasis Type="Italic">Crotalus durissus ruruima</Emphasis> Snake Venom

Cdr-12 and Cdr-13 isoforms of PLA₂, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on μ-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA₂ was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry .The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA₂. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13 This sequence shows high identity values when compared to other D49 PLA₂s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA₂ isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. In vitro, Cdr-12 and Cdr-13 isoforms of PLA₂, caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro. Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA₂ family, which presents the typical bioactivities described for such proteins.     

Structural and Biological Characterization of Two Crotamine Isoforms IV-2 and IV-3 Isolated from the <Emphasis Type="Italic">Crotalus durissus cumanensis</Emphasis> Venom

In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its “in vitro” neurotoxic, myotoxic and lethality (DL₅₀) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (μ-Bondapack C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides, including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These activities were modulated by the presence of positively charged amino acid residues. The LD₅₀ of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route.     

Biological and Structural Characterization of Crotoxin and New Isoform of Crotoxin B PLA<Subscript>2</Subscript> (F6a) from <Emphasis Type="Italic">Crotalus durissus collilineatus</Emphasis> Snake Venom

A new crotoxin B isoform PLA₂ (F6a), from Crotalus durissus collilineatus was purified from by one step reverse phase HPLC chromatography using μ-Bondapack C-18 column analytic. The new crotoxin B isoform PLA₂ (F6a), complex crotoxin, the catalytic subunit crotoxin B isoform PLA₂ (F6a) and two crotapotin isoforms (F3 and F4), were isolated from the venom of Crotalus durissus collilineatus. The crotapotins isoforms F3 and F4 had similar chemical properties, the two proteins different in their ability to inhibit of isoforms of PLA₂ (F6 and F6a). The molecular masses estimated by MALDI-TOF mass spectrometry were: crotoxin B: 14,943.14 Da, crotapotin F3: 8,693.24 Da, and crotapotin F4: 9 314.56 Da. The new crotoxin B isoform PLA₂ (F6a) contained 122 amino acid residues and a pI of 8.58. Its amino acid sequence presents high identity with those of other PLA₂s, particularly in the calcium binding loop and active site helix 3. It also presents similarities in the C-terminal region with other myotoxic PLA₂s. The new crotoxin B isoform PLA₂ (F6a) contained 122 amino acid residues, with a primary structure of HLLQFNKMIK FETRRNAIPP YAFYGCYCGW GGRGRPKDAT DRCCFVHDCC YGKLAKCNTK WDFYRYSLKS GYITCGKGTW CEEQICECDR VAAECLRRSL STYRYGYMIY PDSRCRGPSE TC. A neuromuscular blocking activity was induced by crotoxin and new crotoxin B isoform PLA₂ (F6a) in the isolated mouse phrenic nerve diaphragm and the biventer cervicis chick nerve-muscle preparation. Whole crotoxin was devoid of cytolytic activity upon myoblasts and myotubes in vitro, whereas new crotoxin B isoform PLA₂ (F6a) was clearly cytotoxic to these cells.     

Structural Characterization and Neuromuscular Activity of a New Lys49 Phospholipase A2 Homologous (Bp-12) Isolated from Bothrops pauloensis Snake Venom

Bp-12 was isolated from Bothrops pauloensis snake venom in only one chromatographic step in reverse phase HPLC on μ-Bondapack C-18. The molecular mass of 13,789.56 Da was determined by mass spectrometry. The amino acids composition showed that Bp-12 presented high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA₂. The sequence of Bp-12 contains 122 amino acid residues: SLFELGKMIL QETGKNPAKS LGAFYCYCGW GSQGQPKDAV DRCCYVHKCC YKKITGCNPK KDRYSYSWKD KTLVCGEDNS CLKELCECDK AVAICLRENL NTYNKKYRYF LKPLCKKADA AC, with a pI value of 8.55 and with a high homology with Lys49 PLA₂ from other snake venoms. In mouse phrenic nerve-diaphragm, the time needed for 50% paralysis was: 45 ± 6 min (1.4 μM) and 16 ± 6 min (3.6 μM). Bp-12 can induce indirect and directly blocked evoked twitches, even in the preparations in which Ca²⁺ is replaced by Sr²⁺, being the addition of d-tubocurarine required for direct blocking. These results identify Bp-12 as a new member of the Lys49 PLA₂ family and shows that this toxin might contribute to the effects of the crude venom on the neuromuscular junction.     

Isolation and preliminary enzymatic characterization of a novel PLA2 from Crotalus durissus collilineatus venom

A crotoxin homolog was purified from the Crotalus durissus collilineatus venom using molecular exclusion and reverse-phase HPLC. This crotoxin contained one PLA₂ (Cdcolli III F6) and four crotapotin isoforms, whereas crotoxin from Crotalus durissus terrificus venom had three PLA₂ isoforms and two crotapotin isoforms. SDS-PAGE showed that the C. d. collilineatus PLA₂ and crotapotin had relative molecular mass of 15 and 9 kDa, respectively. Neither the PLA₂ (Cdcolli III F6) nor the crotapotins (Cdcolli III F3 and F4) had any neurotoxicity in mouse phrenic nerve-diaphragm preparations when tested alone. However, when PLA₂ and crotapotin were coincubated before testing, the neurotoxicity was restored to a level similar to test in the venom in native crotoxin. The two crotapotins (Cdcolli III F3 and F4) differed in their ability to inhibit PLA₂ activity, perhaps because of variations in their affinities for this enzyme. Cdcolli III F6 showed allosteric enzymatic behavior, with maximal activity at pH 8.3 and 36°C. Full PLA₂ activity required the presence of a low Ca²⁺ concentration and was inhibited by Cu²⁺ and Zn²⁺ and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. These results indicate that crotoxin from C. d. collineatus venom is very similar enzymatically to crotoxin from C. d. terrificus.     

Isolation and enzymatic characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom

A novel basic phospholipase A₂ (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY . . .) showed a high degree of homology with basic D49 PLA₂ myotoxins from other Bothrops venoms. Bj IV had high PLA₂ activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45°C. Full PLA₂ activity required Ca²⁺ but was inhibited by Cu²⁺ and Zn²⁺, and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA₂ was similar to that in the basic PLA₂ of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA₂ could partly explain the low PLA₂ activity of Bothrops venoms.     

Preparation and some properties of cholesterol oxidase from Rhodococcus sp. R14-2

Rhodococcus sp. R_14-2, isolated from Chinese Jin-hua ham, produces a novel extracellular cholesterol oxidase (COX). The enzyme was extracted from fermentation broth and purified 53.1-fold based on specific activity. The purified enzyme shows a single polypeptide band on SDS-PAGE with an estimated molecular weight of about 60 kDa, and has a pI of 8.5. The first 10 amino acid residues of the NH₂-terminal sequence of the enzyme are A-P-P-V-A-S-C-R-Y-C, which differs from other known COXs. The enzyme is stable over a rather wide pH range of 4.0–10.0. The optimum pH and temperature of the COX are pH 7.0 and 50°C, respectively. The COX rapidly oxidizes 3β-hydroxysteroids such as cholesterol and phytosterols, but is inert toward 3α-hydroxysteroids. Thus, the presence of a 3β-hydroxyl group appears to be essential for substrate activity. The Michaelis constant (Km) for cholesterol is estimated at 55 μM; the COX activity was markedly inhibited by metal ions such as Hg²⁺ and Fe³⁺ and inhibitors such as p-chloromercuric benzoate, mercaptoethanol and fenpropimorph. Inhibition caused by p-chloromercuric benzoate, mercuric chloride, or silver nitrate was almost completely prevented by the addition of glutathione. These suggests that -SH groups may be involved in the catalytic activity of the present COX.     

Biological and Structural Characterization of a New PLA<Subscript>2</Subscript> from the Crotalus durissus collilineatus Venom

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A₂ (PLA₂) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80 with crotalic PLA₂s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50 if compared to other sources of PLA₂s such as from the Bothrops snake venom. Also, this PLA₂ induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and q-BPB showed a similar reduction. The q-BPB did not reduce significantly the myotoxic activity induced by the PLA₂, but the anhydrous acetic acid treatment and the pre-incu-bation of PLA₂ with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiflorae (Gram-negative), which was drastically reduced by incubation of this PLA₂ with q-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or b-wing region may be involved in the binding of this PLA₂ to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.     

Biochemical,Pharmacological and Structural Characterization of a New PLA<Subscript>2</Subscript> from <Emphasis Type="Italic">Crotalus durissus terrificus</Emphasis> (South American Rattlesnake) Venom

A new PLA₂ (F16) was purified from Crotalus durissus terrificus venom by molecular exclusion chromatography followed by analytical reverse phase HPLC. The PLA₂ (14.86 kDa by MALDI-TOF mass spectrometry) had an amino acid sequence of SLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGRRRPKDATDRCCFVHDCCYEKVTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNGYMFYPDSRCRGPSETC, and showed highly conserved Ca²⁺-binding and catalytic sites. F16 showed allosteric behavior with 10 mM Ca²⁺ and had temperature and pH optima of 25°C and 7.9, respectively. F16 (10 μg/ml) produced neuromuscular blockade in chick biventer cervicis preparations in the absence and presence of crotapotin, indicating that crotapotin was not essential for neuromuscular action in this preparation. In contrast, in mouse phrenic nerve-diaphragm preparations, the neuromuscular blockade produced by the same concentration of toxin was dependent on crotapotin. Pre-incubation with heparin markedly reduced the neurotoxicity of F16. These results show that the biochemical and structural properties of F16 are similar to those of the PLA₂ isoforms F15 and F17, but that the neurotoxicity and the requirement for crotapotin to form the crotoxin complex varies according to the neuromuscular preparation.     

Molecular modeling studies of the DCCD-treated cytochrome bc1 complex: predicted conformational changes and inhibition of proton translocation

Dicyclohexylcarbodiimide (DCCD) binds covalently to an acidic amino acid located in the cd loop connecting membrane-spanning helices C and D of cytochrome b resulting in an inhibition of proton translocation in the cytochrome bc ₁ complex with minimal effects on the steady state rate of electron transfer. Single turnover studies performed with the yeast cytochrome bc ₁ complex indicated that the initial phase of cytochrome b reduction was inhibited 25–45% in the DCCD-treated cytochrome bc ₁ complex, while the rate of cytochrome c ₁ reduction was unaffected. Simulations by molecular modeling predict that binding of DCCD to glutamate 163 located in the cd2 loop of cytochrome b of chicken liver mitochondria results in major conformational changes in the protein. The conformation of the cd loop and the end of helix C appeared twisted with a concomitant rearrangement of the amino acid residues of both cd1 and cd2 loops. The predicted rearrangement of the amino acid residues of the cd loop results in disruptions of the hydrogen bonds predicted to form between amino acid residues of the cd and ef loops. Simultaneously, two new hydrogen bonds are predicted to form between glutamate 272 and two residues, aspartate 253 and tyrosine 272. Formation of these new hydrogen bonds would restrict the rotation and protonation of glutamate 272, which may be necessary for the release of the second electrogenic proton obtained during ubiquinol oxidation in the bc₁ complex.     

Determination of the Amino Acid Sequence of a New Phospholipase A<Subscript>2</Subscript> (MIDCA1) Isolated from <Emphasis Type="Italic">Micrurus dumerilii carinicauda</Emphasis> Venom

A new Phospholipase A₂ (PLA₂) from Micrurus dumerilii carinicauda venom was isolated and its primary structure determined. This new PLA₂ showed a low enzymatic activity when compared with other PLA₂s and it is moderately basic with an isoelectric point of 8.0. Its amino acid sequence showed the presence of 120 amino acid residues and its sequence was: NLIQFLNMIQCTTPGREPLVAFANYGCYCGRGGSGTPVDELDRCCQVHDNCYDTAKKVFGCSPYFTMYSYDCSEGKLTCKDNNTKCKAAVCNCDRTAALCFAKAPYNDKNYKIDLTKRCQ. The structural model of MIDCA1, when compared with other strong neurotoxic PLA₂s, such as Naja naja, showed significant differences in the β-wing and neurotoxic sites, despite the high level of amino acid sequence similarity. These observations indicate a dissociation between the biological and catalytic activity of this new PLA₂, supporting the view that other regions of the protein are involved in the biological effects.     

The pet genes of Rhodospirillum rubrum: cloning and sequencing of the genes for the cytochrome bc1-complex

Summary A cytochrome bc ₁-complex of Rs. rubrum was isolated and the three subunits were purified to homogeneity. The N-terminal amino acid sequence of the purified subunits was determined by automatic Edman degradation. The pet genes of Rhodospirillum rubrum coding for the three subunits of the cytochrome bc ₁-complex were isolated from a genomic library of Rs. rubrum using oligonucleotides specific for conserved regions of the subunits from other organisms and a heterologous probe derived from the genes for the complex of Rb. capsulatus. The complete nucleotide sequence of a 5500 by SalI/SphI fragment is described which includes the pet genes and three additional unidentified open reading frames. The N-terminal amino acid sequence of the isolated subunits was used for the identification of the three genes. The genes encoding the subunits are organized as follows: Rieske protein, cytochrome b, cytochrome c ₁. Comparison of the N-terminal protein sequences with the protein sequences deduced from the nucleotide sequence showed that only cytochrome c ₁ is processed during transport and assembly of the three subunits of the complex. Only the N-terminal methionine of the Rieske protein is cleaved off. The similarity of the deduced amino acid sequence of the three subunits to the corresponding subunits of other organisms is described and implications for structural features of the subunits are discussed.Abbreviations BSA bovine serum albumin - SDS sodium dodecylsulphate - Rs Rhodospirillum - Rb Rhodobacter - Pc Paracoccus - Rps Rhodopseudomonas The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X55387     

Strong homology between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase of two species of Acetabularia and the occurrence of unusual codon usage

Summary The complete nucleotide sequence of the genes encoding the Rieske FeS, the cytochrome b and the cytochrome c ₁ subunits of the ubiquinol-cytochrome c ₂ oxidoreductase from the photosynthetic purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequences are presented. These three genes, fbcF, fbcB and fbcC, are located at contiguous sites of the genome. The DNA-deduced amino acid sequences are compared with known primary structures of corresponding proteins from other purple photosynthetic bacteria, as well as mitochondria, cyanobacteria and chloroplasts.Abbreviations BSA bovine serum albumin - Rb Rhodobacter - Rps Rhodopseudomonas     

Analysis of the <Emphasis Type="Italic">MAT1-2-1</Emphasis> gene of <Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis>

A single MAT1-2-1 gene was identified from a mating pair of the filamentous ascomycete Colletotrichum lindemuthianum. The MAT1-2-1 genes from both mating partners carried an open reading frame (ORF) of 870 bp encoding a putative protein of 290 amino acids that includes the highly conserved high mobility group (HMG) domain typical of the fungal MAT1-2-1 genes. Three introns were confirmed within the C. lindemuthianum ORF, two of which were found to be conserved relative to a previously reported MAT1-2-1 gene from C. gloeosporioides. The amino acid sequence of the HMG domain from C. lindemuthianum MAT1-2-1 was also compared with those from other ascomycetes. These results suggest that although the MAT1-2-1 genes are highly conserved among ascomycetes, the mechanism which defines mating partners in the genus Colletotrichum is distinct to the idiomorph system described for other members of this phylum.     

Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs

Three new, unique cDNA sequences encoding isoforms of calmodulin (CaM) were isolated from an Arabidopsis cDNA library cloned in gt10. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis CaM gene family distinct from the three DNA sequences previously reported. ACaM-4 and -6 encode full-length copies of CaM mRNAs of ca. 0.75 kb. The ACaM-5 sequence encodes a partial length copy of CaM mRNA that is lacking sequences encoding the amino-terminal 10 amino acids of mature CaM and the initiator methionine. The derived amino acid sequence of ACaM-5 is identical to the sequences encoded by two of the previously characterized ACaM cDNAs, and is identical to TCH-1 mRNA, whose accumulation was increased by touch stimulation. The polypeptides encoded by ACaM-4 and -6 differ from that encoded by ACaM-5 by six and two amino acid substititions, respectively. Most of the deduced amino acid sequence substitutions in the Arabidopsis CaM isoforms occurred in the fourth Ca²⁺-binding domain. Polymerase chain reaction amplification assays of ACaM-4, -5 and -6 mRNA sequences indicated that each accumulated in Arabidopsis leaf RNA fractions, but only ACaM-4 and -5 mRNAs were detected in silique total RNA. The six different CaM cDNA sequences each hybridize with unique Eco RI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. Our results suggest that CaM isoforms in Arabidopsis may have evolved to optimize the interaction of this Ca²⁺-receptor protein with specific subsets of response elements.     

cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A₂ (PLA₂), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1–414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA₂ protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA₂ protein, suggesting the presence of multiple forms in the starfish PLA₂. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in β-wing region when aligned with the sequence of mammalian pancreatic PLA₂. In phylogenetic tree, the starfish PLA₂ should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and β-wing regions may account for the specific properties of the starfish PLA₂, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA₂ from porcine pancreas.     

Amino acid sequence of piratoxin-I, a myotoxin fromBothrops pirajai snake venom, and its biological activity after alkylation withp-bromophenacyl bromide

The complete sequence of the 121 amino acid residues of piratoxin-I (PrTX-I), a phospholipase A₂ (PLA₂)-like myotoxin fromBothrops pirajai snake (Bahia jararacussu) venom, is reported. From the sequence, anM _r of 13,825 and an approximatepI of 8.3 were calculated. PrTX-I shows a high sequence homology with Lys-49 myotoxins from other bothropic (∼95%) and nonbothropic (∼80%) venoms, but only 70–75% homology w hen aligned with the catalytically active Asp-49 PLA₂s. When compared with bothropstoxin-I fromBothrops jararacussu, which is morphologically almost identical toB. pirajai, only two changes out of 121 total amino acid residues have been observed. The approximate minimal lethal doseLD ₅₀ (mice, i.p., 24 hr) of PrTX-I was 8 (6.8–9.1) mg/kg, and the minimal edematogenic dose (MED) in a rat paw model was 39.5±1.8 ug. After alkylation of His-48 withp-bromophenacyl bromide, the MED was 40.1±1.9 ug, but up to 4LD ₅₀ were unable to cause death in any of a group of eight mice after 72 hr. Therefore the edematogenic activity was retained and apparently did not involve His-48, suggesting that at least two biologically active sites are present in PrTX-I.     

A multicopy suppressor ofnin1-1 of the yeastSaccharomyces cerevisiae is a counterpart of theDrosophila melanogaster diphenol oxidase A2 gene,Dox-A2

NIN1 is an essential gene for growth of the yeastSaccharomyces cerevisiae and was recently found to encode a component of the regulatory subunit of the 26S proteasome. Thenin1-1 mutant is temperature sensitive and its main defect is in G₁/S progression and G₂/M progression at non-permissive temperatures. One of the two multicopy suppressors ofnin1-1, SUN2 (SUppressor of Nin1-1), was found to encode a protein of 523 amino acids whose sequence is similar to those ofDrosophila melanogaster diphenol oxidase A2 and the mouse mast-cell Tum^– transplantation antigen, P91A. The C-terminal half of Sun2p was found to be functional as Sun2p at 25° C, 30° C, and 34° C but not at 37° C. The open reading frame (ORF) of theDrosophila diphenol oxidase A2 gene (Dox-A2) was obtained from a lambda phage cDNA library using the polymerase chain reaction technique. TheDox-A2 ORF driven by theTDH3 promoter complemented the phenotype of a strain deleted forsun2. ThisDox-A2-dependent strain was temperature sensitive and accumulated dumb-bell-shaped cells, with an undivided nucleus at the isthmus, after temperature upshift. This morphology is similar to that ofnin1-1 cells kept at a restrictive temperature. These results suggest thatSUN2 is a functional counterpart ofDox-A2 and that these genes play a pivotal role in the cell cycle in each organism.