Isolation and partial characterization of a polypeptide from Phoneutria nigriventer spider venom that relaxes rabbit corpus cavernosum in vitro

The venom of the Brazilian spider Phoneutria nigriventer was fractionated using a C18 microBondapack reverse-phase high-performance liquid chromatography column. The resulting fractions were assayed in the rabbit perfused corpus cavernosum tissue to identify those fractions responsible for the corpus cavernosum relaxation. Two fractions (A and B) with retention times of 18.1 and 36.7 min, respectively, induced relaxation of corpus cavernosum strips. Fraction A was selected for further biochemical characterization. Repurification of this fraction revealed the presence of a polypeptide (named PNV4) which migrates in sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a single band consistent with a mol. wt close to 16,600. The amino acid composition of PNV4 showed the presence of 147 residues, a high content of Cys and a calculated mol. wt of 17,213 + Trp. The N-terminal amino acid sequence of PNV4 determined for its first 48 residues was AELTSCFPVGHECDGDASNCNCCGDDVYCGCGWGRWNCKCKVADQSYA.     

Investigation of the effect of PhTx2, from the venom of the spider Phoneutria nigriventer, on the release of [3H]-acetylcholine from rat cerebrocortical synaptosomes

Venoms from spiders are an important source of toxins that can help on the dissection of mechanisms involved in neurotransmission. Among them is the venom of the spider Phoneutria nigriventer that contains several toxic fractions with different targets in mammals and/or insects. We here report that one of these fractions (PhTx2) is able to evoke acetylcholine release from rat cortical synaptosomes and that this effect is dependent on extracellular calcium and is inhibited by the sodium channel blocker tetrodotoxin.     

Cloning and sequence analysis of a cDNA encoding salmon (Onchorhynchus keta) liver transglutaminase

We isolated cDNA clones encoding a transglutaminase (TGase: EC 2.3.2.13) from a salmon (Onchorhynchus keta) cDNA library prepared from the liver. In the cDNA sequence combined, an open reading frame coding for a protein of 680 aa was found. The deduced sequence showed a considerable similarity (62.4%) to that of red sea bream TGase. By comparison of sequence similarity to other TGases, the structure of salmon TGase was like tissue type TGases, rather than membrane-associated type or plasma type TGases. As a structural feature of salmon TGase, 3 aa residues were substituted in the 25 aa sequence around the active site Cys residue, which is conserved among several tissue type TGases. The critical residues thought to form the catalytic-center triad (Cys272, His331, and Asp301) were found in the highly conserved region, but the region surrounding Tyr511, which corresponds to the residue participates in hydrogen-bond interactions of active center domain, was less similar to other TGases, except for red sea bream TGase. These finding suggests that the overall structure of fish TGase resembles tissue-type TGases, but has some unique structure.     

Purification and characterization of a kinin-releasing and fibrinogen-clotting serine proteinase (KN-BJ) from the venom of Bothrops jararaca, and molecular cloning and sequence analysis of its cDNA

Two forms of a proteinase, KN-BJ 1 and 2, were purified to homogeneity from the venom of Bothrops jararaca. In SDS/PAGE reduced KN-BJ 1 and 2 migrated as single bands with molecular masses of 38 kDa and 39 kDa. The two enzymes have similar N-terminal amino acid sequences and specific activities on synthetic chromogenic substrates, and both release bradykinin from bovine low-molecular-mass kininogen. KN-BJ 1 and KN-BJ 2 clot fibrinogen with specific activities of 245 NIH U/mg and 219 NIH U/mg, releasing only fibrinopeptide A. The amidolytic, kinin-releasing and coagulant activities are inhibited by phenylmethylsulfonyl fluoride, demonstrating that KN-BJ is a serine proteinase. Benzamidine derivatives, which are competitive inhibitors of trypsin-like proteinases, also inhibited the amidolytic activity of KN-BJ. A cDNA clone (HS104, 2.2 kb) has been isolated from a cDNA library of B. jararaca venom glands with an ORF of 771 bp. The deduced amino acid sequence contains segments that are identical to the sequences of the N-terminus and three tryptic peptides of KN-BJ 2. Therefore, the cDNA is believed to represent the gene of KN-BJ 2. The deduced amino acid sequence indicates that KN-BJ 2 is synthesized as a prezymogen of 257 amino acids with a putative signal peptide of 18 amino acids and an activating peptide of six amino acid residues. The sequence of 233 amino acids representing the mature enzyme exhibits high similarity to sequences of serine proteinases isolated from crotalid venoms.     

Cloning and sequence analysis of cDNA encoding a putative juvenile hormone esterase from the Colorado potato beetle

In the Colorado potato beetle, Leptinotarsa decemlineata, reproduction and diapause are mediated by the juvenile hormone (JH) titer in the hemolymph. This titer is controlled by JH synthesis in the corpora allata and by JH degradation. The main pathway of JH degradation is by JH esterase in the hemolymph. The native JH esterase appeared to be a dimer consisting of two 57 kDa subunits (Vermunt et al., 1997). The 57 kDa subunit of JH esterase was digested with endoproteinase Lys-C and the digestion products were separated by reversed phase HPLC. Three different peptides were collected and sequenced. The amino acid sequence of one peptide showed high similarity to fragments of other insect esterases. Based on the amino acid sequence of these peptides, degenerate primers were constructed for RT-PCR. A PCR product of 1.3 kb was obtained and sequenced. This product was used to screen a cDNA library for a complete cDNA copy and to analyze the messenger RNA from larvae and adult beetles. The size of the messenger RNA was 1.7 kb. The complete amino acid sequence of the protein was deduced from the nucleotide sequence of overlapping clones from a cDNA library and a 5'RACE product. An open reading frame (ORF) of 1545 base pairs encoded a 57 kDa protein with a predicted pI of 5.5. The ORF contained the sequence of the three peptides. It showed no significant homology to other proteins present in databases, but it did contain several functional esterase motifs.     

Cloning and sequence analysis of cDNA encoding rat carboxypeptidase D

Carboxypeptidase D (CPD) is a recently described 180-kD enzyme with carboxypeptidase E-like enzymatic properties. CPD has been proposed to be present in the secretory pathway and to contribute to peptide hormone processing in the Cpe(fat)/Cpe(fat) mouse, which lacks functional CPE. Sequence analysis of cDNA clones encoding rat CPD show the protein to contain an amino-terminal signal peptide, three carboxypeptidase-like domains, a putative transmembrane domain, and a 60-amino-acid cytoplasmic tail. Whereas active site, substrate-binding, and metal-binding residues of other metallocarboxypeptidases are conserved in the first two domains of CPD, several of the critical residues are not conserved in the third domain; this third domain is not predicted to form an active carboxypeptidase. The overall homology between rat CPD and the duck homolog gp180 is high, with 75% amino acid identity. The three carboxypeptidase domains show 66%, 83%, and 82% amino acid identity between rat CPD and duck gp180. Homology is also high in the transmembrane domain (86%) and in the cytoplasmic tail (97%). The mouse Cpd gene maps to the medial portion of chromosome 11, approximately 45.5 cM distal to the centromere. Northern blot analysis of CPD mRNA shows major bands of approximately 8 and 4 kb in many rat tissues, and additional species ranging from 1.4 to 5 kb that are expressed in some tissues or cell lines. CPD mRNA is detectable in most tissues examined, and is most abundant in hippocampus, spinal cord, atrium of the heart, colon, testis, and ovaries. In situ hybridization of CPD mRNA shows a distribution in many cells in rat brain and other tissues, with high levels in hippocampus, olfactory bulb, and the intermediate pituitary. The broad distribution is consistent with a role for CPD in the processing of many peptides and proteins that transit the secretory pathway.     

cDNA cloning and sequence analysis of the bovine adrenocorticotropic hormone (ACTH) receptor

We isolated five independent cDNAs of nearly 3000 bp for the bovine ACTH receptor by screening adrenal cortex cDNA libraries with a PCR cloned cDNA fragment. The deduced receptor sequence includes 297 residues (M(r) = 33,258) with 81% identity with the human ACTH receptor, and shows seven hydrophobic transmembrane domains. The calculated M(r) of the receptor is smaller than the 40-45 kDa observed in crosslinking studies with labeled ACTH. Since the bovine and human receptors have two glycosylation motifs in the N-terminus, the difference may result from glycosylation of the receptor. Analysis of the sequences of both bovine and human receptors revealed a single protein kinase. A phosphorylation motif located in the third intracellular loop (Ser-209) juxtaposed to a protein kinase C phosphorylation motif (Thr-204). Thus, the involvement of protein kinase A and C pathways in ACTH action may be mediated in part by phosphorylation of the ACTH receptor at these motifs. The 3'-untranslated region of the bovine cDNA is > 2000 bp and includes two inverse repeats giving an extensive and strong secondary structure to the ACTH receptor RNA.     

Purification and some properties of two phospholipases and a toxin from the venom of desert cobra (Walterinnesia aegyptia)

An acidic and a basic phospholipase A (PLA) and a toxin have been purified from the venom of W. aegyptia using a TSK G 3000 SW gel filtration column and a Mono Q ion-exchange column. Both phospholipases and the toxin were found to be in a homogeneous state on SDS-PAGE and their purity was verified by isoelectric focusing. The acidic PLA showed higher enzymatic activity and concentration in venom when compared to basic PLA. The acidic and the basic PLA showed 16.5 +/- 0.5 Kd and 14.5 +/- 0.5 Kd molecular weights respectively, while the toxin showed 12.0 +/- 0.5 Kd molecular weights on SDS-PAGE. The isoelectric points for the acidic and basic PLA were at pH 4.5 and 7.8 respectively. The toxin was focused in the basic region at pH 10. Both phospholipases constitute about 20% of the venom protein and were nontoxic. However, only basic PLA when mixed with the toxin reduced the time required by the toxin alone to kill the mice, while the toxin was highly lethal to mice and its LD50 was 0.4 micrograms/g of body weight of mouse. The toxin showed on phospholipase activity.     

Cloning and DNA sequence analysis of an aac(3)-Vb gene from Serratia marcescens

The AAC(3)-V resistance mechanism is characterized by high-level resistance to the aminoglycosides gentamicin, netilmicin, 2'-N-ethylnetilmicin, and 6'-N-ethylnetilmicin and moderate resistance levels to tobramycin. Serratia marcescens 82041944 contains an AA(3)-V resistance mechanism as determined from aminoglycoside resistance profiles. This strain, however, does not exhibit hybridization with a probe derived from the previously cloned aac(3)-Va gene, (R. Allmansberger, B. Br?u, and W. Piepersberg, Mol. Gen. Genet. 198:514-520, 1985). High-pressure liquid chromatography analysis of the acetylation products of sisomicin carried out by extracts of S. marcescens 82041944 have demonstrated the presence of an AAC(3) enzyme. We have cloned the gene encoding this acetyltransferase and have designated it aac(3)-Vb. Nucleotide sequence comparisons show that the aac(3)-Va and aac(3)-Vb genes are 72% identical. The predicted AAC(3)-Vb protein is 28,782 Da. Comparisons of the deduced amino acid sequences show 75% identity and 84% similarity between the AAC(3)-Va and AAC(3)-Vb proteins. The use of a DNA fragment internal to the aac(3)-Vb as a hybridization probe demonstrated that the aac(3)-Vb gene is very rare in clinical isolates possessing an AAC(3)-V mechanism.     

Purification, properties, and N-terminal amino acid sequence of a kallikrein-like enzyme from the venom of Lachesis muta rhombeata (Bushmaster)

An analytical procedure was developed to measure bromate residues in baked goods using a sequence of clean-up procedures followed by high performance liquid chromatography (HPLC) with a post-column reaction for oxidants. Deionized water was used to extract bromate from bread samples. The extract was treated with a C-18 solid phase extraction column to remove lipids, a cation exchange column with the silver cation to remove chloride, and an ultrafiltration membrane to remove proteins. Further treatment of the extract with the sodium form of a propylsulphonic acid ion exchange column was necessary to remove the silver that leached from the silver column. The method had a detection limit of 3 ng/g in baked goods. Recoveries of bromate from breads ranged from 73 to 86% at a fortified bromate level of 5-100 ng/g. Pullman-type white bread, produced by a sponge and dough method, was prepared in our laboratory for measurement of residual bromate. The dough was scaled in three different weights at different specific volumes (3.8, 4.1, 4.3), and samples of each of the three weights were baked for six different baking times ranging from 24 to 34 min. When bromate at a level of 25 mg/kg was added to flour, no residual bromate was detected in any of the samples, regardless of weight and baking time.     

Snake venom toxins. The amino-acid sequence of a short-neurotoxin homologue from Dendroaspis polylepis polylepis (black mamba) venom

The conformation of 5'-nucleotides in the active site of glycogen phosphorylase b has been deduced from linewidth measurements of protons H-1', H-8 and H-2. It is shown by selective deuteration of the purine ring in position 8 that the orientation of the base is anti in the case of strong activators like AMP and syn in that of weak activators like IMP. The orientation correlation time of the nucleotides in the active site is nearly that of the enzyme, i.e. 160 ns at 21 degrees C.     

Cloning and sequence analysis of the gene (eprA1) encoding an extracellular protease from Aeromonas hydrophila

A gene (eprA1) encoding the extracellular protease of Aeromonas hydrophila AH1 has been cloned and sequenced. Nucleotide sequence analysis of eprA1 predicted a single open reading frame (ORF) of 1038 bp encoding a 346 amino acid (aa) polypeptide, with a potential 21-aa signal peptide. When the eprA1 gene was expressed in minicells, one major band of approx. 37 kDa was identified, while protease activity staining experiments identified a caseinolytic band of approx. 29 kDa determined by SDS-PAGE analysis of the minicells. The deduced C-terminal aa region (Arg-290 to Gly-313) showed sequence homology to partial C-terminal sequences of other zinc metalloproteases including Penicillium citrinum metalloprotease (PlnC), Aspergillus oryzae metalloprotease (NpII), Aspergillus flavus metalloprotease (MepA), and Aspergillus fumigatus metalloprotease (Mep20), particularly with respect to zinc-binding residues.     

Cloning, sequencing and analysis of cDNA encoding bovine intercellular adhesion molecule-1 (ICAM-1)

Intercellular adhesion molecule-1 (ICAM-1) is an inducible glycoprotein that interacts with the leukocyte beta 2-integrins, LFA-1 and Mac-1. We have isolated and analyzed a cDNA clone coding for the putative bovine ICAM-1 gene and compared it with known comparative sequences from other species as well as bovine ICAM-3. The 3398-bp bovine ICAM-1 cDNA sequence codes for 535 amino acids and shows 57% homology with human ICAM-1 and 47% homology with bovine ICAM-3 at the amino acid levels. The predicted number and positions of cysteine residues in bovine ICAM-1 are all conserved among species including bovine ICAM-3. It has two arginine-glycine-aspartate (RGD) sites in the extracellular region and a serine residue in the cytoplasmic tail. Northern blot results show that the bovine ICAM-1 gene is expressed in stimulated leukocytes whereas bovine ICAM-3 is expressed predominantly in resting neutrophils.     

Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum malic enzyme

The nucleotide sequence of a full-length cDNA encoding NAD(+)-malic enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2269 bases comprises a 5'-leader, a single open reading frame of 1851 bases, and the complete 3'-noncoding region of 340 bases. The first 12 amino acids of the translated sequence are hydrophobic, typical of mitochondrial translocation signals, and do not appear in the purified mature protein. The mature protein contains 605 amino acids and has a molecular mass of 68,478 Da. The amino acid sequences of tryptic peptides from the purified protein and also the N-terminal sequence show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the ascarid protein with the human and rat liver NAD(+)-malic enzymes reveals highly conserved regions interrupted with long stretches of lesser homologous sequences. Structural motifs such as the putative nucleotide binding domains and also the malate binding site are clearly identified by alignment of the three protein sequences.     

Cloning, expression and sequence analysis of the SphI restriction-modification system

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5'-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3' end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.