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Instituto de Biologia Unicamp






ABSTRACTA IB (2) - 1999


P001-99 Purification, crystallization and preliminary crystallographic study of a Kunitz-type trypsin inhibitor from Delonix regia seeds

Polikarpov I, Golubev AM, Perles LA, Pando SC, Novello JC,
Marangoni S*

The Kunitz-type trypsin inhibitor from seeds of flamboyant (Delonix regia) has been purified to homogeneity. Plate-like crystals suitable for X-ray analysis have been grown by the hanging-drop method using PEG 6000 as a precipitant. The crystals belong to space group P2(1)2(1)2(1) with unit-cell parameters a = 32.15, b = 69.39, c = 72.54 Angstrom. X-ray diffraction data have been collected to 2.95 A resolution. The structure has been solved by molecular replacement using the known structures of trypsin inhibitors from Erythrina caffra seeds and from soya beans as search models. Acta Crystallographica Section D-Biological Crystallography 55: 1611-1613, 1999 IF= 2.118

P002-99 The amino acid sequence of ribitol dehydrogenase-F, a mutant enzyme with improved xylitol dehydrogenase activity

Homsi-Brandeburgo MI, Toyama MH, Marangoni S*, Ward RJ, Giglio JR, Hartley BS

A mutant ribitol dehydrogenase (RDH-F) was purified from Klebsiella aerogenes strain F which evolved from the wild-type strain A under selective pressure to improve growth on xylitol, a poor substrate used as sole carbon source. The complete amino acid sequence of RDH-F showed the mutations Q60 for E60 and V215 for L215 in the single polypeptide chain of 249 aa residues. Structural modeling based on homologies with two other microbial dehydrogenases suggests that E60 --> Q60 is a neutral mutation, since it lies in a region far from the catalytic site and should not cause structural perturbations. In contrast, L215 --> V215 lies in variable region II and would shift a loop that interacts with the NADH cofactor. Another improved ribitol dehydrogenase, RDH-D, contains an A196 --> P196 mutation that would disrupt a surface alpha-helix in region II. Conformational changes in this region may be responsible for the improved xylitol specificity. Journal of Protein Chemistry 18(4): 489-495 , 1999 IF= 1.060

P003-99 Crystal structure of neurotoxin Ts1 from Tityus serrulatus provides insights into the specificity and toxicity of scorpion toxins

Polikarpov I, Matilde MS, Marangoni S*, Toyama MH, Teplyakov A

The crystal structure of neurotoxin Ts1, a major component of the venom of the Brazilian scorpion Tityus serrulatus, has been determined at 1.7 Angstrom resolution. It is the first X-ray structure of a highly toxic anti-mammalian beta-toxin. The folding of the polypeptide chain of Ts1 is similar to that of other scorpion toxins. A cysteine-stabilised alpha-helix/beta-sheet motif forms the core of the flattened molecule. All residues identified as functionally important by chemical modification and site-directed mutagenesis are located on one side of the molecule, which is considered as the Na+ channel recognition site. The distribution of charged and non-polar residues over this surface determines the specificity of the toxin-channel interaction. Comparison to other scorpion toxins shows that positively charged groups at positions 1 and 12 as well as a negative charge at position 2 are likely determinants of the specificity of beta-toxins. The contribution of the conserved aromatic cluster to the interaction might be relatively small. Comparison of Ts1 to weak beta-toxins from Centruroides sculpturatus reveals that a number of basic amino acid residues located on the face of the molecule opposite to the binding surface may account for the high toxicity of Ts1.Journal of Molecular Biology 290: 175-184, 1999 IF=5.673

P004-99 Crystallization and preliminary X-ray diffraction studies of piratoxin III, a D-49 phospholipase A(2) from the venom of Bothrops pirajai

Wen-Hwa L, Toyama MH, Soares AM, Giglio JR, Marangoni S*, Polikarpov I

Crystals of piratoxin III (PrTX-III), a phospholipase A(2) (PLA(2), E.C., phosphatide sn-2 acylhydrolase) isolated from Bothrops pirajai were obtained using the vapour-diffusion technique. X-ray diffraction data have been collected to 2.7 A resolution. The enzyme was crystallized in the space group C2 with unit-cell parameters a = 60.88, b = 100.75, c= 48.19 Angstrom, beta = 123.89 degrees. Angstrom molecular-replacement solution of the structure has been found using bothropstoxin I from the venom of B. jararacussu as a search model. Acta Crystallographica Section D - Biological Crystallography 55: 1229-1230, 1999 IF= 2.118

P005-99 Purification and amino acid sequence of MP-III 4R D49 phospholipase A(2) from Bothrops pirajai snake venom, a toxin with moderate PLA(2) and anticoagulant activities and high myotoxic activity

Toyama MH, Costa PD, Novello JC, de Oliveira B, Giglio JR, da Cruz-Hofling MA, Marangoni S*

MP-III 4R PLA(2) was purified from the venom of Bothrops pirajai venom (Bahia's jararacussu) after three chromatographic steps which started with RP-HPLC. The complete amino acid sequence of MP-III 4R PLA(2) from Bothrops pirajai was determined in reduced and carboxymethylated MP-m 4R and in the isolated peptides from clostripain and protease V8 digestion. MP-m 4R is a D49 PLA(2) with 121 amino acid residues and has a molecular weight estimated at 13,800 Da, with 14 half-cysteines. This protein showed moderate PLA(2) and anticoagulant activity. This PLA(2) does not have a high degree of homology with other bothropic PLA(2)-like myotoxins (similar to 75%) and nonbothropic myotoxins (similar to 60%). MP-III 4R is a new PLA(2), which was isolated using exclusively analytical and preparative HPLC methods.. Clinical manifestations indicate pain of variable intensity at the site of toxin injection. Myotoxicity was observed in gastrocnemius muscle cells after exposure to MP-III 4R. A high frequency (70%) of muscle fibers was affected. Journal of Protein Chemistry 18(3): 371-378, 1999 IF= 1.060

006-99 Effects of Bothrops pirajai venom on the mouse extensor digitorum longus (EDL) muscle preparation

Costa PD, Toyama MH, Marangoni S*, Rodrigues-Simioni L, da Cruz-Hofling MA

The effects of Bothrops pirajai snake venom on the mouse extensor digitorum longus (EDL) preparation were examined using myographic, histopathological and biochemical assays. B. pirajai venom (10, 25 or 50 mu g/ml) dose dependently and irreversibly blocked the contractile response of indirectly stimulated EDL muscle. Histopathological analysis of EDL muscle incubated with venom showed dose-dependent damage with a loss of the normal tissue structure and the appearance of highly dark, edematous fibers together with myofibrils in various stages of condensation. Loss of muscle cells occurred at high doses of venom. In non-stimulated EDL, the venom (10 and 50 mu g/ml) caused a time-dependent release of CK which was maximal after 120 min. A component(s) present in the B. pirajai venom may have a direct myolytic action on the skeletal muscle. Toxicon 37(8): 1143-1153, 1999 IF= 1.150

P007-99 yTsTX-IV, a short-chain four-disulfide-bridged neurotoxin from Tityus serrulatus venom which acts Ca2+-Activated K+ channels: structure and functions

Novello JC, Arantes EC, Varanda WA, Oliveira B, Giglio JR, Marangoni S*

The amino acid sequence of TsTX-IV, a neurotoxin isolated from Tityus serrulatus scorpion venom was determined. The complete sequence showed 41 amino acid residues, which account for an estimated mw of 4520, and 8 half-cystine residues which cross-link the toxin molecule with four disulfide bonds. The molecular weight determined by mass spectrometry was 4518, Comparison of this sequence with those from other scorpion toxins showed a resemblance with toxins which act on different types of K+ channels, TsTx-IV was able to block Ca2+-activated K+ channels of high conductance. TsTX-IV is the first four-disulfide-bridged short toxin from T. serrulatus so far completely sequenced. Toxicon 37(4): 651-660 , 1999 IF= 1.150

P008-99 Growth Phase-Dependent Subcellular Localization of Nitric Oxide Synthase in Maize Cells

Ribeiro Jr EA , Cunha FQ, Tamashiro WMSC, Martins IS*

A protein band of approximately 166 kDa was detected in the soluble fraction of root tips and young leaves of maize seedlings, based on Western blot analysis using antibodies raised against mouse macrophage NOS and rabbit brain NOS. NOS activity was present in the soluble fractions, as determined by L-[U-14C]-citrulline synthesis from L-[U-14C]-arginine. Immunofluorescence demonstrated that the maize NOS protein is present in the cytosol of cells in the division zone and is translocated into cell nucleus at the elongation zone of maize root tips. The results thus show existence of a NOS enzyme in maize tissues, with the localization of this protein depending on the phase of cell growth. FEBS Letters 445: 283-286, 1999 IF= 3.504

P009-99 A Critical Analysis of Microcalorimetric Methodology for Drug-Induced Haemolysis

Malheiros SVP, Meirelles NC*, Volpe PLO

The interaction of trifluoperazine (TFP), dibucaine (DBC) and praziquantel (PZQ), with erythrocyte membranes was studied by measuring the drug partition coefficient between membrane and water, and also by monitoring their haemolytic activity with microcalorimetric techniques. TFP presented the highest membrane partition and best haemolytic efficacy, followed by DBC and PZQ. The heat effects of drug-induced haemolysis obtained by using a thermal activity monitor (TAM) were-18,2 and 3,6 pJ/cell for TFP and DBC, respectively. We were unable to obtain measurable heat effect for PZQ-induced haemolysis. The discrepancy in heart effect values of drug-induced lysis indicates that different pathways are involved in haemolysis establisment. Misunderstandings concerning the effect of drug-induced haemolysis, found in recent literature, lead us to critically analyzing the microcalorimetric methodology and comparing the heart effect values obtained in this work and those available in literature. Thermochimica Acta 328: 121-127, 1999 IF= 0.717

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