Descripción: Fil:González, N.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Descripción: Fil:Chelala, C.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Descripción: Fil:Tigier, H.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Descripción: Fil:Ielpi, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Descripción: 1. 1. Uroporphyrinogen carboxy-lyase (EC 4.1.1.d), the enzyme catalysing the decaroxylation of uroporphyrinogen to coproporphyrinogen, has been isolated from normal chicken erythrocytes. The enzyme was purified 220-fold with a yield of 24% from haemolysate supernatant by DEAE-cellulose batch treatment, (NH4)2SO4 fractionation and chromatography on DEAE-cellulose. 2. 2. The purified material appears to be homogeneous in polyacrylamide gel disc electrophoresis. 3. 3. The enzyme was heat labile and inhibited by sodium salt; the activity was enhanced by EDTA, GSH and boiled rat-liver extract. 4. 4. The influence of these chemical and physical agents on the removal of the first and second carboxyl groups from uroporphyrinogen was compared; the second group was more susceptible to these agents. 5. 5. The possibility that one or several enzymes were involved in the stepwise decarboxylation of uroporphyrinogen is discussed. 6. 6. The general name of porphyrinogen carboxy-lyase for the enzyme system is proposed because of the different porphyrinogens it can decarboxylate. © 1970.

Descripción: The chvA gene product of Agrobacterium tumefaciens is required for virulence and attachment of bacteria to plant cells. Three chvA mutants were studied. In vivo, they were defective in the synthesis, accumulation, and secretion of beta-(1-2)glucan; however, the 235-kilodalton (kDa) protein known to be involved in the synthesis of beta-(1-2)glucan (A. Zorreguieta and R. Ugalde, J. Bacteriol. 167:947-951, 1986) was present and active in vitro. was present and active in vitro. Two molecular forms of cyclic beta-(1-2)glucan, designated types I and II, were resolved by gel chromatography. Type I beta-(1-2)glucan was substituted with nonglycosidic residues, and type II beta-(1-2)glucan was nonsubstituted. Wild-type cells accumulated type I beta-(1-2)glucan, and chvA mutant cells accumulated mainly type II beta-(1-2)glucan and a small amount of type I beta-(1-2)glucan. Inner membranes of wild-type and chvA mutants formed in vitro type II nonsubstituted beta-(1-2)glucan. A 75-kDa inner membrane protein is proposed to be the chvA gene product. chvA mutant inner membranes had increased levels of 235-kDa protein; partial trypsin digestion patterns suggested that the 235-kDa protein (the gene product of the chvB region) and the gene product of the chvA region form a complex in the inner membrane that is involved in the synthesis, secretion, and modification of beta-(1-2)glucan. All of the defects assigned to the chvA mutation were restored after complementation with plasmid pCD522 containing the entire chvA region.

Descripción: Although the existence of O-linked oligosaccharide residues in glycoproteins of Plasmodium falciparum has been shown, the existence of N- linked glycoproteins is still a matter of controversy and skepticism. This report demonstrates the unequivocal presence of N-linked glycoproteins in P. falciparum, principally in the ring and young trophozoite stages of the intraerythrocytic cycle. These glycoproteins lose their capacity to bind to concanavalin A-Sepharose after treatment of cultures with tunicamycin under conditions that do not affect protein synthesis. When the glycoproteins were treated with N-Glycanase®, oligosaccharides were released. It was possible to identify an N-linked glycoprotein of >200 kDa in the ring stage and also N-linked glycoproteins in the range of 200-30 kDa in the trophozoite stage. Treatment of trophozoites with 12 μM tunicamycin inhibited differentiation to the schizont stage. To our knowledge, this is the first report in the literature unequivocally showing N-linked glycoproteins in trophozoites of P. falciparum as well as their importance for the differentiation of the intraerythrocytic stages of this parasite.

Descripción: Comparative studies on fatty acid and protein composition of the endosperm and embryo of palmito (Euterpe edulis Martius) were conducted using gas-liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On a dry weight basis, the embryo contained extremely lower amounts of lipids and proteins than did the endosperm, which was associated with the scarce lipid and protein bodies previously reported in axis and cotyledon. The fatty acid composition also exhibited differences between both tissues: (I) the fatty acid diversity was greater in embryo than in endosperm; (II) embryo and endosperm contained predominantly linoleic, palmitic, oleic and stearic acids even though the relative values were different for each tissue. As compared to other palm species, the higher fatty acid unsaturation in Euterpe edulis seed could be involved in the previously reported short longevity and recalcitrant behavior during storage. Proteins of both tissues were heterogeneous in molecular mass. Some proteins were tissue-specific, but other were common, among them a highly glycosylated protein which migrated at about 55 kDa. We hypothesize that the latter, also reported in all previously studied palm species, is one of the proteins characterizing the Arecaceae family.

Descripción: Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 μg) with Freund adjuvant. Groups of six mice (20 ± 2 g) were inoculated with 0.5 ml i.p. of C. d. t. venom (4 μg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms.

Descripción: 1. 1.|Porphobilinogenase has been isolated and purified from cow liver and its components, porphobilinogen deaminase and uroporphyrinogen isomerase, have been separated from each other and purified. 2. 2.|The effect of NH4+ was studied. The deaminase exhibited classical Michaelis-Menten kinetics in the absence or presence of NH4+, which at high concentrations behaved as a noncompetitive inhibitor of the deaminase. As expected from Hill plots, n = 1 both in the absence or presence of NH4+. Instead, when activity of porpho bilinogenase is plotted versus porphobilinogen concentration, sigmoid curves are obtained; but the presence of NH4+ at different concentrations altered the kinetic parameters of this enzymic system, again showing normal kinetics. In addition, n values were found to be 2 for porphobilinogen per porphobilinogenase molecule and 1 in the presence of NH4+ which behaves as a competitive inhibitor of the isomerase. Results are discussed in relation to the allosteric theories of monod et al.1,2 and liver porphobilinogenase is proposed to be an allosteric protein. 3. 3.|The presence of an ultrafiltrable factor which stimulates uroporphyrinogen formation from porphobilinogen has been revealed. © 1969.

Descripción: 1. 1. A method for the isolation and purification of porphobilinogenase, porphobilinogen deaminase and uroporphyrinogen isomerase from avian erythrocytes is described. 2. 2. Some properties of the isolated enzymes were studied. The optimal pH for porphobilinogenase and deaminase was 7.4. Purified porphobilinogenase was resolved into three bands on starch gel electrophoresis. The molecular weight of the purified enzymes was determined by gel filtration. The presence of porphobilinogen or NH4 + at certain concentrations afforded protection against heat inactivation of isomerase, the heat labile enzyme. Initial porphyrin formation by porphobilinogenase was linear with time. 3. 3. The action of various compounds added to the system was studied. Thiol reagents inhibited both porphobilinogenase and deaminase, indicating the presence of thiol groups essential for activity. NH4 +, hydroxylamine, adenine, ADP, ATP, some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited deaminase. © 1971.

Descripción: 1. 1. Preliminary experiments with Euglena gracilis indicated that an endogenous factor which modified enzymic synthesis of porphyrinogens from PBG, was present in Homogenates (H) and Supernatant (S) fractions. 2. 2. When H or S was stored at 4-6°C, enzymic activity underwent an apparent spontaneous activation, increasing by as much as 7.5-8 times after 14 and 22 days of aging respectively. 3. 3. Experiments were carried out to detect, isolate and identify this factor. S and H were heated and the effect of the protein free supernatants (Hø,Sø) on activity were tested. By gel filtration of H and S, a low molecular weight compound (FH, FS) was separated, and the activity of the eluated protein (PrH, PrS) was enhanced 10-12 times. 4. 4. Addition of either Hø, Sø, FH or FS to different E. gracilis preparations increased their activities, suggesting the existence of a low molecular weight, heat-stable factor which would act stimulating enzymic synthesis of porphyrinogens. However some differences in the properties of the factor present in Hø or Sø and that present in FH or FS were observed. 5. 5. Studies on the FH and FS, confirmed that the factor was heat-stable, upon storage at 4°C its activation properties were not modified; but they were destroyed by basic or acid treatment. 6. 6. The same degree of activation as that produced by FH and FS on H, S, PrH and PrS, was obtained by replacing the factor solution by 10-7M folic acid or 10-3-10-2M Gluthation (GSH); however, neither the factor nor folic acid or GSH has any effect on the pellet enzyme, either bound to the membrane or solubilized by means of a chaothropic agent. 7. 7. The potential use of this factor in the treatment of acute porphyrias was indirectly investigated, by treating acute intermittent porphyria patients in early or acute attack with folic acid; after its oral administration at a dose of 30 mg daily for not longer than 10 days, both biochemical and clinical recovery followed. 8. 8. A scheme to explain the role of this factor in acting and controlling porphobilinogenase activity in E. gracilis is proposed. © 1981.

Descripción: Enzyme preparations from Xanthomonas campestris incubated in the presence of UDP-[14C]GlcA and Mg2+ produced a lipophilic galacturonide with unusual properties. It was easily degraded by both mild acid treatment (0.01 M-HCl, 100°C, 10 min) and mild alkali treatment (0.06 M-NaOH, room temperature, 5 min) releasing free [14C]galacturonic acid. The galacturonide appeared to be a single compound with one negative charge, as judged by TLC, paper electrophoresis and chromotography, LH-20 gel filtration and DEAE-cellulose column chromatography. Competition experiments indicated that the true glycosyl donor was UDP-GalA, in agreement with the detection of UDP-GlcA-4-epimerase activity in the crude enzyme preparation. The transglycosidase activity was located mainly in the membrane fraction. UDP inhibited the reaction and even produced some loss of label, suggesting an easily reversible reaction. UMP had almost no effect.

Descripción: A peptide from hindguts of the Triatoma hematophagous Chagas insect vector activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes to metacyclic trypomastigotes. Hindguts were obtained from insects fed 2 days earlier with chicken blood. Purification was performed by gel filtration and HPLC on C18 and C4 columns. SDS/PAGE of the purified peptide showed a single band of about 10 kDa. The following sequence was determined for the 20 amino-terminal residues of this peptide: H 2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln-Gln-Ala-Trp-Glu-Lys- Ala-Ala-Ser-His. This sequence is identical to the amino terminus of chicken αD-globin. On a Western blot, the peptide immunoreacted with a polyclonal antibody against chicken globin D. A synthetic peptide corresponding to residues 1-40 of the αD-globin amino terminus also stimulated adenylyl cyclase activity and promoted differentiation. This 125I-labeled synthetic peptide bound specifically to T. cruzi epimastigote cells. Activation of epimastigote adenylyl cyclase by the hemoglobin-derived peptide may play an important role in T. cruzi differentiation and consequently in the transmission of Chagas disease.