Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition.

Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified >133-fold with a total yield of 9.7%. A molecular mass of 11.7kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1µM, a kcat of 10s(-1) and a catalytic efficiency of 3.2×10(6)M(-1)s(-1). The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins.

A method for the isolation of tegument syncytium mitochondria from Taenia crassiceps cysticerci and partial characterization of their aerobic metabolism.

Heterogeneous populations of mitochondria have been described in helminths. Mitochondria from different tissues have been isolated in adult organisms. However, in larvae, due to their small size, isolation from tissues has not been feasible. A method for the isolation of tegumental mitochondria from the larval stage of Taenia crassiceps is described. After solubilization of the plasma membrane with saponin, tegumental mitochondria were purified by a simple and rapid protocol of differential centrifugation, which allowed the retention of suitable quantities of well-preserved mitochondria, as judged by biochemical and ultrastructural parameters. Respiratory activity evoked by exogenous NADH was negligible, but its oxidation increased several-fold after sonication of intact mitochondria. Other substrates, e.g., succinate and malate-glutamate, were oxidized at high rate, leading to the formation of a H+ gradient across the inner mitochondrial membrane, which, in turn, supported oxidative phosphorylation. These results indicate that tegumental mitochondria carry out aerobic metabolism.

[In situ studies of myoinositol-1-P synthase in wild and inos- strains of Neurospora crassa]. / Estudios "in situ" de la mioinositol 1-P sintasa en cepas silvestre e inos- de Neurospora crassa.

The biosynthetic pathway for myo-inositol consist of two enzymatic steps: first, the cycloaldolization of glucose-6P to L-myo-inositol-IP followed by its hydrolysis to form free myo-inositol. The former reaction is catalyzed by myo-inositol-IP synthase (MIPS) while, a phosphatase is responsible for the hydrolysis step. Depending on its degree of purification and storage age, MIPS activity us to be, from partial to fully, dependent on added NAD. Therefore, we decided to study the kinetic properties of the enzyme within the cell, specially its requirements for free NAD. To this purpose, a method was designed for the assay of MIPS-activity in situ, using toluene permeabilized mycelia. MIPS-activity "in situ" was fully displayed in the absence of added NAD; on the contrary, the purified enzyme showed only 33% of that activity displayed when NAD was included in the assay. Thus, it seems that the native enzyme contains tightly bound NAD, instrumental for its activity, and that during purification or storage, the coenzyme is progressively lost, rendering the NAD-dependent enzyme, as was previously envisage. In addition, the in situ assay method for MIP-Synthase was applied to several mutants of N. crassa having the inosphenotype. Our results showed that only in 3 of 14 cases analyzed the phenotype could be clearly associated to the lack of MIP-synthase activity. Indeed most of the mutants analyzed showed significant levels (from 5 to 21%) of MIP-synthase, when compared to the activity shown by the RL-21 WT strain. Finally, all the mutants and WT strains were zymographically analyzed for phosphatase activity and showed close to equal strong reaction levels.