[In situ studies of myoinositol-1-P synthase in wild and inos- strains of 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.
Departamento de Bioquímica, Fac. de Medicina, U.N.A.M.
I P
IP
Del Arenal Mena
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Flores
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Escamilla
D002463:Cell Membrane Permeability / Q000187:drug effects; D005656:Fungal Proteins / Q000378:metabolism*; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism; D007294:Inositol / Q000096:biosynthesis*; D007700:Kinetics; D016296:Mutagenesis; D007296:Myo-Inositol-1-Phosphate Synthase / Q000235:genetics / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009492:Neurospora crassa / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D014050:Toluene / Q000494:pharmacology
D005656:Fungal Proteins; D005958:Glucosephosphates; D009243:NAD; D014050:Toluene; D007294:Inositol; D019298:Glucose-6-Phosphate; D007296:Myo-Inositol-1-Phosphate Synthase
Estudios "in situ" de la mioinositol 1-P sintasa en cepas silvestre e inos- de Neurospora crassa.