Costante Ceccarini

The biochemical relationship between trehalase and trehalose during growth and differentiation in the cellular slime mold, Dictyostelium discoideum☆

Abstract 1. 1. The hydrolytic enzyme trehalase (trehalose i -glucohydrolase, EC 3.2.1.28) was studied during the life cycle of the cellular slime mold Dictyostelium discoideum. The enzyme is found at the amoebae stage but at aggregation it is preferentially released and can be recovered in the external medium. 2. 2. Once trehalase is released, the cells are capable of synthesizing the enzyme de novo only upon feeding Escherichia coli. 3. 3. Aggregation per se is not necessary for this release but conditions suitable for aggregation must be satisfied. 4. 4. Upon spore germination the enzyme reappears and the non-reducing disacharide, trehalose, is converted to glucose. Glucose gradually disappears from amoebae so that by aggregation both glucose and trehalose are detected in very small quantities. It is suggested that the breakdown of trehalose is necessary for spore germination.

Trehalase from Dictyostelium discoideum: purification and properties.

The hydrolytic enzyme, trehalase, was isolated and purified approximately 90-fold from the cellular slime mold Dictyostelium discoideum. The purified trehalase has an optimal temperature of 45�C and shows maximum activity at pH 5.5 in citrate buffer. Its Michaelis constant is 1.2x 10-3M.

Thyroid hormone effect on α-fetoprotein and albumin coordinate expression by a human hepatoma cell line

The action of triiodothyronine on the production of alpha-fetoprotein and albumin in serum-free cultures of Hep G2 human hepatoma cells was examined. Our data showed that a marked inhibition (up to 8-fold) of alpha-fetoprotein secretion and an increase in albumin (up to 4-fold) are produced by 10(-8) M triiodothyronine. These effects were slow in their onset and for completion required 20-25 days of treatment with the hormone. However, an exposure of the cells to triiodothyronine for only the first 4 h was sufficient to affect, in a similar way, the secretion of alpha-fetoprotein and albumin when measured 15 days after treatment. The secretion of the two proteins parallels their intracellular levels. The decrease in alpha-fetoprotein production can be explained by a reduction of the RNA coding for the protein. The same is essentially true also for albumin increased secretion and related mRNA expression.

Fractionation of ovalbumin glycopeptide AC-C by high-pressure liquid chromatography Determination of structure by 1H-NMR spectroscopy

We describe for the first time a method for the separation of intact ovalbumin glycopeptides by high-pressure liquid chromatography (HPLC). The separation was achieved using two reverse-phase columns connected in series and eluting with an isocratic solvent system at acid pH containing 1-hexane sulfonate. Ovalbumin glycopeptide fraction AC-C has been separated into at least four distinct glycopeptides. High resolution 1H-NMR spectroscopy has confirmed the reported structure of the two major species. We also extend our structural studies to the two other glycopeptides and establish the structure of a previously unreported ovalbumin glycopeptide, Man3GalGlcNAcAsn.

Determination of the structure of ovalbumin glycopeptide AC-B by 1H nuclear magnetic resonance spectroscopy at 500 MHz.

Three unique, unmodified ovalbumin glycopeptides were separated to homogeneity by high-pressure liquid chromatography. The nuclear magnetic resonance data, at 500 MHz, confirmed the structure of two of the three species and for the first time established the presence of a Man8GlcNAc2Asn glycopeptide in ovalbumin. This compound was a single homogeneous isomeric form out of three possible compounds expected as processing intermediates.

Identification of a novel glycopeptide species that correlates with differentiation of F9 cells

The high-molecular-weight fucosyl glycopeptides of differentiated F9 cells have been analyzed. We found that these high-molecular-weight surface structures contain two components with different molecular weights, the largest of which, peak I, has never before been reported. The material eluting in this peak seems to contain only acidic species. Removal of sialic acid from both the peak I and the peak II species does not eliminate the differences in molecular weight, indicating that the two species have more profound structural differences than can be accounted for by sialic acid. Since peak I glycopeptides were found both in differentiated F9 cells and in two parietal endoderm cell lines, we suggest that its presence is related to parietal endoderm differentiation.