Hydrophobic properties of NAD glycohydrolase from neurospora crassa conidia and interaction with dioxane.

NAD glycohydrolase (NADase, EC 3.2.2.5) from Neurospora crassa conidia shows marked hydrophobic properties which are related to the self inhibition of the enzyme. Both aliphatic amines and carboxylic acids are able to inhibit noncompetitively the catalytic activity of the enzyme and the inhibition depends on the non-polar moiety of the substances. Also dioxane is an inhibitor of NAD glycohydrolase even though it apparently increases the specific activity of the enzyme. This effect can be explained by the fact that NADase is present as a dimer when the enzyme is concentrated or at high temperature, and dioxane binds the enzyme breaking the hydrophobic bonds in the dimeric enzyme and yielding the most active monomeric form which is only slightly inhibited by the organic solvent.

Phosphorylation of Opaque2 changes diurnally and impacts its DNA binding activity.

In the maize endosperm, the Opaque2 (O2) basic leucine zipper transcriptional activator regulates the expression of a subset of the zein seed storage protein gene family. Immunodetection of wild-type or mutant O2 polypeptides fractionated by SDS-PAGE resolved a closely spaced doublet migrating in the 68- to 72-kD range, whereas by using isoelectric focusing, seven to nine isoforms were detected for each allele. Phosphatase treatment simplified the protein patterns to a single band corresponding to the nonphosphorylated component. In vivo and in vitro labeling confirmed that O2 can be phosphorylated. In protein gel blots probed with DNA, only the nonphosphorylated and hypophosphorylated O2 polypeptides were able to bind an oligonucleotide containing the O2 binding sequence. Upon in situ dephosphorylation of the focused isoforms by phosphatase treatment of the isoelectric focusing filter, the hyperphosphorylated forms acquired DNA binding activity. The ratio among the various isoforms remained constant throughout the developmental stages of endosperm growth but changed from daytime to nighttime, with a significant increase of the hyperphosphorylated forms during the night period. These results indicate that O2 exists in vivo as a pool of differently phosphorylated polypeptides and demonstrate that O2 DNA binding activity is modulated by a phosphorylation/dephosphorylation mechanism that appears to be influenced by environmental conditions.

Expression of PTP35, the murine homologue of the protein tyrosine phosphatase-related sequence IA-2, is regulated during cell growth and stimulated by mitogens in 3T3 fibroblasts.

Protein tyrosine phosphatases (PTPases) have been implicated in the control of cell proliferation and differentiation. To isolate new members of this family potentially involved in cell growth regulation, we looked for PTPase sequences differently expressed in proliferating or quiescent NIH 3T3 fibroblasts. The full-length cDNA of one of these growth-regulated genes, named PTP35, was isolated from a 3T3 library and found to encode the murine IA-2 PTPase-related sequence. Endogenous PTP35 mRNA steady-state levels were found to be strictly regulated during cell growth in 3T3 fibroblasts, being high in actively cycling cells and barely detectable in density-arrested cells. Both PTP35 mRNA and protein levels could be induced in quiescent cells by mitogenic stimulation. The growth factor specificity and kinetics of this induction were analyzed in detail.

Influence of mucin type on polymer-mucin rheological interactions.

There are numerous in vitro methods with which to investigate the mucoadhesive properties of polymers. One recent method is based on the measurement of rheological interactions between polymer and mucin, which implies the use of mucins isolated from the mucous tissue. The extraction and purification of glycoprotein fraction, which is responsible for rheological interaction, can modify the native structure of mucin or spoil it with exogenous substances. Therefore the particulars of the mucin employed (origin, purification grade, the effect of further treatments such as freezing or freeze-drying) are likely to be critical for the interaction. The aim of this work was to compare some commercial mucins of differing origin and grade of purification for their rheological interaction with well-known mucoadhesive polymers (polyacrylic acid and sodium carboxymethylcellulose). For polyacrylic acid, which is sensitive to ions, we found rheological interaction to be strongly influenced by mucin type. The removal of ions, with dialysis, improved the interaction. For sodium carboxymethylcellulose, which is less sensitive to ions, rheological interaction proved to be less dependent on mucin type and improved upon glycoprotein solubilization.

Purification of NAD glycohydrolase from Neurospora crassa conidia by a polyclonal immunoadsorbent.

NAD glycohydrolase from Neurospora crassa conidia was purified by affinity chromatography on a column of polyclonal antibodies bound to an agarose matrix. The procedure was easy, non-denaturating and suitable for repetitive use of the gel. The enzyme obtained appeared homogeneous by sodiumdodecyl sulphate-polyacrylamide gel electrophoresis.