Purification and Characterization of a Human Brain Galectin-1 Ligand

Abstract: Our previous studies have characterized an endogenous lectin from human brain identified as galectin-1. A soluble ligand of galectin-1 was purified from human brain by affinity chromatography and preparative electrophoresis. The purified ligand (termed HBGp82, for human brain galectin-1-binding polypeptide of 82,000 daltons) has an apparent molecular mass of 82 kDa and is glycosylated by N -linked biantennary complex structures. HBGp82 was partially characterized by microsequencing of peptide fragments. Similar peptides were found in a heat shock of protein of 90,000 daltons, hsp90. However, comparison of apparent molecular weights and matrix-assisted laser desorption mass spectrometry clearly showed that HBGp82 differs to some degree from hsp90.     

Partial purification of a phosphoethanolamine methyltransferase from rat brain cytosol

The conversion of phosphoethanolamine to phosphocholine requires 3 separate N-methyltransferases. We had previously purified the enzyme catalyzing the last methylation, phosphodimethylethanolamine N-methyltransferase. We have successfully purified the enzyme catalyzing the initial methylation of phosphoethanolamine. A 434 fold purified enzyme from rat brain was obtained by the sequential use of ammonium sulfate fractionation, Q-Sepharose fast flow column chromatography and a -aminoethyl agarose column chromatography. The pH optimum was 11 or greater, the Km value for phosphoethanolamine was 167.8±41.7 M and the Vmax was 487.3±85 mmoles/mg/hr. The kinetics for S-adenosyl-methionine, the methyldonor, has characteristics of cooperative binding with a Km of 1.805±0.59 mM and a Vmax of 16.9±3.6 moles/mg/hr. The activity was stimulated 6 fold by 2.5 mM MnCl₂ and inhibited by DZA and S-adenosylhomocysteine. These results reinforce the early in vivo observations which had provided suggestive evidence for the existence of a pathway for the methylation of phosphoethanolamine to phosphocholine in rat brain.Abbreviations used Adomet S-adenosylmethionine - AdoHcy S-adenosyl-homocysteine - CAPS 3-(cyclohexyl)amino-1-propanesulphonic acid - Cho choline - 3-DZA 3-deazaadenosine - Etn ethanolamine - N-MT N-methyltransferase - PEG polyethyleneglycol - PMSF phenylmethanesulphonyl fluoride - PEtn phosphoethanolamine - PCho phosphocholine - PMe₂Etn phosphodimethylethanolamine - PtdCho phosphatidylcholine - PtdEtn phosphatidylethanolamine     

Effects of the organophosphate insecticide,monocrotophos, on acetylcholinesterase activity in the nile tilapia fish (Oreochromis niloticus) brain

The neurotoxic effects of monocrotophos on the brain of the nile tilapia fish (Oreochromis niloticus) were examined, using a static bioassay under laboratory conditions. By probit analysis the 96 h LC₅₀ value of monocrotophos was 4.9 mg/l. After 96 h exposure to acute levels of monocrotophos, the brain acetylcholinesterase (AChE) activity decreased progressively as the concentration of monocrotophos increased. In addition, four weeks following transfer to toxicant-free water after exposure to 1 mg monocrotophos, nile tilapia fish brain regained 95% of control AChE activity. The results indicate that inhibition of AChE activity in fish exposed to monocrotophos may serve as an indicator of hazard due to application of this chemical in the natural environment.Special issue dedicated to Dr. Robert Balazs.     

Demonstration of high opioid-like activity in isolated peptides from wheat gluten hydrolysates

Because of a possible relationship between schizophrenia and celiac disease, a condition in some individuals who are sensitive to wheat gluten proteins in the diet, there has been interest in observations that peptides derived from wheat gluten proteins exhibit opioid-like activity in in vitro tests. To determine the origin of the peptides exhibiting opioid activity, wheat proteins were fractionated by size (gel filtration), by charge differences (ion exchange chromatography) and by differences in hydrophobicity (reversed-phase HPLC). These fractions were hydrolyzed by pepsin or pepsin and trypsin and the resulting peptides separated by gel filtration chromatography. The separated peptides were tested for opioid-like activity by competitive binding to opioid receptor sites in rat brain tissue in the presence of tritium-labeled dihydromorphine. The peptides showed considerable differences in activity; while some peptides exhibited no activity, 0.5 mg of the most active peptides were equivalent to 1 nM of morphine in the binding assay. The most active peptides were derived from the gliadin fraction of the gluten complex.     

Incorporation of3H-thymidine into DNA and the activity of alkaline phosphatase in zinc-deficient fetal rat brains

The incorporation of³H-thymidine into DNA in the brains of the 17-day and 20-day old rat fetuses was significantly reduced by maternal zinc restriction during pregnancy. The activity of the enzyme thymidine kinase (EC 2.7.1.21) was similarly reduced in the zine-deprived fetal brains on days 14 and 20 of gestation, but not on day 17. Fetal brain alkaline phosphatase (EC 3.1.3.1) was significantly depressed by maternal zinc deprivation on days 17 and 20 of pregnancy. The data suggest an association between thymidine kinase and the reduced incorporation of³H-thymidine into DNA in the brains of 20-day old fetuses but not in animals on day 17. Alkaline phosphatase was however depressed at this stage. The suggestion is made that because of the complexity of brain development, future biochemical studies in this area should concern specific structures in the brain at particular critical stages during neurogenesis.     

Glycosaminoglycan synthesis by mammary tumor spheroids

Multicellular aggregates of tumorigenic mouse mammary epithelial cells contain a hyaluronate-rich matrix, both at the aggregate periphery and within the growing spheroid. It is proposed that the establishment of a hyaluronaterich matrix is essential to spheroid growth in vitro, and that the spheroid is a good model system for analysis of this aspect of early tumor development.