Proliferative stimulation of lymphocytes by calmodulin binding proteins isolated from amniotic fluid.

In view of the role of calmodulin and calmodulin binding proteins in modulating the second messenger functions of Ca2+, we studied the presence of such proteins in amniotic fluid, which may be considered as a dynamic medium for promoting foetal growth. Affinity chromatography of amniotic fluid proteins revealed the presence of calmodulin binding proteins in samples obtained either at 28 or 36 wk of pregnancy. The relative content of these proteins increased in amniotic fluid from 1.5 mg/g total protein at 28 wk to 3.6 mg/g at full term of pregnancy. Culturing murine splenocytes in presence of the isolated calmodulin binding proteins (10 micrograms protein/10(6) cells) resulted in nearly 4-fold enhancement of 3H-thymidine incorporation into them as compared to controls. In comparison, similar incorporation of the radiolabel into lymphocytes obtained from cord blood was enhanced only by 2 fold in presence of calmodulin binding proteins, though at a much lower protein concentration (50 ng/10(6) cells). SDS-PAGE on 12.5 per cent gels of eluates obtained from calmodulin-agarose columns, followed by overlay of corresponding western blots with biotinylated calmodulin revealed the presence of a 68 kDa calmodulin binding protein in samples collected either at 28 wk or at full-term of pregnancy. In addition, the amniotic fluid also contained 83 kDa calmodulin binding protein at 28 wk. This first-time demonstration of mitogenic, calmodulin binding proteins in amniotic fluid suggests that such mitogens may participate in promoting foetal growth.

Isolation and characterization of a copper containing protein from blue cell walls of Neurospora crassa.

Culturing Neurospora crassa in presence of toxic amounts of copper (0.63 mM) resulted in blue coloured mycelia and cell walls. Significant amounts (approximately 45%) of total mycelial copper were associated with cell wall isolates under conditions of copper toxicity. Hence, such blue cell walls were analysed to identify specific ligands involved in copper binding. While decuprification of the blue cell walls with 8-hydroxy quinoline (8 HQ) did not alter their copper binding abilities, similar treatment with EDTA (10 mM) decreased such abilities indicating that EDTA treatment lead to loss of copper binding ligands from cell walls. Treatment of blue cell walls with 8 HQ followed by EDTA resulted in the solubilization of a copper binding protein (relative MW approximately 14 kDa) which was associated with phosphate and carbohydrate moieties. On amino acid analysis, this protein was found to be devoid of free thiol groupings but enriched in acidic and basic amino acids, distinguishing it from classical intracellular metal binding proteins such as metallo-thioneins and phytochelatins that are inducively synthesized under conditions of metal toxicity. The biological significance of the isolated wall-bound copper binding protein, which appears to be a normal constituent of cell walls, is discussed in relation to cytoplasmic metal binding proteins and mechanism(s) adapted by fungi in countering metal toxicity.

An enzymic method for the determination of chitin and chitosan in fungal cell walls.

The free and N-acetyl glucosamine contents, serving as a measure of the amounts of chitosan and chitin respectively, were determined in the chitinase hydrolysates of the cell wall of a wild strain of Neurospora crassa. Chitinase, obtained from cultures of Serratia marcescens, could hydrolyse the cell wall completely apart from being capable of hydrolysing preparations of chitin and chitosan. The free and N-acetyl glucosamines, released by chitinase hydrolysis, were determined by a modified Morgan-Elson reaction carried out in the presence and absence of acetic anhydride. The method is capable of estimating chitin and chitosan contents in as little as 100 μg of cell wall material.

The effect of copper on histidine biosynthesis in Neurospora crassa.

The inhibition of growth of a wild strain of Neurospora crassa by Cu2+ is counteracted by histidine, histidine methyl ester, histidinol and Mn2+. In the presence of Cu2+, the total free amino acid content decreased by 30%. The decreased free amino acid pools of arginine, histidine and tyrosine were restored on the addition of Mn2+. Histidinol phosphate phosphatase showed a decrease in activity in the presence of Cu2+. This inhibition was reversed on the addition of excess Mn2+.. The data suggest that copper toxicity in the mould is due to suppression of histidine biosynthesis.