The cyst wall of Colpoda steinii. A substance rich in glutamic acid residues

Abstract

The cyst wall of Colpoda steinii has been isolated and its chemical nature examined. It had a nitrogen content 13.9 + 0.2% (S.D.) and an ash 8.6 + 1.6% (S.D.). After lipid and hot-acid extraction there was a variable residual phosphorus of 0.19-0.64%. The protein nature, indicated by infrared and ultraviolet absorption, was confirmed when 100 [mu]g of hydrolyzed wall gave a ninhydrin colour equivalent to that given by 0.88-1.01 /imoles of glycine. Hexosamine, hexose, pentose, lipid and dipicolinic acid were absent. Paper chromatography of hydrolysates, besides showing the presence of the usual protein amino acids and three unidentified ninhydrin-reacting spots, indicated the presence of large amounts of glutamic acid. Estimated by chromatography, the amount present was 52.9 [plus or minus]0.6 (S.D.) g/100 g of ash-free wall; manometric estimation of L-glutamic acid with L-glutamate l-carboxy-lyase gave 46.5 [plus or minus] 0.9 (S.D.) g/100 g. Free carboxyl groups were estimated by titration as 0.159 [plus or minus] 0.011 (S.D.) mole/100 g and those present as amide as 0.154[plus or minus]0.004 (S.D.) mole/100 g, and the total was compared with the dicarboxylic acid content 0.360[plus or minus]0.010 (S.D.) mole/100 g. After treatment with 98% formic acid 25-30% of the wall material could be extracted by 0.05 [image]-sodium carbonate solution (extract 1); after treatment of the residue with performic acid a further 62-63% based on the original weight could be extracted by 0.05 [image]-sodium carbonate (extracts 2). The average values found for the glutamic acid contents were 21.7 g/100 g for extract 1 and 58.0 g/100 g for extract 2. The cysteic acid content of whole oxidized wall was about 5.8 g/100 g and of extract 2 also about 5.8 g/100 g. The glutamic acid and cysteic acid contents of the final residue were also investigated. The significance of these extraction experiments in relation to the wall structure is discussed.