Teruyoshi Matoba

Modified colorimetric ninhydrin methods for peptidase assay.

Abstract Four colorimetric procedures suitable for the determination of peptidase activity on peptides having a free α- or e-amino group are described. Two of the methods (A and B) are modifications of the conventional ninhydrin method described by S. Moore and W. H. Stein ((1948) J. Biol. Chem.176, 367–388; (1954) Ibid.211, 907–913); the heating time is shortened to 5 min at 100°C and the pH of the buffer in the reagent is lowered to 4.0. Method A differs from method B in buffer concentration. The other two methods (C and D) are modifications of the Cd-ninhydrin method described by A. P. Tsarichenko ((1966) Nauch. Tr. Krasnodar. Gos. Pedagog. Inst.70, 86–88, as cited in Chem. Abs.67, 79479c); the water content in the reagent is reduced to 1 20 of the original reagent and the sample is heated for 5 min at 84°C. Method C differs from method D in the ratio of sample to reagent. In contrast to the free amino acids which are sufficiently colored, various peptides and amino acid derivatives except for the glycylpeptides give only a faint color with these methods. These four methods are not only useful for the determination of peptidase activity on peptides (e.g., Leu-Gly and tert-butyloxycarbonyl-glycyl-lysyl-leucine), but are also useful for the determinations of amidase activity on amino acid amides (e.g., Leu-NH3) and esterase activity on amino acid esters (e.g., tyrosine ethyl ester).

Lysosomal enzyme activities in the central vacuole of the internodal cells of Nitella

Abstract The contents of the central vacuole (cell sap) were collected from internodal cells of Nitella by a modification of the procedure described by Tazawa (1964). Most of the acid phosphatase and carboxypeptidase, which are marker enzymes for lysosomes, were detected in the cell sap. In contrast, most of the catalase and cytochrome c oxidase were detected in parts of the cell other than the cell sap; this indicates there is relatively little contamination of the separated cell sap by the cytoplasm. It is suggested that the central vacuole of a mature plant cell possesses some of the properties of a lysosome.