1. The subcellular distribution of peptidase activities in the normal human jejunum against glycine and leucine homopeptides has been investigated with an analytical fractionation technique.
2. An 8000 g-min supernatant was prepared from homogenates of Crosby capsule biopsy specimens and subjected to isopycnic centrifugation in a Beaufay automatic zonal rotor.
3. The distribution of subcellular organelles in the gradient was established by measurement of organelle-specific marker enzymes.
4. A sensitive fluorimetric assay for glycine peptidase was developed and used for the localization of peptidase activity with peptides composed of from two to five glycine residues as substrates.
5. Glycine peptidase activity was located in the cytosol and in the brush-border membrane but the distribution of activity varied markedly with the chain-length of substrate; the longer the peptide the greater the proportion of activity associated with the brush border. Leucine peptidase showed a similar variation in cytosol—brush border distributions.
6. The results are consistent with concepts that suggest absorption and intracellular hydrolysis of small peptides and brush-border digestion of larger peptides.