324 Sweeney and Walkereral months at -20°C. The enzyme is reported to be stable at pH 7.0 at
temperatures up to 65°C, and is stable between pH 3.5 and 11.0 at room
temperature for at least 3 h (15).
2.2. 7. Activation/Inhibition
The enzyme is not affected by sulfhydryl reagents, has no require-
ments for divalent metal ions (unlike the cytosolic leucine aminopep-
tidase), is stable in the presence of trypsin, and is active in 6M urea. It
is not inhibited by PMSF, DFP, or PCMB. It is, however, irreversibly
denatured by alcohols and acetone, and 0.5M guanidinium chloride,
but cannot be precipitated by trichloroacetic acid (15). It is inhibited
by 1,10-phenanthroline (10M) (16).
2.3. Prolidase (Porcine Kidney)
2.3.1. Alternative Names
These are imidodipeptidase, proline dipeptidase, aminoacyl L-pro-
line hydrolyase, and peptidase D.
2.3.2. Specificity
The enzyme is highly specific, and cleaves dipeptides with a prolyl
or hydroxyprolyl residue in the carboxyl terminal position (18,30). It
has no activity with tripeptides (19). The rate of release is inversely
proportional to the size of the amino terminal residue (19). The enzyme's
activity depends on the nature of the amino acids bound to the imino
acid. For optimal activity, amino acid side chains must be as small as
possible and apolar to avoid steric competition with the enzyme recep-
tor site. The enzyme has the best affinity for alanyl proline and glycyl
proline. Experimental data have also suggested that prolidase may
only cleave the trans-form of the peptide bond (31). Active site modeling
studies have also been performed (20).
2.3.3. Molecular Mass
The mol mass, determined by SDS-PAGE, is 110,000 Da for the
native protein and 53,000 Da for the reduced form. The enzyme is there-
fore thought to consist of two chains linked by disulfide bonds (30).
Prolidase is a glycoprotein containing about 0.5% carbohydrate (32).
2.3.4. pH Optimum
The enzyme has optimal activity at pH 6-8, but it is normally used
at pH 7.8-8.0 (33).