322 Sweeney and Walkerincubated at 37°C for 1 h. The solution is then cooled, and the absorbance
recorded at 570 nm. The amount of ]3-naphthylene can be determined
using a standard curve (23). Using this assay, the enzyme is normally
supplied with a specific activity of 100-350 U/mg protein, where 1 U
hydrolyzes 1 nmol of L-pyroglutamic acid ~l-naphthylamide to L-pyroglu-
tamic acid and ]3-naphthylamine/min. The enzyme has also been assayed
using (Pro-3H)-thyroliberin as substrate (24), and by HPLC methods
that use peptides (containing pyroglutamate residues) as substrates (25).
2.1.6. Stability
The lyophilized enzyme is stable at 4°C for months. The enzyme is
generally unstable. Its stability is enhanced by sucrose and EDTA in
some commercial preparations. It may be reconstituted in solutions
containing 5 mMDTT and 10 mMEDTA, and stored at-20°C, or it can
be used for a maximum of 1 wk, if stored at 4°C. The enzyme is stable
in 1M urea and 0.1M guanidine hydrochloride (21). Podell and Abraham
(26) have found the enzyme to be extremely unstable above room
temperature, and it was found that deblocking did not occur at 37°C.
It was found that an initial incubation at 4°C followed by a second
incubation at room temperature (see Section 3.1.) was necessary to
ensure maximum enzyme stabilizing conditions. Air oxidation causes
severe reduction in enzyme activity, and therefore, incubations involv-
ing the enzyme should be carried out under nitrogen.
2.1. Z Activation/Inhibition
Podell and Abraham (26) have studied factors affecting the stability
of the enzyme and have developed a buffer (deblocking buffer; see
Section 3.1.) that is compatible with the use of the enzyme. The enzyme
requires a thiol compound for activation and is inactivated by thiol-block-
ing compounds, such as iodoacetamide, and divalent metal ions, such as
Hg 2+. Activity can be restored by short incubations with mercapto-
ethanol. Dithiothreitol and EDTA are included in the deblocking buffer
to overcome inactivation (6).
2.2. Aminopeptidase M
(Porcine Kidney Microsomes)
2.2.1. Alternative Names
These are amino acid arylamidase, microsomal alanyl aminopepti-
dase, and o~-aminoacyl peptide hydrolase.