Rat proestrus uterine fluid contains a large molecular-weight protein complex with metalloendopeptidase activity.

A neutral metalloendopeptidase has been identified in the secretion of the rat proestrous uterus. The native form of the enzyme secreted in the uterus is a high-mol-wt protein complex, which barely migrates in 3% native polyacrylamide gel electrophoresis. In 2% SDS, the enzyme resolves into 380-, 210-, and 110-kDa subunits. The 380- and 210-kDa proteins demonstrate peptidase activity in SDS-PAGE as detected by an enzyme overlay membrane impregnated with Suc-Ala-Ala-Phe-AFC. The uterine peptidase is substantially inhibited by EDTA, EGTA, 1,10 phenanthroline, thiorphan, DTT, and sodium dodecyl sulfate (SDS). The inhibition by 1,10 phenanthroline is reversed by CO2+. The uterine enzyme has a pH optimum of 7.0, and readily hydrolyzes acetyl tetraalanine and Suc-Ala-Ala-Leu-pNA by releasing alanylalanine and Leu-pNA, respectively. Kinetic analysis of the peptidase using Suc-Ala-Ala-Leu-pNA as substrate yielded Km and Vmax values 0.9 mM and 4.3 microM/min/3.5 microg protein, respectively. Negative staining of the purified uterine metalloprotease demonstrated spherical particles made up of several subunits. The earlier work had indicated that the uterine fluid metalloendopeptidase adhered to the head region of the ejaculated spermatozoa. It is very likely that this peptidase may be involved in assisting the fertilization process.