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Human erythrocyte membrane acid proteinase (EMAP): sidedness and relation to cathepsin D
Abstract:
An acid proteinase purified from human erythrocyte membranes (Yamamoto, K. & Marchesi, V.T. (1984) Biochem. Biophys. Acta 790, 208-218), now termed "EMAP," was further characterized with respect to its localization and relation to cathepsin D. The membrane-associated form of EMAP was shown to be latent by demonstrating that no activity was detectable in both resealed (right-side-out) ghosts and inside-out vesicles in the absence of detergents. The enzyme associated with the inside-out vesicles was unstable when exposured to acidic pH between 4.0 and 4.5, whereas the enzyme associated with the resealed ghosts was stable in the wide pH range of 3.7 to 9.0. Tryptic digestion produced the loss of activity for the enzyme associated with the inside-out vesicles but not the resealed ghosts. The antibody to rat spleen cathepsin D, which cross-reacted weakly but detectably with EMAP, selectively bound to the inside-out vesicles. These results indicate the location of EMAP on th inner surface of the membranes. Comparison of a number of enzymatic properties of EMAP with rat cathepsin D showed significant differences between these two enzymes. EMAP was less stable in the pH range of 3.5 to 6.0 than cathepsin D. The enzymes were distinguished from each other by differences in their elution profiles on DEAE-Sephacel and chromatofocusing columns and by differences in the extent of inhibition by a few specific inhibitors. Both enzymes revealed significant differences in the amino acid composition and specific activity towards bovine hemoglobin. The immunological relationship between these two enzymes is discussed.
Insights
Erythrocyte membrane-associated proteinase (EMAP) is located on the inner membrane surface and exhibits distinct enzymatic properties compared to rat cathepsin D, suggesting it is a unique enzyme.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- An acid proteinase, termed EMAP, was previously purified from human erythrocyte membranes.
- Further characterization is needed to understand EMAP's localization and relationship to cathepsin D.
Purpose of the Study:
- To characterize the localization of EMAP on human erythrocyte membranes.
- To compare the enzymatic properties of EMAP with rat spleen cathepsin D.
- To investigate the immunological relationship between EMAP and cathepsin D.
Main Methods:
- Assessed EMAP activity in resealed and inside-out erythrocyte vesicles under various pH conditions and with detergent.
- Investigated the effect of tryptic digestion on EMAP activity in different vesicle preparations.
- Utilized antibodies against rat spleen cathepsin D for immunochemical analysis and compared enzymatic properties (stability, inhibition, amino acid composition, substrate specificity) with rat cathepsin D.
Main Results:
- EMAP is latent in the membrane and located on the inner surface of erythrocyte membranes, as evidenced by pH stability, tryptic digestion, and antibody binding.
- EMAP exhibits significant differences in stability, elution profiles, inhibitor sensitivity, amino acid composition, and substrate specificity compared to rat cathepsin D.
- A weak but detectable cross-reactivity exists between EMAP and antibodies to rat cathepsin D.
Conclusions:
- EMAP is an acid proteinase localized to the inner surface of human erythrocyte membranes.
- EMAP is enzymatically and immunologically distinct from rat cathepsin D, suggesting it is a unique enzyme.
- The findings provide insights into the functional role and identity of membrane-associated proteinases in erythrocytes.