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High molecular weight aspartic endopeptidase generates a coronaro-constrictory peptide from the beta-chain of
N Barkhudaryan1, J Kellermann, A Galoyan
1Max-Planck Institute of Biochemistry, Martinsried, Germany.
Insights
High molecular weight aspartic endopeptidase, not cathepsin D, processes beta-globin to yield a specific peptide fragment. This suggests distinct biological roles for these brain enzymes in peptide processing.
Area of Science:
- Biochemistry
- Enzymology
- Neuroscience
Background:
- Brain aspartic proteinases, including cathepsin D and high molecular weight (HMW) aspartic endopeptidase, play roles in peptide processing.
- Calmodulin-binding coronaro-constrictory peptides are derived from the beta-chain of globin.
Purpose of the Study:
- To investigate the specific influence of brain cathepsin D and HMW aspartic endopeptidase on the processing of beta-globin-derived peptides.
- To determine which enzyme generates the fragment 31-40 of the beta-chain of bovine hemoglobin.
Main Methods:
- Enzymatic digestion of beta-globin using cathepsin D and HMW aspartic endopeptidase at 37°C, pH 3.5, for 4-10 hours.
- Separation of resulting peptides via reversed-phase high-performance liquid chromatography (HPLC).
- Identification of peptides using amino acid analysis and Edman degradation.
Main Results:
- HMW aspartic endopeptidase specifically cleaved the beta-chain of bovine hemoglobin at the Leu30-Leu31 and Phe40-Phe41 bonds.
- This cleavage by HMW aspartic endopeptidase generated the fragment 31-40.
- Cathepsin D did not produce this specific fragment under the tested conditions.
Conclusions:
- Significant differences in specificity and activity exist between brain cathepsin D and HMW aspartic endopeptidase, likely due to structural variations.
- HMW aspartic endopeptidase is implicated in the in vivo processing of coronaro-constrictory peptides via limited proteolysis of hemoglobin or similar proteins.
Abstract:
Studying the influence of brain cathepsin D (EC 3.4.23.5) and high molecular weight (HMW) aspartic endopeptidase (EC 3.4.23.-) on the processing of hypothalamic calmodulin-binding coronaro-constrictory peptide factors from the beta-chain of globin it was found that only HMW aspartic endopeptidase generates the fragment 31-40 of the beta-chain of bovine hemoglobin (Hb) by cleavage of the Leu30-Leu31 and Phe40-Phe41 bonds. Digestion of the beta-chain of globin was performed at 37 degrees C at an enzyme/substrate ratio of 1:80 at pH 3.5 using different times of incubation (from 4 h to 10 h). The resulting peptides were separated by reversed-phase high-performance liquid chromatography (HPLC) and then identified by amino acid analysis and Edman degradation. The differences in specificity and activity of these two brain aspartic proteinases could be explained by their different structural features. Our finding provides evidence for a different biological function of these two enzymes. Data obtained give us reason to suppose that HMW aspartic proteinase probably can participate in the processing of the coronaro-constrictory peptide in vivo by limited proteolysis of Hb or Hb-like protein.