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Multiple forms of medicinal leech destabilase-lysozyme.
L L Zavalova1, I I Artamonova, S N Berezhnoy
1Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, 117997, Moscow, Russian Federation. leech@humgen.siobc.ras.ru
Biochemical and Biophysical Research Communications
|June 6, 2003
Summary
Destabilase-lysozyme enzyme preparations contain multiple protein forms, with only one matching the Ds3 gene product. Researchers identified heterogeneity in the wild-type sequence and a novel acidic form, P-Ac.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Three genes (Ds1, Ds2, Ds3) encode destabilase-lysozyme isoforms.
- Previous studies indicated only one isoform matched CNBr fragments, suggesting limited heterogeneity.
Purpose of the Study:
- To characterize the molecular heterogeneity of destabilase-lysozyme.
- To identify specific protein forms corresponding to the Ds3 gene product.
- To discover novel destabilase-lysozyme variants.
Main Methods:
- Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry to determine molecular masses.
- Cyanogen bromide (CNBr) peptide mapping to analyze protein sequence heterogeneity.
- N-terminal sequencing to identify novel forms.
Main Results:
- The enzyme preparation comprises at least three forms with distinct molecular masses (12677.6, 12839.7, 12938.2 Da), each with seven disulfide bridges.
- Only the 12839.7 Da form aligns with the calculated mass for the Ds3 gene product.
- Heterogeneity was observed at positions 64 and 67 in the wild-type sequence, with one variant matching the Ds3 protein.
- A new acidic form, P-Ac, with a unique N-terminal sequence was identified.
Conclusions:
- Destabilase-lysozyme exhibits significant molecular heterogeneity.
- The Ds3 gene encodes a specific destabilase-lysozyme isoform.
- A novel acidic destabilase-lysozyme variant (P-Ac) has been discovered.