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Isolation and characterization of two proteins possessing Hpa II methylase activity
Abstract:
Two proteins exhibiting Hpa II methylase activity have been purified to homogeneity from Haemophilus parainfluenzae and their physical and catalytic properties have been studied. Separation of the two Hpa II methylase activities was achieved by DEAE-Sephadex A-50 chromatography. In subsequent steps, each methylase was purified separately by chromatography on Sephacryl S-200, phosphocellulose, and hydroxylapatite. The proteins have molecular weights of 38,500 +/- 1,000 (Hpa II) and 41,500 +/- 1,000 (Hpa II') as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Sedimentation equilibrium analyses of the native proteins yield molecular weights of 38,800 +/- 3,000 and 42,200 +/- 3,000 for Hpa II and Hpa II', respectively, indicating that both enzymes are composed of a single subunit. Furthermore, both methylases exhibit identical specificity in the methylation of the nucleotide sequence dC-C-G-G in simian virus 40 (SV40) DNA and in a short synthetic oligonucleotide duplex. Although pH, temperature, and salt optima are the same for both enzymes, homogeneous Hpa II' methylase is more stable than Hpa II methylase. Preliminary peptide mapping indicates that the two enzymes are structurally related, suggesting the possibility that Hpa II' methylase may represent a precursor form of Hpa II methylase.
Insights
Two Hpa II methylase enzymes were purified from Haemophilus parainfluenzae. These enzymes show identical DNA methylation specificity, but Hpa II
Area of Science:
- Molecular Biology
- Enzymology
- Biochemistry
Background:
- Haemophilus parainfluenzae possesses methylase activity, crucial for DNA modification.
- Understanding DNA methyltransferases aids in deciphering gene regulation and DNA repair mechanisms.
Purpose of the Study:
- To purify and characterize two distinct Hpa II methylase proteins from Haemophilus parainfluenzae.
- To investigate the physical and catalytic properties of these purified methylases.
- To explore the relationship between the two identified Hpa II methylase forms.
Main Methods:
- Proteins were purified using a multi-step chromatography approach including DEAE-Sephadex A-50, Sephacryl S-200, phosphocellulose, and hydroxylapatite.
- Molecular weights were determined using SDS-polyacrylamide gel electrophoresis and sedimentation equilibrium analysis.
- Enzyme specificity was assessed using simian virus 40 (SV40) DNA and synthetic oligonucleotide duplexes.
Main Results:
- Two homogeneous Hpa II methylase proteins, Hpa II and Hpa II', were successfully purified.
- Both enzymes possess identical molecular weights (approx. 38.5 kDa for Hpa II, 41.5 kDa for Hpa II') and are single subunits.
- Hpa II and Hpa II' exhibit identical specificity for methylating the dC-C-G-G sequence, with Hpa II' showing greater stability.
Conclusions:
- The study successfully isolated and characterized two Hpa II methylases from H. parainfluenzae.
- Structural and functional similarities suggest Hpa II' may be a precursor to Hpa II methylase.
- These findings contribute to the understanding of DNA methyltransferase diversity and function.