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Pressure acceleration of proteolysis: A general mechanism

View Article: PubMed Central - PubMed

ABSTRACT

Remarkable acceleration of enzymatic proteolysis by pressure at kbar range is reported with ubiquitin as substrate and α-chymotrypsin as enzyme. The acceleration is interpreted in terms of the shift of conformational equilibrium in ubiquitin from the non-degradable folded conformer to the enzyme-degradable unfolded conformer by pressure because of the lower volume of the latter, while the enzymatic activity of α-chymotrypsin is still largely retained. This mechanism is considered generally applicable to most globular proteins and the method of pressure-accelerated proteolysis will have an enormous potential utility in systems wherever efficient removal of proteins is needed.

No MeSH data available.


SDS-PAGE patterns for ubiquitin subjected to enzymatic hydrolysis with α-chymotrypsin at 37°C for 50 min at following pressures. a. 1bar, b. 500 bar, c. 1000 bar, d. 1500 bar, e. 2000 bar, f. 2500 bar, g. 3500 bar. The solution contained ubiquitin (31 μM) and α-chymotrypsin (3 μM) in 10 mM Tris-HCl buffer, pH 7.0. Lanes for ubiquitin alone and α-chymotrypsin alone are also shown for comparison.
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f1-4_29: SDS-PAGE patterns for ubiquitin subjected to enzymatic hydrolysis with α-chymotrypsin at 37°C for 50 min at following pressures. a. 1bar, b. 500 bar, c. 1000 bar, d. 1500 bar, e. 2000 bar, f. 2500 bar, g. 3500 bar. The solution contained ubiquitin (31 μM) and α-chymotrypsin (3 μM) in 10 mM Tris-HCl buffer, pH 7.0. Lanes for ubiquitin alone and α-chymotrypsin alone are also shown for comparison.

Mentions: Figure 1 shows the result of SDS-PAGE analysis of ubiquitin subjected to digestion with α-chymotrypsin in solutions containing ubiquitin (31 μM) and α-chymotrypsin (3 μM) in 10 mM Tris-HCl buffer (pH 7.0) for 50 min at various pressures from 1 bar to 3500 bar at 37°C. At 1 bar, no degradation products were detected (Fig. 1a). When we applied pressure, the situation dramatically changed. At 500 bar the band for intact ubiquitin started getting smaller and continued to decrease at 1000 and 1500 bar. At 2000 bar, the ubiquitin band nearly disappeared, showing that ubiquitin was almost degraded. The pressure enhancement of proteolysis of ubiqutin with α-chymotrypsin is quite dramatic. The result also indicates that α-chymotrypsin retains its catalytic activity at 2 kbar or higher, meaning that its activity is not totally destroyed by this range of pressure.


Pressure acceleration of proteolysis: A general mechanism
SDS-PAGE patterns for ubiquitin subjected to enzymatic hydrolysis with α-chymotrypsin at 37°C for 50 min at following pressures. a. 1bar, b. 500 bar, c. 1000 bar, d. 1500 bar, e. 2000 bar, f. 2500 bar, g. 3500 bar. The solution contained ubiquitin (31 μM) and α-chymotrypsin (3 μM) in 10 mM Tris-HCl buffer, pH 7.0. Lanes for ubiquitin alone and α-chymotrypsin alone are also shown for comparison.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036607&req=5

f1-4_29: SDS-PAGE patterns for ubiquitin subjected to enzymatic hydrolysis with α-chymotrypsin at 37°C for 50 min at following pressures. a. 1bar, b. 500 bar, c. 1000 bar, d. 1500 bar, e. 2000 bar, f. 2500 bar, g. 3500 bar. The solution contained ubiquitin (31 μM) and α-chymotrypsin (3 μM) in 10 mM Tris-HCl buffer, pH 7.0. Lanes for ubiquitin alone and α-chymotrypsin alone are also shown for comparison.
Mentions: Figure 1 shows the result of SDS-PAGE analysis of ubiquitin subjected to digestion with α-chymotrypsin in solutions containing ubiquitin (31 μM) and α-chymotrypsin (3 μM) in 10 mM Tris-HCl buffer (pH 7.0) for 50 min at various pressures from 1 bar to 3500 bar at 37°C. At 1 bar, no degradation products were detected (Fig. 1a). When we applied pressure, the situation dramatically changed. At 500 bar the band for intact ubiquitin started getting smaller and continued to decrease at 1000 and 1500 bar. At 2000 bar, the ubiquitin band nearly disappeared, showing that ubiquitin was almost degraded. The pressure enhancement of proteolysis of ubiqutin with α-chymotrypsin is quite dramatic. The result also indicates that α-chymotrypsin retains its catalytic activity at 2 kbar or higher, meaning that its activity is not totally destroyed by this range of pressure.

View Article: PubMed Central - PubMed

ABSTRACT

Remarkable acceleration of enzymatic proteolysis by pressure at kbar range is reported with ubiquitin as substrate and α-chymotrypsin as enzyme. The acceleration is interpreted in terms of the shift of conformational equilibrium in ubiquitin from the non-degradable folded conformer to the enzyme-degradable unfolded conformer by pressure because of the lower volume of the latter, while the enzymatic activity of α-chymotrypsin is still largely retained. This mechanism is considered generally applicable to most globular proteins and the method of pressure-accelerated proteolysis will have an enormous potential utility in systems wherever efficient removal of proteins is needed.

No MeSH data available.