Refolding of Fully Reduced Bovine Pancreatic Trypsin

Ohshima, Yuji; Suzuki, Yusuke; Nakatani, Akihiro; Nohara, Daisuke

doi:10.1263/jbb.106.345

Cited by 6 publications

(7 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, because the S196A mutants exhibit significantly higher apparent T m (54–55°C instead of 48–49.5°C), denaturation of the proenzyme is likely accompanied by autolysis, as observed during the folding experiments of other proteases [40]. This phenomenon was more observed for the P8A and P-A mutants which display a slightly higher proteolytic activity than the other proDer p 3 zymogens.…”

Section: Discussionmentioning

confidence: 89%

See 1 more Smart Citation

The Proline-Rich Motif of the proDer p 3 Allergen Propeptide Is Crucial for Protease-Protease Interaction

et al. 2013

View full text Add to dashboard Cite

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen.

show abstract

Section: Discussionmentioning

confidence: 89%

“…In good agreement with our findings, Craik and collaborators [38], [39] demonstrated that the recombinant expression of an active trypsin in E. coli did not require the presence of a propeptide. More recently, the refolding of chemically denaturated bovine pancreatic trypsin was successfully performed without a propeptide [40].…”

Section: Discussionmentioning

confidence: 99%

The Proline-Rich Motif of the proDer p 3 Allergen Propeptide Is Crucial for Protease-Protease Interaction

et al. 2013

View full text Add to dashboard Cite

show abstract

“…It is also known that the refolding recoveries of trypsin and trypsinogen are also extremely low since significant amounts of aggregation and degradation also occur during this process. Indeed, the refolding recovery of trypsin from the reduced form was reported to be 12% [13,14]. To solve this problem, the refolding of trypsin was carried out in an immobilized state or on an inhibitor-immobilized resin [14].…”

Section: Resultsmentioning

confidence: 99%

Degradation-Suppressed Cocoonase for Investigating the Propeptide-Mediated Activation Mechanism

et al. 2022

View full text Add to dashboard Cite

Cocoonase is folded in the form of a zymogen precursor protein (prococoonase) with the assistance of the propeptide region. To investigate the role of the propeptide sequence on the disulfide-coupled folding of cocoonase and prococoonase, the amino acid residues at the degradation sites during the refolding and auto-processing reactions were determined by mass spectrometric analyses and were mutated to suppress the numerous degradation reactions that occur during the reactions. In addition, the Lys8 residue at the propeptide region was also mutated to estimate whether the entire sequence is absolutely required for the activation of cocoonase. Finally, a degradation-suppressed [K8D,K63G,K131G,K133A]-proCCN protein was prepared and was found to refold readily without significant degradation. The results of an enzyme assay using casein or Bz-Arg-OEt suggested that the mutations had no significant effect on either the enzyme activity or the protein conformation. Thus, we, herein, provide the non-degradative cocoonase protein to investigate the propeptide-mediated protein folding of the molecule. We also examined the catalytic residues using the degradation-suppressed cocoonase. The point mutations at the putative catalytic residues in cocoonase resulted in the loss of catalytic activity without any secondary structural changes, indicating that the mutated residues play a role in the catalytic activity of this enzyme.

show abstract

“…Derived from the hepatopancreas, intestine, and blind pyloric sac of fish, trypsin is the main endogenous protein hydrolase of the fish gut [28], which selectively hydrolyses peptide bonds in proteins made up of the carboxyl groups of lysine or arginine. In vertebrates, it functions as a digestive enzyme [29], which breaks down proteins in food and promotes nutrient absorption. In the pancreas, trypsin is synthesized as precursor trypsinogen and then secreted as a component of pancreatic juice into the duodenum, where it is activated by enterokinase or self-catalysis.…”

Section: Introductionmentioning

confidence: 99%

Mechanisms of Digestive Enzyme Response to Acute Salinity Stress in Juvenile Yellowfin Tuna (Thunnus albacares)

Zhang,

Yang,

et al. 2023

Animals

View full text Add to dashboard Cite

This study investigates the effect of a sudden change in salinity for 48 h on the digestive enzyme activity of juvenile yellowfin tuna. The treatment included a control salinity of 32‰ in natural seawater and an experimental salinity of 29‰. Acute stress experiments were carried out on 72 juvenile yellowfin tuna (646.52 ± 66.32 g) for 48 h to determine changes in digestive enzyme activity in different intestinal sections over time (0 h, 12 h, 24 h, 48 h). The activities of pepsin, trypsin, α-amylase, lipase, and chymotrypsin in the digestive organs (stomach, foregut, and pyloric ceca) of juvenile yellowfin tuna were measured. Pepsin and pancreatic protease in the experimental group were significantly lower than in the control group (p < 0.05). α-amylase showed a fluctuating trend of decreasing and then increasing, and its activity trend was pyloric ceca > foregut > stomach. The lipase activity of gastric tissues decreased at the beginning and then increased, reaching a minimum at 24 h (2.74 ± 1.99 U·g protein−1). The change of lipase in the pyloric ceca and foregut was increasing and then decreasing. The lipase activity trend was pyloric ceca > foregut > stomach. The chymotrypsin showed a decreasing and increasing trend and then stabilized at 48 h with a pattern of pyloric ceca > foregut > stomach. Similarly, the gut villi morphology was not significantly altered in the acutely salinity-stressed compared to the non-salinity-stressed. This study suggests that salinity may change the digestive function of juvenile yellowfin tuna, thereby affecting fish feeding, growth, and development. On the contrary, yellowfin tuna is highly adapted to 29‰ salinity. However, excessive stress may negatively affect digestive enzyme activity and reduce fish digestibility. This study may provide a scientific basis for a coastal aquaculture water environment for yellowfin tuna farming, which may guide the development and cultivation of aquaculture.

show abstract

Refolding of Fully Reduced Bovine Pancreatic Trypsin

Cited by 6 publications

References 16 publications

The Proline-Rich Motif of the proDer p 3 Allergen Propeptide Is Crucial for Protease-Protease Interaction

The Proline-Rich Motif of the proDer p 3 Allergen Propeptide Is Crucial for Protease-Protease Interaction

Degradation-Suppressed Cocoonase for Investigating the Propeptide-Mediated Activation Mechanism

Mechanisms of Digestive Enzyme Response to Acute Salinity Stress in Juvenile Yellowfin Tuna (Thunnus albacares)

Contact Info

Product

Resources

About