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Differences in PAR-2 activating potential by king crab (Paralithodes camtschaticus), salmon (Salmo salar), and bovine (Bos taurus) trypsin.

Larsen AK, Kristiansen K, Sylte I, Seternes OM, Bang BE - BMC Res Notes (2013)

Bottom Line: During purification king crab trypsin displayed stronger binding capacity to the anionic column used in fast protein liquid chromatography compared to fish trypsins, and was identified as a slightly bigger molecule.Measurements of enzymatic activity yielded no obvious differences between the trypsins tested.These preliminary results indicate that electrostatic interactions could be of importance in binding, cleavage and subsequent activation of PAR-2.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Occupational- and Environmental Medicine, University Hospital North Norway, Tromsø, Norway.

ABSTRACT

Background: Salmon trypsin is shown to increase secretion of the pro-inflammatory cytokine interleukin (IL)-8 from human airway epithelial cells through activation of PAR-2. Secretion of IL-8 induced by king crab trypsin is observed in a different concentration range compared to salmon trypsin, and seems to be only partially related to PAR-2 activation. This report aim to identify differences in the molecular structure of king crab trypsin (Paralithodes camtschaticus) compared to salmon (Salmo salar) and bovine trypsin (Bos taurus) that might influence the ability to activate protease-activated receptor-2 (PAR-2).

Results: During purification king crab trypsin displayed stronger binding capacity to the anionic column used in fast protein liquid chromatography compared to fish trypsins, and was identified as a slightly bigger molecule. Measurements of enzymatic activity yielded no obvious differences between the trypsins tested. Molecular modelling showed that king crab trypsin has a large area with strong negative electrostatic potential compared to the smaller negative areas in bovine and salmon trypsins. Bovine and salmon trypsins also displayed areas with strong positive electrostatic potential, a feature lacking in the king crab trypsin. Furthermore we have identified 3 divergent positions (Asp196, Arg244, and Tyr247) located near the substrate binding pocket of king crab trypsin that might affect the binding and cleavage of PAR-2.

Conclusion: These preliminary results indicate that electrostatic interactions could be of importance in binding, cleavage and subsequent activation of PAR-2.

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Related in: MedlinePlus

Purified king crab trypsin differs in size from purified sardine trypsin. 250 ng of purified king crab and sardine trypsin were run on a SDS-PAGE gel and stained with silver staining for detection. The result shows that purified king crab trypsin (lane 7 and 8) is a slightly bigger molecule residing in the 28 – 30 kDa area compared to the purified sardine trypsin (lane 3 and 4) at 24 – 25 kDa. Additional purified fish trypsins tested (anchovy (lane 5 and 6), yellow tail, jacopever, spotted mackerel (not shown)) displayed similar size as purified sardine trypsin. Lane 1, 2 and 10 contain protein standards, respectively 10 μl SeeBlue®,10 μl Mark 12™, 16 μl SeeBlue®. Lane 9 is empty.
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Figure 1: Purified king crab trypsin differs in size from purified sardine trypsin. 250 ng of purified king crab and sardine trypsin were run on a SDS-PAGE gel and stained with silver staining for detection. The result shows that purified king crab trypsin (lane 7 and 8) is a slightly bigger molecule residing in the 28 – 30 kDa area compared to the purified sardine trypsin (lane 3 and 4) at 24 – 25 kDa. Additional purified fish trypsins tested (anchovy (lane 5 and 6), yellow tail, jacopever, spotted mackerel (not shown)) displayed similar size as purified sardine trypsin. Lane 1, 2 and 10 contain protein standards, respectively 10 μl SeeBlue®,10 μl Mark 12™, 16 μl SeeBlue®. Lane 9 is empty.

Mentions: King crab trypsin is reported to be in the size range of 23 – 29 kDa (Kislitsyn et al., [21]; Rudenskaya et al., [20]). Our results from SDS-PAGE reveal a protein in the 28 – 29 kDa range (Figure 1) compared to sardine trypsin at 24 kDa [22] (Figure 1), and salmon trypsin at 23, 7 – 25 kDa [23,27].


Differences in PAR-2 activating potential by king crab (Paralithodes camtschaticus), salmon (Salmo salar), and bovine (Bos taurus) trypsin.

Larsen AK, Kristiansen K, Sylte I, Seternes OM, Bang BE - BMC Res Notes (2013)

Purified king crab trypsin differs in size from purified sardine trypsin. 250 ng of purified king crab and sardine trypsin were run on a SDS-PAGE gel and stained with silver staining for detection. The result shows that purified king crab trypsin (lane 7 and 8) is a slightly bigger molecule residing in the 28 – 30 kDa area compared to the purified sardine trypsin (lane 3 and 4) at 24 – 25 kDa. Additional purified fish trypsins tested (anchovy (lane 5 and 6), yellow tail, jacopever, spotted mackerel (not shown)) displayed similar size as purified sardine trypsin. Lane 1, 2 and 10 contain protein standards, respectively 10 μl SeeBlue®,10 μl Mark 12™, 16 μl SeeBlue®. Lane 9 is empty.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733831&req=5

Figure 1: Purified king crab trypsin differs in size from purified sardine trypsin. 250 ng of purified king crab and sardine trypsin were run on a SDS-PAGE gel and stained with silver staining for detection. The result shows that purified king crab trypsin (lane 7 and 8) is a slightly bigger molecule residing in the 28 – 30 kDa area compared to the purified sardine trypsin (lane 3 and 4) at 24 – 25 kDa. Additional purified fish trypsins tested (anchovy (lane 5 and 6), yellow tail, jacopever, spotted mackerel (not shown)) displayed similar size as purified sardine trypsin. Lane 1, 2 and 10 contain protein standards, respectively 10 μl SeeBlue®,10 μl Mark 12™, 16 μl SeeBlue®. Lane 9 is empty.
Mentions: King crab trypsin is reported to be in the size range of 23 – 29 kDa (Kislitsyn et al., [21]; Rudenskaya et al., [20]). Our results from SDS-PAGE reveal a protein in the 28 – 29 kDa range (Figure 1) compared to sardine trypsin at 24 kDa [22] (Figure 1), and salmon trypsin at 23, 7 – 25 kDa [23,27].

Bottom Line: During purification king crab trypsin displayed stronger binding capacity to the anionic column used in fast protein liquid chromatography compared to fish trypsins, and was identified as a slightly bigger molecule.Measurements of enzymatic activity yielded no obvious differences between the trypsins tested.These preliminary results indicate that electrostatic interactions could be of importance in binding, cleavage and subsequent activation of PAR-2.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Occupational- and Environmental Medicine, University Hospital North Norway, Tromsø, Norway.

ABSTRACT

Background: Salmon trypsin is shown to increase secretion of the pro-inflammatory cytokine interleukin (IL)-8 from human airway epithelial cells through activation of PAR-2. Secretion of IL-8 induced by king crab trypsin is observed in a different concentration range compared to salmon trypsin, and seems to be only partially related to PAR-2 activation. This report aim to identify differences in the molecular structure of king crab trypsin (Paralithodes camtschaticus) compared to salmon (Salmo salar) and bovine trypsin (Bos taurus) that might influence the ability to activate protease-activated receptor-2 (PAR-2).

Results: During purification king crab trypsin displayed stronger binding capacity to the anionic column used in fast protein liquid chromatography compared to fish trypsins, and was identified as a slightly bigger molecule. Measurements of enzymatic activity yielded no obvious differences between the trypsins tested. Molecular modelling showed that king crab trypsin has a large area with strong negative electrostatic potential compared to the smaller negative areas in bovine and salmon trypsins. Bovine and salmon trypsins also displayed areas with strong positive electrostatic potential, a feature lacking in the king crab trypsin. Furthermore we have identified 3 divergent positions (Asp196, Arg244, and Tyr247) located near the substrate binding pocket of king crab trypsin that might affect the binding and cleavage of PAR-2.

Conclusion: These preliminary results indicate that electrostatic interactions could be of importance in binding, cleavage and subsequent activation of PAR-2.

Show MeSH
Related in: MedlinePlus