Effect of cationic flaxseed protein hydrolysate fractions on the in vitro structure and activity of calmodulin-dependent endothelial nitric oxide synthase.
The objective of this study was to determine the in vitro effects of cationic flaxseed protein hydrolysate fractions on calmodulin (CaM) structure as well as the activity of CaM-dependent endothelial nitric oxide synthase (eNOS). Flaxseed protein isolate was hydrolyzed with alcalase, and two peptide fractions were isolated by cation-exchange chromatography. Fraction I, eluted first from the column, and fraction II contained 42% and 51% contents of basic amino acids, respectively. Fractions I and II reduced the activity of CaM-dependent eNOS through a mostly mixed-type inhibition mode. Fraction II was at least ten times more effective as an eNOS inhibitor when compared to fraction I, as evident from the IC(50) (concentration of protein hydrolysate that reduced eNOS activity by 50%) values. Fluorescence spectroscopy showed that the tyrosine residues in CaM were increasingly exposed upon addition of fraction I, while the opposite was the case when fraction II was added. Circular dichroism studies showed that fractions I and II reduced the alpha-helix content but increased the rigidity of the active calcium/CaM complex. We concluded that ability of the protein hydrolysate fractions to change the secondary and tertiary structures of CaM may explain their ability to reduce activity of CaM-dependent eNOS.
Omoni
Adetayo O
AO
Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada.
Aluko
Rotimi E
RE
D002147:Calmodulin / Q000737:chemistry* / Q000494:pharmacology*; D002412:Cations; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019597:Flax / Q000737:chemistry*; D005453:Fluorescence; D007700:Kinetics; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000494:pharmacology*; D011492:Protein Hydrolysates / Q000494:pharmacology*; D017433:Protein Structure, Secondary / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D002147:Calmodulin; D002412:Cations; D004791:Enzyme Inhibitors; D010940:Plant Proteins; D011492:Protein Hydrolysates; D052250:Nitric Oxide Synthase Type III