Kinetics of the inhibition of calcium/calmodulin-dependent protein kinase II by pea protein-derived peptides.
Calcium/calmodulin-dependent protein kinase II (CaMKII) catalyzes the phosphorylation of various cellular proteins and excessive activities have been implicated in the pathogenesis of various chronic diseases. We hypothesized that positively charged peptides can be produced through enzymatic hydrolysis of pea proteins; such peptides could then bind to negatively charged calmodulin (CaM) at a physiological pH level and inhibit CaMKII activity. Pea protein isolate was hydrolyzed with an alkaline protease (alcalase) and filtered through a 1000-mol wt cutoff membrane. The permeate, which contained low-molecular weight peptides, was used to isolate cationic peptides on an SP-Sepharose column by ion exchange chromatography. Separation of the permeate on the SP-Sepharose column yielded two fractions with net positive charges that were subsequently used for enzyme inhibition studies. Fraction I eluted earlier from the column and contained lower contents of lysine and arginine than Fraction II, which eluted later. Results show that both peptide fractions inhibited CaMKII activity mostly in a competitive manner, although kinetic data suggested that inhibition by Fraction II may be of the mixed type. Kinetic analysis (K(m) and K(i)) showed that affinity of peptides in Fraction II for CaM was more than that in Fraction I, which was directly correlated with the higher inhibitory properties of Fraction II against CaMKII. The results suggest that it may be possible to use pea protein-derived cationic peptides to modulate CaMKII activities.
Li
Huan
H
Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Canada.
Aluko
Rotimi E
RE
D000596:Amino Acids / Q000032:analysis; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D018532:Pisum sativum / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification / Q000494:pharmacology
D000596:Amino Acids; D010446:Peptide Fragments; D010940:Plant Proteins; D047428:Protein Kinase Inhibitors; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases