Characterization of the elastase inhibitor properties of alpha-crystallin and the water-insoluble fraction from bovine lens.
Alpha-crystallin exhibits variable inhibition of several members of the chymotrypsin family of proteinases. Complete inhibition of elastase was obtained by the addition of either alpha-crystallin or a sonicated preparation of the water-insoluble fraction from bovine lens. Little or no inhibition was seen, however, with either beta-crystallin or bovine serum albumin under the same conditions. Complete binding of elastase was demonstrated by Sephadex G-100 gel filtration chromatography, and a direct correlation between binding and inhibition was obtained. This observation permitted us to do a Scatchard analysis of the inhibition data. Scatchard plots for the binding of elastase gave a biphasic response suggesting two separate binding sites. These sites had Kd values of 15 and 40 nM for alpha-crystallin and 6 and 42 nM for the bovine water-insoluble fraction. Similarly, a Dixon plot exhibited a Ki value of 3 nM and was consistent with non-competitive inhibition. One mole of alpha-crystallin (8 x 10(5) Da), or an equivalent amount of water-insoluble protein, bound from 13 to 19 mol of elastase which were about equally divided between the higher and lower affinity sites. Saturation studies confirmed 20 and 16 elastase binding sites per 8 x 10(5) Da for alpha-crystallin and water-insoluble protein, respectively. DFP-elastase was capable of binding to alpha-crystallin suggesting that a proteolytic cleavage was not required for complex formation. Stability measurements showed a linear return to 60% of the original activity over a 30-min period. Therefore, the interaction between elastase and alpha-crystallin resembles that of a heterologous protease:inhibitor complex in both binding and stability.
Ortwerth
B J
BJ
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Olesen
P R
PR
D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D012995:Solubility; D014867:Water
D003459:Crystallins; D014867:Water; D010196:Pancreatic Elastase