These research efforts are aimed, on a biochemical and biophysical level, at determining whether or not the enzyme pyruvate kinase (PK) changes its isozyme form, hence properties, during the processes of age and cataract formation, particularly in the diabetic case. Ample evidence is available from studies on non-lenticular tissues that shows that PK occurs in an altered state in many disease situations; but, this has never been addressed for the case of cataract disease in the lens. Data has been acquired primarily from the rat lens model that shows that both the activity level of PK and its kinetic response to substrate are modified during the course of aging and cataract formation. Studies involving PK isolation and isozyme forms of PK are present at different critical stages of age and cataract development. Techniques have already been developed to purify the enzyme and the isozyme form of PK will be determined from its kinetic response to PEP (S0.5 Hill coefficient and Vmax) in the presence and absence of certain effectors that have been used to distinguish one form of PK from the other, its characteristic molecular mass and subunit size, its pI, and its immunochemical cross reactivity. The effect of phosphorylation and oxidation on these parameters will be measured to see if either of these post translational modifications shift the isozyme pattern of PK in a manner consistent with that seen in age and cataract development.These experiments are to involve the Sprague-Dawley rat lens; human epithelial cell lines and lens that have been cultured or incubated in the presence of high glucose; and human lens sections that have been derived from normal human lenses of different ages and from cataracts. The immediate goal of this proposal is to provide a complete biochemical characterization of PK in the normal, the aged and the cataractous lens. The long range objective of these plans are to provide a biochemical description of the form of PK in the diseased state so that, one day, it can be converted to the form established for the normal state.
