The enzyme NAD kinase (NADK, ATP:NAD 2'-phosphotransferase, EC 2.7.1.23) is widely distributed, catalyzes the only known reaction leading to the production of NADP, and acknowledged to play a central role in the metabolism of many tissues. Its presence in brain and retinal tissue has been established but little or no follow up on these reports has appeared. Its presence in lens is suspected, but not yet established. The long range objectives of this research are pointed to a better understanding of the fundamental properties of this enzyme in neural tissue. Our initial experiments will focus on the soluble protein fraction (crystallins) of bovine lens since this constitutes some 90% of the total protein. Purification/fractionation will be carried out using established gel chromatography protocols then as needed, ion-exchange and affinity chromatography. Rate measurements and subsequent kinetic characterization of the candidate isolate(s) will employ enzymatic cycling fitted to a centrifugal fast analyzer. Lens exhibits a major energy dependency on anaerobic glycolysis, maintains a strong reducing environment involving the glutathione peroxidase/reductase system, and funnels some glucose (5%) through the pentose pathway. It is the immediate and specific aim of this proposal to answer the question, what is the origin of lenticular NADP? Does it arise from the action of NADK? Or does an NADH kinase coupled with glutathione reductase satisfy the needs of glucose 6-phosphate dehydrogenase for NADP? Taken cumulatively, these characteristics provide strong inference that an NAD(H) kinase is present. Our understanding of the fundamental chemistry associated with several of the degenerative processes in lens will remain seriously deficient until the questions raised above are resolved.
