Increased eNOS monomer and decreased eNOS dimer and zinc content are associated with decreased eNOS activity in tissues of diabetic LDLR-KO mice. (a) Representative Western blot of eNOS protein in tissues from control and diabetic LDLR-KO mice. Tissue homogenate proteins were separated by normal-temperature SDS-PAGE under reducing conditions, blotted, and stained with a polyclonal antibody against eNOS. Blot shown is representative of three independent experiments. (b) Increased eNOS monomers are accompanied by a decrease in eNOS dimer in the tissues obtained from diabetic LDLR-KO mice. The eNOS protein in control or diabetic tissue homogenates was purified, separated by low-temperature SDS-PAGE under reducing conditions (without boiling), and detected by Coomassie staining. Results shown represent four independent experiments. (c) Decreased zinc content is associated with inhibition of NO synthesis and decreased eNOS dimer in diabetic LDLR-KO mice. The zinc content of eNOS from diabetic LDLR-KO mouse tissues was expressed as a percentage of that in normoglycemic control LDLR-KO animals. L-citrulline formation was determined in homogenates from control and diabetic animals and expressed as a percentage of that in tissues from control animals (n = 7, *P < 0.05). The assay was performed as described in Methods, and the rates of L-citrulline formation were determined to be 47 ± 11, 56 ± 19, and 43 ± 16 pmol/mg/min in liver, heart, and kidneys, respectively, of normoglycemic control LDLR-KO animals.