Fig. 3.

Cellular BH₄deficiency alters mitochondrial biopterin balance. A) Enriched/isolated fractions of mitochondria were obtained from sEnd.1 cells as outlined in ‘Experimental Procedures’, and their purity confirmed by Western blot analysis using anti-SDH, -VDAC, -cytochrome C, and -GAPDH antibodies. Importantly, these proteins were observed in the mitochondrial fraction, and GAPDH in the cytosolic-, without contamination of the mitochondrial fraction. B) Mitochondrial biopterin levels were determined using HPLC with electrochemical and fluorescent detectors as previously. BH₄, BH₂ and biopterin levels were all significantly attenuated in mitochondria isolated from BH₄ deficient vs. NS control cells, together with C) a lower BH₄:BH₂+B ratio, and D) a higher percentage of oxidised biopterins (BH₂+B). n =3 (*, p<0.05).