Figure 1.

Old and new BHMT forms. (A) Amino acid sequences (red) from BHMT forms associated with autophagosomal membranes, previously mapped by MALDI-TOF tryptic fingerprinting.³⁹ Blue lines indicate amino acids thought to be involved in binding of the homocysteine substrate; yellow lines indicate an involvement in betaine binding and green lines indicate participation in catalysis.⁸⁶ Grey lines indicate sites involved in dimerization (316–349) and tetramerization (381–407).⁸⁷ (B) A frozen-thawed cytoplasmic extract (postnuclear supernatant) from rat hepatocytes was solubilized in an SDS-containing lysis buffer, fractionated by SDS-PAGE and immunoblotted with an N-terminal BHMT antibody. In addition to full-length BHMT (p45), several BHMT fragments (10–33 kDa) are detected. (C) Separation of BHMT forms by liquid-phase isoelectric focusing. A frozen-thawed cytoplasmic extract (postnuclear supernatant) from rat hepatocytes was fractionated by liquid-phase isoelectric focusing (LP-IEF) on a mini-Rotofor into 20 fractions of different pH values as indicated. Each Rotofor fraction was further fractionated by gel electrophoresis and immunoblotted with the N-terminal BHMT antibody. The figure is a composite of three separately stained blots from two different gels.