Analytical Subcellular Fractionation of Human Granulocytes with Reference to the Localization of Vitamin B12-Binding Proteins

Abstract

1. A granulocyte-rich fraction was isolated from blood of a patient with chronic granulocytic leukaemia and from blood of a normal subject and the cells were disrupted in isotonic sucrose. The nuclei were removed by low-speed centrifugation and the post-nuclear supernatant was fractionated by centrifugation on sucrose density gradients. 2. The subcellular organelles in the gradients were detected with marker enzymes by the use of highly sensitive assay techniques. Similar results were obtained with granulocytes from both subjects. 3. Unsaturated vitamin B₁₂-binding proteins were almost exclusively localized to the specific granules. Chromatographic analysis of these proteins showed them to have approximately equal proportions of transcobalamins I and III. Vitamin B₁₂ assayed microbiologically could not be detected in the specific granule fractions.