Fig. 1. Separation of peroxisomal subcompartments from activated spheroplasts. (A)
Outline of procedure. After metabolic labeling, activated spheroplasts are
subjected to osmotic lysis at pH 5.5. An organellar pellet (PEL) and
supernatant (SUP) are generated by centrifugating the lysate at 20,000
g. (There are no prespins.) The pellet is resuspended in
buffer at pH 9.0 to lyse peroxisomes, and then spun at 100,000
g, generating a supernatant containing peroxisomal matrix
(MAT) and a pellet containing peroxisomal membrane (MEM). Analysis of markers
in a typical experiment is shown in B-D. (B) Formaldehyde dehydrogenase, a
cytosolic enzyme. There is little contamination of cytosol in the matrix. (C)
Coomassie Blue staining indicates the integrity of peroxisomes after cell
lysis, and the subsequent release of matrix. (D) Immunoblot with anti-Pmp47,
an integral peroxisomal membrane protein.
