The metabolic fate of putrescine labelled in vivo was investigated after administration of a trace (10-7 M) of L-[14C]ornithine to exponentially growing mycelia of Neurospora crassa, followed by a large chase (2 mM) of L-[12C]ornithine. The specific radioactivities of putrescine and spermidine were determined during the chase period by reaction with [3H]dansyl chloride of known specific radioactivity and isolation of the dansyl-derivatives by thin-layer chromatography. Radioactivity remained in the putrescine pool for over 2 h during the chase period. This suggests that putrescine is largely sequestered (80% or more) in vivo. The metabolic sequestration of polyamines may be a significant factor in the regulation of polyamine synthesis. © 1982.

A new and inexpensive glass-bead blender allows rapid, easy, and controlled cell breakage of Neurospora, with good organellar survival. The yield of mitochondria and vacuoles is comparable to or better than methods involving sand grinding or snail-gut digestion of cell walls. A method for removing cell wall fragments from a crude homogenate is described. Isolation of mitochondria and vacuoles from the crude homogenate with little cross-contamination is accomplished by density-gradient centrifugation in a fixed-angle rotor (Sorvall).