Some pyrogen could be extracted from disrupted cells, but never more than a fraction (<25%) of that released from incubated whole cells. The yield could be slightly improved by lowering the pH (to 3.5) and by increasing the volume of extraction fluid.

Virtually all of the preformed pyrogen that could be extracted from sucroselysed cells was found in their cytoplasmic fraction. Contrary to the results of Herion et al. (3), none could be detected in the granular (or lysosomal) fraction. Likewise, all efforts to recover pyrogen from the membrane-nuclear fraction were unsuccessful.

In keeping with the finding that preformed pyrogen is contained in the cytoplasmic fraction were the observations that practically all of the aldolase, a cytoplasmic enzyme, and very little of the acid phosphatase, a granular enzyme, were lost from the cells during the release of pyrogen. Lysozyme, an enzyme stored in both the granules and the cytoplasm, was partially released from the cells under the same circumstances.

Neither the release of pyrogen nor its slight intracellular buildup that precedes release (4) were affected by concentrations of puromycin that block protein synthesis in the cells and prevent their activation. Hence, it is concluded that the release process, which also involves the formation of active pyrogen (4), does not require protein synthesis, whereas activation of the cells, which may involve the synthesis of an inactive precursor (2), does.