New fracture-labelling method: alkaline phosphatase in unstimulated human neutrophils

We show a new freeze-fracture enzyme cytochemistry technique, i.e. fracture-labelling by enzyme cytochemistry. Freeze-fracture replication is carried out first and subsequently enzyme molecules in the split-membrane halves of cellular membranes are labelled with enzyme cytochemical markers. A replica-digestion treatment before the cytochemical reactions is a key step in this method. Triton X-100, saponin, and ultrasonication provided adequate cleaning of the replicas with good preservation of enzyme activity. Enzyme cytochemical fracture-labelling was applied to the study of alkaline phosphatase (ALPase)-positive granules in unstimulated human neutrophils. ALPase activity was found primarily on the exoplasmic membrane halves of intracellular small granules. Using X-ray microanalysis, we confirmed that the electron-dense deposit on replicas was the reaction product demonstrating ALPase activity. The results obtained by this method should provide unique information for the understanding of structure and function of biological membranes.