Previous studies on nerve phosphates (Gerard and Wallen, '29) were concerned primarily with the creatine-phosphate and acid soluble fractions. These were amplified for invertebrate nerve (Engel and Gerard, '35) and by creatine analyses of vertebrate nerve and muscle (Gerard and Tupikova, '38). The present work extends the investigation to other phosphate fractions of frog nerve and muscle, especially the pyrophosphate and the acid insoluble ones ; and we have also examined the distribution of phosphorus compounds along nerve in relation to the claim that a definite gradient exists.
METHODSThe preparation and handling of tissue and the main analytic procedures have been described elsewhere (Gerard and Tupikova, '38 ; Engel and Gerard, ' 3 5 ) . A number of control tests were made, all of which affirmed the validity of our procedures : color comparisons with varying strengths of acid and molybdate agreed, within the range of our conditions ; ashing in 2 N or 10 N sulphuric acid gave the same total phosphate values ; no phosphate was gained or lost by contact with filter papers ; triplicate determinations on the same material made in a few cases of 'labile' and for total acid soluble phosphate, agreed within 2 % ; inorganic phosphate added to muscle was recovered entirely in the inorganic fraction. T~ILIS, in two paired experiments, 0.16 mg., P was added to one muscle, none to its mate. After some hours in oxygen, P was distributed in the control muscles : inorganic 20 mg. per cent, creatine-phosphate 59, pyrophosphate 21. The expected values for the treated muscles were, respectively: 71, 59 and 21, and those found ; 70,58,20. Pyrophosphate is determined by the difference between measurements with and without 7 minutes' boiling in normal HC1. Six entire hydrolysis curves showed the same values after 7 and 15 minutes' boiling, so that the 7-minute value may fairly be assumed to represent pyrophosphate rather than other carbohydrate phosphates (Engel and 'Aided by a grant from the Rockefeller Foundation to The University of Chicago.