Summary
Various methods of disrupting liver cells and preparing microsomes were studied. The quality and yield of the microsomes in the preparations was judged by the NADPH-dependent N- and p-hydroxylation of N-ethylaniline, protein content, and cyanide-sensitive respiration.
The smallest content of inert proteins and mitochondrial fragments, and therefore the highest activity per mg microsomal protein, was found in microsomes prepared from the pulp obtained by forcing liver through a 1 to 3 mm mesh screen. Homogenizing the pulp in a rotating homogenizer did not increase the yield of hydroxylating activity but only the inert protein, mitochondrial fragments, and factors causing auto-inhibition.
The first extract from the diluted pulp or homogenate prepared by centrifugal fractionation contained less than half the total activity. Further extracts from the homogenized 9000×g sediment yielded microsomal fractions of considerable activity.
In determining the total hydroxylating activity of several microsomal fractions prepared from liver pulp or homogenate, attention had to be paid to the auto-inhibition observed by increasing the content of microsomes in the suspension.
The use of water, 0.15 M KCl, or 0,25 M sucrose solution in place of 0.1 M phosphate solution for diluting or homogenizing the liver pulp did not increase the yield of microsomes.
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von Jagow, R., Kampffmeyer, H. & Kinese, M. The preparation of microsomes. Naunyn - Schmiedebergs Arch 251, 73–87 (1965). https://doi.org/10.1007/BF00245731
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DOI: https://doi.org/10.1007/BF00245731