1. Is there a difference between the pattern of acid phosphatase and
uricase?
2. Is there a difference between the pattern of acid phosphatase and
cathepsin?
3. Is there a difference between the pattern of acid phosphatase and
cytochrome oxidase?
4. Is there a difference between the pattern of acid phosphatase and
glucose-6-phosphatase?
5. Why is sucrose used in the homogenization medium? How about water?
6. What would be the expected result if a different centrifugation
speed has been selected?
7. Why the fractionation steps only enrich for organelles and cannot
yield a pure fraction of organelles?
Expected results:
The enzyme distribution pattern shows a novel fraction containing acid
hydrolases (some enzyme assays have been performed at acidic pH, pH 5).
This experiment is similar to the one originally described in 1955 for
the discovery of the lysosome. By density gradient centrifugation,
the organelle containing uricase can be separated from the lysosome as
a separate organelle (peroxisome). Dr. Christian de Duve, who made
this discovery together with his colleagues, was awarded a Nobel prize
in 1974 for his discovery of lysosomes and peroxisomes. Lysosomes
serve a wide range of cellular degradative processes and the lack of lysosomal
enzymes, such as acid phosphatase, leads to lysosomal storage diseases.
References:
C. DeDuve et al. 1955 Tissue fractionation studies 6. Intracellular
distribution patterns of enzymes in rat liver tissue. Biochem J 60, 604.
H.G. Hers & F. van Hoof 1973 Lysosomes and Storage Diseases, Academic
Press, New York.
J. Lindsten 1992 Nobel Lectures Physiology or Medicine 1971-1980, World
Scientific, Singapore.
J.B. Lloyd & R.W. Mason 1996 Biology of the Lysosome. Plenum, New
York.
D.L. Spector et al. 1998 Cells; A Laboratory Manual, Cold Spring Harbor
Press, Cold Spring Harbor, New York.
L.A. Seidman & C.J. Moore 2000 Basic Laboratory Methods for Biotechnology,
Prentice Hall, New Jersey.