The distribution and some of the properties of phosphodiesterase II were studied in homogenates of rat intestinal mucosa in an attempt to elucidate its role in the nucleic acid metabolism of this tissue. In most of the experiments the p-nitrophenyl ester of thymidine 3'-phosphate was used as a substrate for phosphodiesterase.
During the work, evidence was accumulated which indicated that phosphodiesterase II of intestine was lysosomal in origin. For instance, when the tissue was suspended (or homogenized) in media of differing tonicity, the phosphodiesterase II activity in the hypotonic preparations increased markedly over a period of 96 hours. Other investigators have shown that this "osmotic activation" is a characteristic of lysosomal enzymes.
Subsequently, homogenates of mucosal tissue were fractionated by differential centrifugation and the subcellular fractions obtained were identified by known enzyme markers. The distribution of phosphodiesterase II in the fractions was most similar to that of the marker for lysosomes - acid phosphatase. However a large proportion of the phosphodiesterase II activity, greater than that of acid phosphatase, was found in the supernatant solution remaining after the final high-speed centrifugation step. The highest specific activity for phosphodiesterase II was found in the "light mitochondrial" and "final supernatant" fractions. Similar results were obtained when homogenates or nuclei-free homogenates were fractionated by sucrose density-gradient centrifugation. The distribution patterns of phosphodiesterase II and acid phosphatase were again similar and the particles to which phosphodiesterase II were bound exhibited the highest acid phosphatase activity. An attempt was made to confirm these results by "purifying" lysosomes from intestinal mucosa using a combined differential centrifugation and density-gradient centrifugation technique. During the purification, the specific activities of phosphodiesterase II and acid phosphatase increased parallel with each other and the "purified lysosomal" fractions exhibited the highest specific activities for these enzymes. However the total activities of the two enzymes recovered in the purified fractions were quite small, indicating considerable loss in the discarded soluble fractions.
Other workers have shown that homogenization ruptures lysosomes in certain fragile tissues, resulting in high soluble activities of the enzymes contained in these particles. It would seem possible therefore that in intestinal mucosa phosphodiesterase II is located in lysosomes in vivo, since most of its activity was found to be distributed between the lysosomal and soluble fractions of homogenates of this tissue.
The phosphodiesterase II of intestine was most active at pH values around neutrality. A second substrate, 2,4-dinitro-phenyl thymidine 3'-phosphate, which was used in only"a few experiments because of its limited availability, was hydrolyzed at a rate faster than that of p-nitrophenyl thymidine 3'-phosphate. Little or no change in the activity of the enzyme was observed in the presence of Mg++, Ca++ or EDTA, but Zn++, Cu++ and Hg++ inhibited markedly. The enzyme was most active at a temperature of 58° and only 27% of the activity was lost on heating the preparation for 1 hour at 55°. The Michaelis constant for the enzyme with p-nitrophenyl thymidine 3'-phosphate was 4.5 x 10(-4) M at 37°, and the activation energy for the phosphodiesterase II catalyzed hydrolysis of the same compound was 14.63 kilocalories/ mole. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/34704 |
| Date | January 1970 |
| Creators | Flanagan, Peter Rutledge |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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