Myokinase and three inorganic pyrophosphatases with pH optima of 6.4, 7.2 and 8.4 have been isolated and purified from the thoracic muscle mitochondria of the American cockroach.
Myokinase was purified over 100-fold by heat and acid treatment, ammonium sulfate fractionation and column chromatography on G-75 sephadex. Some properties of the enzyme were determined. These include: a pH optimum of 5.8, an optimum Mg concentration of 3 x 10⁻³ M, a substrate specificity for ADP, and an equilibrium constant of 0.44. In addition, Km and Ki values were determined for each adenosine nucleotide, inhibition studies were conducted, and the enzyme was found to have the greatest stability against heat denaturation at a neutral pH. From the results obtained here it appears that the enzyme apyrase is not responsible for the rapid dephosphorylation of ATP. In addition, this study indicates that a specific triphosphatase coupled with myokinase produces the products AMP and P<sub>i</sub> which are found when crude homogenates are used as the enzyme.
The three inorganic pyrophosphatases were purified by sonication, ultracentrifugation, ammonium sulfate fractionation, and column chromatography on G-75 sephadex, DEAE cellulose, and CM cellulose. The three enzymes were found to have distinct pH optima: one acid, one neutral and one alkaline. The enzyme activated at a neutral pH needed only one-half as much Mg as substrate. The optimum ratio for the other two enzymes was one to one. Substrate specificity studies indicated that the enzymes may be important in the breakdown of polyphosphates and GTP. No other high energy phosphate bonds were acted on. The physiological significance remains obscure although data from this study indicate that an important function of the enzymes may be to remove undesirable pyrophosphate from the mitochondria. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/106282 |
| Date | January 1964 |
| Creators | Mills, Richard Randolph |
| Contributors | Entomology |
| Publisher | Virginia Polytechnic Institute |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | 60 leaves, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 20269346 |
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