The mantle pyruvate kinase (E.C. NO. 2.7.1.40) of Mytilus is shown to be subject to allosteric regulation, being inhibited by L-alanine and activated by D-fructose-1,6-diphosphate. It is also inhibited by adenosine-5'-triphosphate and to a lesser extent by L-phenylalanine and L-valine. Substrate (phosphoenolpyruvate) dependence of the enzyme is sigmoidal, and the sigmoidicity is increased at low pH. D-fructose-l,6-diphosphate at physiological concentrations over-rides inhibition of the enzyme by L-alanine. The result of these modulators acting together is suggested to Indicate that both the reaction catalysed by pyruvate kinase (phosphoenolpyruvate ? pyruvate) and the reaction catalysed by phosphoenolpyruvate carboxykinase (E.C, No, 4,1,1,32) (phosphoenolpyruvate ? oxaloacetate) could proceed together, with the predominance of one reaction over the other possibly being determined by the degree of tissue hypoxia. The value for K0.5s PEP of mantle pyruvate kinase varies seasonally and different patterns of variation are observed in three populations of Mytilus. The changes in substrate affinities are explained in terms of the different gametogenic cycles and ambient temperature regimes of the populations. The values for K0.5S posterior adductor muscle pyruvate kinases of two of the populations show a difference due to ambient temperature. It is concluded that the generation of molecular variants of pyruvate kinase from mantle tissue is related to gametogenesis. The implications of this conclusion are discussed in terms of the seasonal reproductive storage cycles of marine invertebrates. The operation of the anaerobic pathway in the posterior adductor musole of Mytilus is demonstrated under conditions of reduced ambient oxygen tension, viz: 120 mm Hg, 80 mm Hg and 40 mm Hg. Alanine, malate, succinate and glutamate accumulate while the steady-state concentration of "total amino acids" does not change. It is concluded that simultaneous operation of acrobic and anaerobic pathways in a single tissue occurs. The anaerobic pathway is operative also in whole tissues of Mytilus but not apparently in the mantle tissue i.e. a differential response of different tissues to partial anaerobiosis is observed. The anaerobic pathway is increasingly operative with reduction in ambient oxygen tension. The time-course of the substrate accumulations parallels the time-course of physiological acclimation of the whole organism to low oxygen tension. The levels of accumulated substrates decrease as acclimation increases the supplies of oxygen to the tissues, but generally remain in excess of the control concentrations after the process of acclimation has occurred indicating a continuing operation of the anaerobic pathway. Ration and temperature as stressors do not cause the anaerobic pathway to be operative at full air-saturation in the posterior adductor muscle, mantle (ration only) and whole tissues (temperature only), but do have an indirect effect on the requirement for its operation at reduced ambient oxygen tensions. The combination of very low oxygen tension (40 mm Hg) and warm temperature stress (+7C) is lethal and Mytilus is unable to acclimate. This is reflected biochemically in a continuing accumulation of anaerobic substrates without any subsequent decline in concentrations. A seasonal aspect to anaerobic metabolism is also indicated in the data. The amino acid aspartic acid is shown to be involved in anaerobic metabolism. Its concentration at reduced oxygen tensions changes in a reciprocal fashion to those of alanine, glutamate and succinate i.e. as the concentrations of the latter substrates decrease that of aspartic acid increases (and vice-versa). The amino acid alanine does not always accumulate at reduced oxygen tension while the accumulations of malate and succinate appear dependent upon the particular conditions of partial anaerobiosis. A ratio of aspartate: glutamate is proposed as an "index of anaerobic metabolism" and the use of the index as an indicator of sublethal-stress is discussed. The possible metabolic relationship of aspartate and glutamate is discussed and suggestions are made regarding the roles of these amino acids in anaerobic metabolism. Comparisons are made with the current hypotheses of the reaction pathways of anaerobic metabolism. The kinetic, adaptive and in some instances the regulatory properties of the phosphoenolpyruvate-carboxykinases, L-malate dehydrogenases (E.C. NO. 1.1.1.37) and L-glutamate dehydrogenases (E.C. NO. 1.4.1.3) from the posterior adductor muscle and mantle tissues of Mytilus are described. The optimal assay conditions and pH-profiles are demonstrated and comparisons of the enzymes from the two tissues made. No qualitative or quantitative changes in the pyruvate kinases and phosphoenolpyruvate- carboxykinases occur with acclimation to low oxygen tension but a qualitative change in the malate dehydrogenases is indicated. A possible regulatory role for the malate dehydrogenases is also indicated. The properties of all the enzymes studied are discussed in relation to anaerobic metabolism and are also compared with the known properties of other invertebrate and vertebrate enzymes. The results and conclusions of the experimental studies are discussed finally in relation to the possible role of the anaerobic pathway in the ecology of Mytilus and other marine invertebrates.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:463407 |
| Date | January 1975 |
| Creators | Livingstone, David Robert |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/34136 |
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