UBC Theses and Dissertations
Gluconeogenic adaptations in Cancer Magister Guderley, Helga E
The periodic requirement for a new chitincus excskeleton imposes a large biosynthetic lead on the metabolism of crustaceans, with the hypodermis facing the brunt of the load. Since the freshly molted animal is highly susceptible to predation, the mechanisms for efficient gluccnecgenesis in support of chitin synthesis are of definite survival value to the organism. Measurements of enzyme activities in the hypodermis , gill and muscle of Cancer magisrter indicate that the hypodermis and muscle undergo considerable metabolic changes during the melt cycle. Freshmolt hypodermis shows elevated specific activities of both the gluconeogenic and the glycolytic enzymes, while freshmolt muscle shows decreased levels of the glycolytic and increased levels of the gluconeogenic enzymes. Hypodermis maintains a highly gluconeogenic orientation throughout the melt cycle. Phosphoglycerate kinase is considered to be one of the bifuncticnal enzymes in the glycolytic pathway, but the kinetic characteristics of the previously studied enzymes are ill suited for function in a gluconeogenic system. Since the inter melt and freshmolt muscle and hypodernis present a variety of metabolic poises (i. e. ranging 'from highly gluconeogenic tc highly glycolytic), I studied the control of phos|lidglycerate kinase in these tissue,s:. I found that the inte^0^^iusc 1 e enzyme shows kinetics much like those of the mammalian muscle and the yeast enzyme, with a high sensitivity to MgADP/ADP inhibition (MgADP Ki = 1.3 x 10⁻⁵M) and a relatively lew affinity for ATP as a substrate (Km = 2.03 x 10-⁴M). By contrast, the freshmolt hypodermal enzyme shows a considerably decreased sensitivity to Mg ADP/ADP inhibition (Mg ADP Ki = 2 x10-⁴M) and a considerably increased affinity for ATP (Km = 6.8 x 10-⁵M). The freshmolt muscle enzyme also shares these changed affinities. The intermolt hypodernal phosphoglycerate kinase shows the decreased sensitivity to Kg ADP/ADP inhibition but shares the ATP affinity of the intermolt muscle enzyme. The kinetic characteristics of the freshmolt hypodermal and muscle enzymes reduce the susceptibility of the enzymes to inhibition by MgADP and facilitate the reversal of the reaction for gluccneogenesis. The control of pyruvate kinase is integral to the control of both glycolysis and gluconeogenesis. In glycolysis, it forms the second major control site; in gluconeogenesis, it is one of the prime determinants of the rate of gluconeogenesis from lactate and amino acids. Muscle and hypodermal pyruvate kinases from Cancer magister are distinct proteins, on the basis of isoelectric points, kinetic characteristics, and thermal denaturation behavior. In contrast to the phcsphoglycerate kinase system, there are no pronounced differences between freshmolt and intermolt forms. Muscle pyruvate kinase is activated by FDP, inhibited by KgATP, arginine phosphate, Mg2citrate, tryptophan and is also sensitive to some inhibition by alanine, ⍺--glycerolphosphate, Mg-malate and ⍺- ketoglutarate. The muscle enzyme has a high affinity for PEP (Km = 0.1 mM) and the addition of 0.05 mM FDP drops the PEP Km to 0.05 mM. In comparison with other muscle pyruvate kinases, the enzyme is quite sensitive to MgATF inhibition (Ki = 1.8 mM) and shows FDP reversal of the inhibition. Arginine phosphate inhibition is competitive with ADP, and is not reversed by FDP. The reversal of the reaction accounts for only 0.5% of the forward reaction. Although high levels of ATP and arginine phosphate strongly inhibit the reaction, the inhibition is not sufficient to allow net flux through the low levels of the bypass enzymes present in the muscle of freshmolt animals. Thus, muscle pyruvate kinase has kinetic characteristics which suit it for function in the control of glycolysis, but do not allow gluconeogenic flux past the reaction locus. In contrast, hypodermal pyruvate kinase is a consplex protein capable of making large transitions between high activity during oxidation of carbohydrate substrates and virtually no activity during gluconeogenesis from lactate and amino acids. Hypodermal pyruvate kinase exists in twc conformational states, one a high affinity state (PK I) and the ether a low affinity state (PK II), PK I has a Km for PEP of 0.1 mM and a Ka for FDP of 1.3 x 10⁻⁵. PK II has a Km for PEP of 0.55 mM and a Ka for FDP of 9 x 10⁻⁸. For both forms, FDP facilitates the binding of PEP, Eofh forms are sensitive to MgATP inhibition and show FDP reversal of the inhibition, PK II is more sensitive to inhibition by alanine, serine, and Mg2citrate. For PK II, FDP alters the inhibition due to these compounds, changing the interactions between these inhibitors and both PEE and ADP. Incubation of PK II with 0.05 mK FDP produces PK I. Prolonged dialysis of PK I leads to an enzyme with the characteristics of PK II. The levels of FDP associated with PK I are higher than the levels associated with PK II. During gluconeogenesis, the FDP levels in the cell are low. This would shift the equilibrium between the two forms towards PK II. Since physiological levels of PEP, ADP, ATP, alanine, and serine limit PK II activity to less than 0.5% of maximal, considerable flux through the phosphcenolpyruvate carboxykinase and pyruvate carboxylase bypass would be feasible. The rise in FDP levels during inhibition of gluconeogenesis would shift the equilibrium in favor of PK I. This shift would immediately raise pyruvate kinase activity from less than 0.5% to around 50% of maximal activity. This, coupled with the other changes in metabolite levels during an inhibition of gluconeogenesis, would lead to a marked activation of pyruvate kinase activity. These conformational states allow rapid changes in flux through the reaction, and thus would allow flexible and responsive regulation of this important glycolytic and gluconeogenic control site. Thus, both the phcsphoglycerate kinase and pyruvate kinase present in the hypodermis of Cancer magister have special characteristics which facilitate efficient gluconeogenesis. To elucidate the possible importance of ions in regulating the activity of the above enzymes, I measured the levels of sodium, potassium, magnesium, and calcium in the muscle and hypodermis of intermolt and freshmolt animals. I found that the extracellular' space of- the hypodermis is considerably higher than that of the muscle (45% versus 12%) , but that there was no variation between intermolt and freshmolt tissue extracellular space. While freshmolt muscle sodium concentrations were significantly higher than intermolt sodium concentrations, none of the other ions showed significant differences between molt cycle stages. However, there were significant differences between the ionic concentrations in hypodermis and muscle. Hypodermis shewed higher calcium levels and lower potassium levels than muscle in both freshmolt and intermolt animals. Although icnic changes do not play a role in differential regulation of enzyme activity during the molt cycle, the ionic concentrations present in these tissues are such that the ions could set guidelines for the activity of phosphoglycerate kinase, pyruvate kinase, phosphofructokinase, and fructose diphosphatase in these tissues.