Gluconeogenic adaptations in Cancer Magister

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.Science, Faculty ofZoology, Department ofGraduat

Are mitochondria subject to evolutionary temperature adaptation

Thermal tolerance and the respiratory properties of isolated red muscle mitochondria were investigated in Oreochromis alcalicus grahami from the alkaline hot-springs, Lake Magadi, Kenya. Populations of O. a. grahami were resident in pools at 42.8 degrees C and migrated into water reaching temperatures of 44.8 degrees C for short periods. The maximum respiration rates of mitochondria with pyruvate as substrate were 217 and 284natom0 mg(-1) mitochondrial proteinmin-l at 37 degrees C and 42 degrees C, respectively (Q(10) = 1.71). Fatty acyl carnitines (chain lengths C8, C12 and C16), malate and glutamate were oxidised at 70-80 % of the rate for pyruvate. In order to assess evolutionary temperature adaptation of maximum mitochondrial oxidative capacities, the rates of pyruvate and palmitoyl carnitine utilisation in red muscle mitochondria were measured from species living at other temperatures: Notothenia coriiceps from Antarctica(-1.5 to +1 degrees C); summer-caught Myoxocephalus scorpius from the North Sea (10-15 degrees C); and Oreochromis andersoni from African lakes and rivers (22-30 degrees C). State 3 respiration rates had Q(10) values in the range 1.8-2.7. At the lower lethal temperature of O. andersoni (12.5 degrees C), isolated mitochondria utilised pyruvate at a similar rate to mitochondria from N. coriiceps at 2.5 degrees C (30 natom O mg(-1) mitochondrial protein min(-1)). Rates of pyruvate oxidation by mitochondria from M. scorpius and N. coriiceps were similar and were higher at a given temperature than far O. andersoni. At their normal body temperature (-1.2 degrees C), mitochondria from the Antarctic fish oxidised pyruvate at 5.5% and palmitoyl-DL-carnitine at 8.8% of the rates of mitochondria from the hot-spring species at 42 degrees C. The results indicate only modest evolutionary adjustments in the maximal rates of mitochondrial respiration in fish living at different temperatures

Enzyme activity in the aestivating Green-striped burrowing frog (Cyclorana alboguttata)

Green-striped burrowing frogs (Cyclorana alboguttata) can depress their resting metabolism by more than 80% during aestivation. Previous studies have shown that this species is able to withstand long periods of immobilisation during aestivation while apparently maintaining whole muscle mass and contractile performance. The aim of this study was to determine the effect of prolonged aestivation on the levels of metabolic enzymes (CCO, LDH and CS) in functionally distinct skeletal muscles (cruralis, gastrocnemius, sartorius, iliofibularis and rectus abdominus) and liver of C. alboguttata. CS activity was significantly reduced in all tissues except for the cruralis, gastrocnemius and the liver. LDH activity was significantly reduced in the sartorius and rectus abdominus, but remained at control (active) levels in the other tissues. CCO activity was significantly reduced in the gastrocnemius and rectus abdominus, and unchanged in the remaining tissues. Muscle protein was significantly reduced in the sartorius and iliofibularis during aestivation, and unchanged in the remaining muscles. The results suggest that the energy pathways involved in the production and consumption of ATP are remodelled during prolonged aestivation but selective. Remodelling and subsequent down-regulation of metabolic activity seem to target the smaller non-jumping muscles, while the jumping muscles retain enzyme activities at control levels during aestivation. These results suggest a mechanism by which aestivating C. alboguttata are able to maintain metabolic depression while ensuring that the functional capacity of critical muscles is not compromised upon emergence from aestivation. © 2010 Springer-Verla