Urea and salt effects on enzymes from estivating and non-estivating amphibians

Abstract

The effects of urea, cations (K+,NH4,Na+,Cs+,Li+), and trimethylamines on the maximal activities and kinetic properties of pyruvate kinase (PK) and phosphofructokinase (PFK) from skeletal muscle were analyzed in two anuran amphibians, an estivating species, the spadefoot toad Scaphiopus couchii, and a semi-aquatic species, the leopard frog Rana pipiens. Urea, which accumulates naturally to levels of 200-300 mM during estivation in toads, had only minor effects on the Vmax, kinetic constants and pH curves of PK from either species and no effects on PFK Vmax or kinetic constants. Trimethylamine oxide neither affected enzyme activity directly or changed enzyme response to urea. By contrast, high KCl (200 mM) lowered the Vmax of toad PFK and of PK from both species and altered the Km values for both substrates of frog PFK. Other cations were even more inhibitory; for example, the Vmax of PK from either species was reduced by more than 80% by the addition of 200 mM NH4Cl, NaCl, CsCi, or LiCl. High KCl also significantly changed the Km values for substrates of toad lactate dehydrogenase and strongly reduced the Vmax of glutamate dehydrogenase and NAD-dependent isocitrate dehydrogenase in both species whereas 300 mM urea had relatively little effect on these enzymes. The perturbing effect of urea on enzymes and the counteracting effect of trimethylamines that has been reported for elasmobranch fishes (that maintain high concentrations of both solutes naturally) does not appear to apply to amphibian enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

A current perspective on the compensatory effects of urea and methylamine on protein stability and function

“…Insensitiveness of urea effects on O 2 affinity of the hemoglobin of Squalus acanthias towards TMAO was found by Weber and his group (Weber, 1983). Grundy and his co-workers also observed that the perturbing effect of urea on kinetic properties of phosphofructokinase and pyruvate kinase from skeletal muscles of two anuran amphibians, an aestivating species, Scaphiopus couchii, and a semi-aquatic species, the leopard frog Rana pipiens were not counteracted by TMAO (Grundy and Storey, 1994). Besides these examples, in some systems like the contraction of skinned shark muscle fibres (Altringham and Johnston, 1982) and mitochondrial respiratory system, the counteraction system has been demonstrated to be very complex.…”

Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses

“…Inhibition by urea may actually be useful during estivation. However, at least some of their enzymes are more resistant to urea than are enzymes of other species (Grundy and Storey, 1994;Fuery et al, 1997). Thus, as originally noted for elasmobranch proteins (Yancey et al, 1982), there may be more than one way to adapt to high urea.…”

Cryoprotection by urea in a terrestrially hibernating frog

“…The urea-hypometabolism hypothesis garners support from in vitro studies of enzyme kinetics (Cowan and Storey, 2002;Grundy and Storey, 1994;Stewart et al, 2000) but heretofore was not demonstrated at higher levels of organization. Our present findings should be regarded as tentative until confirmed through additional research.…”