Tài liệu Báo cáo khoa học: Temperature and phosphate effects on allosteric phenomena of

Temperature and phosphate effects on allosteric

phenomena of phosphofructokinase from a hibernating

ground squirrel (Spermophilus lateralis)

Justin A. MacDonald1 and Kenneth B. Storey2

1 Department of Biochemistry & Molecular Biology, University of Calgary, AB, Canada

2 Institute of Biochemistry and Department of Biology, Carleton University Ottawa, ON, Canada

Environments with widely differing seasonal tempera￾tures present thermoregulatory challenges to small

mammals who aim to maintain a constant body tem￾perature of about 37
C. Winter is particularly difficult

because energy use in support of homeothermy increa￾ses dramatically in cold weather at the same time as

the food supply declines. For many small mammals,

the only survival solution to this combination of low

food availability and low environmental temperatures

is hibernation [1–3]. The mammalian hibernator aban￾dons homeothermy and allows body temperature to

drop to that of its surroundings (although regulating

body temperature at 0–5
C if ambient temperature

falls below 0
C). The mechanisms that control the

entry into hibernation are still not fully understood

but it is known that an active suppression of basal

metabolic rate occurs (often to only 1–5% of the nor￾mal resting rate), preceding and causing the fall in

body temperature. Hibernation is also facilitated by

the accumulation, during late summer feeding, of huge

reserves of lipids; for example, in ground squirrels,

body mass often increases by 50% or more. These

lipids are the main fuel for winter energy metabolism

during torpor and measurements of respiratory quo￾tients confirm this. Lipid oxidation is supplemented to

some extent by gluconeogenesis from amino acids but

carbohydrate reserves are largely spared to be used

only by tissues and organs that can oxidize little else

Keywords

glycolysis; mammalian hibernation;

metabolic rate depression;

phosphofructokinase; temperature effects

Correspondence

K. B. Storey, Institute of Biochemistry and

Department of Biology, Carleton University,

1125 Colonel By Drive, Ottawa, ON,

K1S 5B6 Canada

E-mail: [email protected]

(Received 7 July 2004, revised 31 August

2004, accepted 14 September 2004)

doi:10.1111/j.1432-1033.2004.04388.x

Temperature effects on the kinetic properties of phosphofructokinase

(PFK) purified from skeletal muscle of the golden-mantled ground squirrel,

Spermophilus lateralis, were examined at 37
C and 5
C, values character￾istic of body temperatures in euthermia vs. hibernation. The enzyme

showed reduced sensitivity to all activators at 5
C, the Ka values for

AMP, ADP, NH4

+ and F2,6P2 were 3–11-fold higher at 5
C than at

37
C. Inhibition by citrate was not affected whereas phosphoenolpyruvate,

ATP and urea became more potent inhibitors at low temperature. While

typically considered an activator of PFK activity, inorganic phosphate per￾formed as an inhibitor at 5
C. Decreasing temperature alone causes the

actions of inorganic phosphate to change from activation to inhibition. We

found that Km values for ATP remained constant while Vmax dropped sig￾nificantly upon the addition of phosphate. Phosphate inhibition at 5
C

was noncompetitive with respect to ATP and the Ki was 0.15 ± 0.01 mm

(n ¼ 4). The results indicate that PFK is less likely to be activated in cold

torpid muscle; PFK is less sensitive to changing adenylate levels at the low

temperatures characteristic of torpor, and PFK is clearly much less sensi￾tive to biosynthetic signals. All of these characteristics of hibernator PFK

would serve to reduce glycolytic rate and help to preserve carbohydrate

reserves during torpor.

Abbreviations

PFK, 6-phosphofructo-1-kinase; F6P, fructose 6-phosphate; F2,6P2, fructose 2,6-bisphosphate.

120 FEBS Journal 272 (2005) 120–128 ª 2004 FEBS