Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues

Abstract

The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 µmol/g wet weight and in muscle led to its increase from 3.64 to 25.1 µmol/g wet weight.Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK) by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

Influence of exercise on the activity and the distribution between free and bound forms of glycolytic and associated enzymes in tissues of horse mackerel

“…Previous studies from our laboratories found that the distribution of enzymes of carbohydrate metabolism between free and bound fractions may be modified by exercise in trout (12,29), anoxia in goldfish (28), and hypoxia in sea scorpion (30). However, whereas in the two first species an activation of glycolysis led to an increase or no change in enzyme binding, in sea scorpion tissues the situation was more complicated.…”

“…However, whereas in the two first species an activation of glycolysis led to an increase or no change in enzyme binding, in sea scorpion tissues the situation was more complicated. With increased glycolytic rate, the bound portion increased in some enzymes, decreased in some and did not change in others (30).…”

“…Exercised tissues were quickly washed in ice-cold 0.9% NaCl, blotted on filter paper, weighed and homogenized in a Potter homogenizer with a glass pestle in 10 volumes of homogenization solution containing 0.25 M sucrose, 10 mM dithiothreitol (DTT), 20 mM NaF, 1 mM EDTA, and 20 mM imidazole-HCl, pH 7.0, at 20 o C; 0.1 mM phenylmethylsulfonyl fluoride (PMSF, a protease inhibitor) was added immediately before homogenization (30). The homogenate was centrifuged for 20 min at 45,000 g at 4 o C and the supernatant was removed and stored on ice.…”

“…Reactions were started by the addition of the specific substrate but, if no spontaneous change in absorbance was observed, subsequent assays were started by the addition of homogenate. Optimal assay conditions were based on those described earlier (30) and adapted to our experiments. Hexokinase (HK, EC 2.7.1.1): 10 mM glucose (*), 0.2 mM NADP, 2 mM ATP, 5 mM MgCl 2 , 0.5 unit (U) glucose-6-phosphate dehydrogenase (G6PDH), and 50 µl supernatant.…”

“…In general, an increase in enzyme binding correlates with an increase in glycolytic flux and vice versa. But in our recent studies with a teleost fish, the sea scorpion, subjected to hypoxia we found that binding of some enzymes increased whereas binding of other enzymes decreased under the same stress conditions (30). Therefore, to determine whether this was an isolated instance or a common response by fish muscle, we undertook a comparable study with another teleost species.…”