Organ-specific regulation of phosphofructokinase during facultative anaerobiosis in the marine whelk Busycotypus canaliculatum

Abstract

The kinetic properties of 6-phosphofructo-1-kinase (PFK) were assessed in five organs (ventricle, radular retractor muscle, gill, hepatopancreas, and kidney) of aerobic and anoxic (21 h in N2-bubbled seawater) whelks, Busycotypus canaliculatum. The enzyme in all organs showed a stable modification of kinetic parameters as a result of exposure of the animal to anoxic conditions. In most cases these changes were consistent with the conversion of the enzyme to a less active form in the anoxic organ. In ventricle, for example, the anoxic enzyme form showed significant changes, including a 36% increase in the value of the substrate affinity constant (S0.5) for Mg∙ATP, a 19% increase in S0.5 fructose-6-phosphate, a 57% increase in the 50% inhibition value (I50) for phosphoenolpyruvate, a 30% increase in I50 citrate, and a fivefold increase in the activator constant (Ka) for fructose-2, 6-bisphosphate, as compared with the aerobic enzyme. Analysis of the time course of anoxia-induced modification of PFK showed that changes to the properties of gill PFK were accomplished within 2 h of the exposure to N2-bubbled seawater, whereas changes to ventricle PFK required up to 8 h. In vitro incubation of ventricle homogenates with Mg∙ATP plus protein kinase second messengers or with Mg2+ plus added protein phosphatases showed that the aerobic enzyme form was modified by protein kinase action with an increase in Ka fructose-2,6-bisphosphate that mimicked the effect of the aerobic to anoxic transition on the enzyme. Phosphatase action on the anoxic enzyme form had the opposite effect. The data suggest that the modification of PFK properties under anoxia in whelk organs is due to protein phosphorylation of the enzyme. Such covalent modification of PFK and other enzymes, notably pyruvate kinase, coordinates the anoxia-induced glycolytic rate depression and overall metabolic arrest that is a prominent feature of facultative anaerobiosis in marine molluscs.