Studies on the Regulation of Enzyme Binding During Anoxia in Isolated Tissues of Busycon Canaliculatum

Abstract

The role of pH and protein kinase second messengers in triggering or potentiating anoxia-linked changes in enzyme binding to particulate matter were evaluated using in vitro incubations of isolated ventricle strips of Busycon canaliculatum (L.) (Prosobranchia, Melongenidae). Incubating whelks under anoxic conditions for 4h reduced the percentage of phosphofructokinase (PFK), aldolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) activity associated with cellular particulate matter. Triose phosphate isomerase, phosphoglycerate kinase, phosphoglyceromutase and enolase showed no changes in enzyme binding when whelks were subjected to anoxic stress in vivo. Incubating isolated ventricle strips in vitro under anoxic conditions simulated the changes seen in vivo in whole, anoxic whelks with respect to the percentage of PFK and PK bound during anoxic stress; both whole-animal studies and isolated tissue studies showed reduced PFK and PK binding after 4 h of anoxic incubation. Tissue pH could be artificially changed by incubating isolated ventricle strips in sea water buffered to a desired pH. This permitted an investigation of the effect of intracellular pH on PFK and PK binding in situ. PFK and PK responded to altered intracellular pH with increased enzyme binding at lower intracellular pH values and decreased enzyme binding at higher intracellular pH values. These binding patterns were exactly the opposite of those observed during anoxia; during anoxia stress, both intracellular pH and the percentage of PFK and PK associated with particulate matter decreased. Addition of the second messenger compounds dibutyryl cyclic AMP, dibutyryl cyclic GMP or phorbol 12-myristate 13-acetate plus the calcium ionophore A23187 had no effect on the percentage of activity bound to subcellular structures measured under either normoxic or anoxic conditions. This study suggests that enzyme binding in vivo is not regulated by changes in intracellular pH or concentrations of protein kinase second messenger compounds during anoxia.