Toshiharu Furusawa, Aya Konishi, Daisuke Sakano, Eiji Kotani, Yukio Sugimura, Janet M. Storey and Kenneth B. Storey
Phosphofructokinase (PFK) was purified from non-diapause eggs (incubated at 25°C) of the silkworm, Bombyx mori, by using hydroxylapatite, Sephacryl S-300, and ATP-agarose column chromatography. PFK activity separated into two distinct peaks on Sephacryl S-300, the molecular masses being 310 and 185 kDa; SDS-PAGE showed that both were composed of 48 kDa subunits. When eggs were cold-acclimated, different profiles occurred; eggs acclimated at 0°C exhibited two broad peaks of PFK activity centered at 310 and 90 kDa, whereas eggs held at 5°C showed activity primarily in a broad peak at 90 kDa. Partially purified PFK showed a sigmoidal saturation curve for its substrate, fructose-6-phosphate (F6P) with an S0.5 of 1.02 ± 0.27 mM at pH 7.0. PFK was activated by fructose-2,6-bisphosphate, AMP and inorganic phosphate, and inhibited by high concentrations of its second substrate, ATP. Diapause eggs showed low and constant PFK activity (about 0.2 U/g eggs) over 9-days after oviposition whereas when diapause initiation was prevented by HCl treatment one day after oviposition, PFK activity had doubled by day 6 and rose to 5-fold higher than initial values by day 9. To determine if the mechanism of this activity increase could be a change in the phosphorylation state of the enzyme, homogenates of day 6 and day 9 eggs were incubated under conditions that promoted the action of cAMP-dependent protein kinase or protein phosphatase and the effect of incubation on the S0.5 for F6P was monitored. Diapause egg PFK showed a high S0.5 of about 4 mM which was unchanged by incubation under phosphorylating conditions and but was strongly reduced under dephosphorylating conditions. PFK from HCl-treated eggs showed a decrease in S0.5 over time to 3.4 mM at day 6 and 0.9 mM at day 10 that was virtually the same as the effect of incubation under dephosphorylating conditions. Hence, the diapause form of the enzyme appears to be the phosphorylated form and the increase in activity when diapause was prevented by HCl-treatment can be linked to dephosphorylation of PFK. Thus, PFK activity in Bombyx eggs appears to be regulated by a variety of factors including temperature effects on the native state of PFK subunits, reversible protein phosphorylation, and the effects of allosteric modulators.