REGULATION OF CYTOSOLIC CARBON METABOLISM IN GERMINATING RICINUS-COMMUNIS COTYLEDONS .1. DEVELOPMENTAL PROFILES FOR THE ACTIVITY, CONCENTRATION, AND MOLECULAR-STRUCTURE OF THE PYROPHOSPHATE-DEPENDENT AND ATP-DEPENDENT PHOSPHOFRUCTOKINASES, PHOSPHOENOLPYRUVATE CARBOXYLASE AND PYRUVATE-KINASE

The activity, concentration, and molecular structure of the ATP- and p yrophosphate-dependent phosphofructokinases (PFK and PFP, EC 2.7.1.11 and 2.7.1.90, respectively), phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) and cytosolic pyruvate kinase (PKc, EC 2.7.1.40) from ca stor seed (Ricinus communis L.) cotyledons were investigated over 20 d of germination. The activities of the four enzymes rapidly increased to maximal values by day 5, and then significantly declined over the s ubsequent 15-d growth period. The activity of PFK consistently exceede d that of any of the other enzymes, whereas PKc activity always surpas sed that of PEPCase. The PEPCase activity was undetectable by day 10. Laser-densitometric quantification of immunoblots probed with polyclon al antibodies against the four enzymes revealed that the developmental changes in enzymatic activity arose from alterations in the relative concentration of each respective protein. There were progressive incre ases and decreases in the ratio of the alpha (68-kDa):beta (64-kDa) su bunits of PFP over the first 5 d and subsequent 15 d of germination, r espectively. The alpha:beta subunit ratio of PFP was positively correl ated with the extent of activation of the enzyme by 2 mu M fructose-2, 6-bisphosphate. There was also an apparent alteration in the subunit c omposition of PEPCase during germination. The PEPCase from cotyledons of imbibed castor seeds consists of a single immunoreactive 100-kDa su bunit, whereas immunologically related 100- and 110-kDa polypeptides w ere observed on immunoblots of extracts prepared from cotyledons of 2- to 18-d germinated seeds. By contrast, over 20 d of seedling growth t he cytosolic isoenzymes of cotyledonary PFK and PK appeared to be unif ormly composed of a single type of subunit of approximately 61.5 and 5 6 kDa, respectively. This study provides a first insight into changes at the molecular level that accompany the surge in cotyledon glycolysi s during castor seed germination, and suggests that the synthesis of f our key glycolytic enzymes is highly regulated in this tissue and that this regulation follows a preset developmental program.