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 pyrophosphate-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 castor 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 subsequent 15-d growth period. The activity of PFK consistently exceeded that of any of the other enzymes, whereas PKc activity always surpassed that of PEPCase. The PEPCase activity was undetectable by day 10. Laser-densitometric quantification of immunoblots probed with polyclonal 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 increases and decreases in the ratio of the alpha (68-kDa):beta (64-kDa) subunits of PFP over the first 5 d and subsequent 15 d of germination, respectively. The alpha:beta subunit ratio of PFP was positively correlated 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 composition of PEPCase during germination. The PEPCase from cotyledons of imbibed castor seeds consists of a single immunoreactive 100-kDa subunit, whereas immunologically related 100- and 110-kDa polypeptides were observed on immunoblots of extracts prepared from cotyledons of 2- to 18-d germinated seeds. By contrast, over 20 d of seedling growth the cytosolic isoenzymes of cotyledonary PFK and PK appeared to be uniformly composed of a single type of subunit of approximately 61.5 and 56 kDa, respectively. This study provides a first insight into changes at the molecular level that accompany the surge in cotyledon glycolysis during castor seed germination, and suggests that the synthesis of four key glycolytic enzymes is highly regulated in this tissue and that this regulation follows a preset developmental program.