Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor‐oil‐plant endosperm and leaf
Authors: PLAXTON William C. 
Published: March 2005

Monospecific antiserum was raised in rabbits to homogeneous cytosolic pyruvate kinase isolated from 5‐day‐old germinating endosperm of the castor oil plant, Ricinus communis. An earlier study demonstrated that the purified enzyme is putatively heterotetrameric, composed of two subunits which migrate as 57‐kDa and 56‐kDa proteins upon sodium dodecyl sulfate/polycrylamide gel electrophoresis [Plaxton, W. C. (1988) Plant Physiol. (Bethesda) 86, 1065–1069]. Both proteins were detected on Western blots of extracts prepared under denafuring conditions from 4–8‐day‐old, but not 0–3‐day‐old, germinating‐endosperm tissue. This suggests that both subunits exist in vivo, and that the large increase in pyruvate kinase activity which occurs around the fourth day of germination is due to an increase in pyruvate kinase concentration.
The cytosolic and plastidic pyruvate kinase isozymes (termed PKc and PKp, respectively) from castor‐oil‐plant developing endosperm and expanding leaf tissue were separated by anion‐exchange chromatography on Q‐Sepharose. The antigenic reaction of the partially purified enzyme preparations to rabbit polyclonal antibodies raised against homogeneous germinating‐castor‐bean PKc was tested by immunoprecipitation and Western blotting. Although developing‐endosperm and leaf PKc appeared to be antigenically very similar to germinating‐endosperm PKc, they differed from the heterotetrameric germinating‐endosperm enzyme by being composed of a single type of subunit with a molecular mass of about 56 kDa. No cross‐reactivity of the PKc antibodies was observed with either developing‐endosperm or leaf PKp, nor with rabbit muscle or Bacillus stearothermophilus pyruvate kinase. Conversely, none of the castor‐oil‐plant pyruvate kinase preparations showed significant cross‐reactivity with antibodies raised against purified yeast or rabbit muscle pyruvate kinases.
To investigate the structural relationship between the two germinating‐endosperm‐PKc subunits, each polypeptide was characterized by amino acid composition analysis and peptide mapping by CNBr fragmentation. The amino acid compositions and CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. Mild tryptic attack of native enzyme led to an approximate 6‐kDa reduction in the apparent molecular mass of both subunits, further indicating sequence similarity between the two polypeptides.
Native molecular masses of the various castor‐oil‐plant pyruvate kinases were estimated by Superose‐6 gel‐filtration chromatography. Developing‐endosperm PKp was found to have a native molecular mass of about 290 kDa. In contrast, all PKc samples had native molecular masses in the range of 240 kDa, implying that leaf and developing‐endosperm PKc are homotetrameric.
The striking similarity between several comparative molecular and immunological characteristics of germinating versus developing castor‐bean‐endosperm PKc, and mammalian erythrocyte versus liver‐type pyruvate kinase is discussed.