STUDIES ON GLYCOPROTEIN ENZYMES OF PEANUT COTYLEDON CELL MEMBRANES : PURIFICATION TO HOMOGENEITY AND CHARACTERIZATION OF SOME GLYCOPOLYPEPTIDES WITH HIGHLY SPECIFIC AMPase ACTIVITY

Abstract

A plasma membrane localized low molecular mass (26.3 Kd) adenosine 5'-monophosphatase (PM-AMPase) and a Golgi apparatus localized high molecular mass (52.4 Kd) adenosine 5'-monophosphatase (GA- AMPase), from cotyledons oi 7 -days old germinating peanut seedlings, have been purified to apparent homogeneity. This is evident by SDS-PAGE and Western blotting. Purification was carried out by selective solubilization of the membrane-bound enzyme with 1.0% CHAPS, at aproteinto- detergent ratio of 2:3 in the presence of Mg2+ ions and EDTA, followed by gel filtration on Sephadex G-1 50 and ion-exchange chromatography on DEAE-cellulose. Both PMAMPase and the GA-AMPase are glycoproteins with 37% and 39% of carbohydrate content, respectively and have optimum pH in the range of 5.0 to 5.5. The K and Vmax values for the PM-AMPase are 0.58 mM and 5.9 u mole Pi/min/mg-protein and that for the GA-AMPase are 1.0 mM and 6.8 (.i mole Pi/min/mg-protein,respectively. The PM-AMPase is highly specific for 5'-AMP. Other nucleotides, phosphorylated sugars and p-nitrophenyl phosphate (p-NPP) were not hydrolyzed. The purified GA-AMPase shows much broader substrate specificity and also catalyzes the hydrolysis of S'-GMP. 5'-UMP and p-NPP at significant rates. The purified enzymes are highly unstable, losing their total activity within 24 h at -20°C. Under similar conditions the partially purified enzyme from Sephadex G-150 column was stable, indicating that some stabilizing factor(s), most likely phospholipids., necessary for the maintenance of the biologically active conformation of the enzyme was lost during enzyme purification by ion-exchange column. This indeed was the case, as is evident from the results obtained on addition of phosphatidylcholine Addition of phosphatidycholine restored nearly 46% of the activity of the enzyme, (i) which had earlier lost about 90% of its activity during storage at -20UC. These results also suggest that the PM- and GA-AMPases occur as complex ot phospholipid and protein. The stability of the purified enzymes was greatly enhanced by 20% glycerol. Thus, the PM-AMPase and GA-AMPase show a great degree of resemblance in their properties. However, the two AMPase, besides having different molecular masses, vary in their immunological response. The polyclonal monospecific antibodies raised against the purified PM-AMPase did not cross react with the purified GA-AMPase as shown by Western blotting, double immunodiffusion and immunoprecipitation experiments. In other words the PM-AMPase and GA-AMPase do not have common determinants and may be structurally unrelated, being most likely the products of two different genes. The GA-AMPase may be a Golgi resident protein which does not migrate to the PM while the PM-AMPase migrates to PM by a default mechanism without being retained in GA. The peanut PM-AMPases antibodies also did not cross react with detergentsolubilized AMPases from PM of other plant sources, for example, soybean, chickpea, wheat and pea. Thus, the plant AMPase are both subcellular as well as speciesspecific in nature. It is proposed that the GA-AMPase and PM-AMPase may be used as model glycoproteins for studying the intracellular transport of proteins in plant cells.