STUDIES ON GLYCOPROTEIN ENZYMES OF PLANT STORAGE TISSUES : PURIFICATION AND CHARACTERIZATION OF ACID PHOSPHATASE ISOENZYMES FROM THE MEMBRANES OF PEANUT COTYLEDON CELLS

Abstract

Highly enriched plasma membrane (PM), Golgi apparatus (GA) and endo plasmic reticulum (ER) fractions were obtained from the germinating cotyledons of peanut (Arachis hypogaea) and were identified by the presence of various marker enzymes. All the three membrane fractions were shown to contain the glycoprotein enzyme acid phosphatase (APase). The PM fraction exhibited the maximum activity of APase, followed by GA and ER, respectively. Since the microsomal membrane fraction of 2-days old cotyledons was found to be devoid of 5'-nucleotidase activity but possessing considerable amount of APase acti vity, it was used as the starting material for purifying APases present in the PM traction. The isoenzymic pattern of APase in the PM fraction was found to change with progressive germination along with changes in the relative levels of the APase isoenzymes. The PM fraction of 0-days and 2-days old coty ledons were found to contain three distinct APase isoenzymes whereas /-days germinated cotyledons contained only two APase isoenzymes. APases were purified from the PM fraction by the selective solubiliza tion of the enzyme in an active and stable form with 0\5% n-octylglucoside at a protein-to-detergent ratio of 2:3 in presence of Mg"* and EDTA, followed by CM-Sephadex C-5G chromatography and gel filtration on Sephadex G-150 resin. The chromatographic step identified and separated three APase isoenzymes named APase I, APase II and APase III. Sodium dodecyl sulfate - polyaerylamide gel

electrophoresis (SDS-PAGE) established the homogeneity of the APase isoenzymes. The purification folds of APase I, APase II dnd APase III were 12.1, 10.6 and 9.9 while their respective molecular weights were 79 Kda, 76 Kda and 66 Kda. The PM-APases gave identical pH-activity profiles with a pH-opt unum of 5.0. The isoenzymes were found to exist in monomeric forms at pH 5.0 consisting of single polypeptide chains, but they associated at pH 7.2 to grve higher dimeric forms consisting of two polypeptide chains of identical molecular weight. The higher d.meric forms subsequently dissociated back into monomers on lowering the pH from 7.2 to 5.0. The «4,trat. specificities of the PM-APases was eiso foood to he pHdepeodeot. MpH 7.2, the ,Pase isoeoz^es showed ,,„(„ ,ui,t„t. specif icit, •owards oodeoMde -sod .„„„ pto.pAit„, „„„„ „ pfl }Q^ ,^ ^ <«.«»», soced M,i„„ specif icit, to„scds „_„„„ ^ ^^f^ T"e ". Md \ax "•'»« <* ««. r. sPsse II sod .Psse III for t„ hydrolljsls «*» were ,.« *,, 0.50 „ d„d „.„ „ ^ ^ ^^ ^^^ ^ ^ 10 OToi/.io/^ sod ,., „20* „„,/*,„/„, c„wtJv.iv, Ortio(lh<„piat. mM_ M« -see Isod ,Psse II eo.petit^I, « i„,iMted „„. ,„ „0_peU_ Mwl,. Pespoose of ,he P„-,Psses t0„„ds ,„,„„ c«t ions aBt ^ _ ^ to be highly differential in nature. Metidioe residues. C.steioe residues also appeared to he involved .«» the to be associated with thfle« PP«M-^ApPd^sceesc. 2tkh«e presence of Cu and Fe was not. detec ted in any of the isoenzymes. »*se I, APase Uand tease UI contained 50,. 27,. and 10, oarhohgdra-e

sugars m case of APase' II and APase III were D-man and D-glu. The nature of peptide-carbohydrate linkage of APase I appeared to be N-glycosidic but O-glycosidic in case of APase II and APase III. Sodium metieriodate strongly inhibited the activities of PM-APases. The APase isoenzymes exhibited a broad range of thermal stability and temperature activity profiles. They also appeared to be quite stable for a considerable period of time when stored at -20"C. Nonensin brought about an increase in the level ot GA-APase but . conco mitant decrease in the level of PM-APase suggesting a blockage in intracellular transport of APases from GA to PH, Gibberellic acid (GA,) treatment brought about an accumulation of APase activity in the PM but fl decredSe t„ APdse activity in GA dnd ER fractioa8t n is 5Ugges,ed thdt Apdses ^^^ ^ the peanut cotyledon cells are intracellularly transported by the route ER -+GA —*PM. Antibodies raised against peanut cotyiedon PM-APases were seen to immunoprecipitate APase activity from ER and GA fractions as well. PM-APases were found to be immunologically related to each other. They were also found to be unmunologically related to an APase isoenzyme (AP-I) present, in >he plasma membranes of pea cotyledons and APase present in the epididymis of ram and assumed to share some common antigenic determinants with them. It is suggested that APases from peanut cotyledon PM, pea cotyledon PM and ram epididymis share common epitopic regions and all of them may have evolved from acommon ancestral gene.