Please use this identifier to cite or link to this item: http://hdl.handle.net/123456789/531
Title: BIOCHEMICAL INVESTIGATIONS ON PLANT MEMBRANES OF STORAGE TISSUES: PURIFICATION AND CHARACTERIZATION OF A GLYCOPROTEIN ACID PHOSPHATASE FROM MEMBRANES OF PISUM SATIVUM COTYLEDONS
Authors: Basbaa, Omar Mohammad
Keywords: BIOCHEMICAL INVESTIGATIONS
PLANT MEMBRANES
GLYCOPROTEIN ACID PHOSPHATASE
PISUM SATIVUM
Issue Date: 1989
Abstract: Various endomembrane fractions have been isolated from 18 h imbibed pea cotyledons by combination of differential and sucrose density gradient centrifugations. Without Mg and in the presence of 3 mM EDTA, organelles with average densities of 1.17 g.cnT3, 1.15 g.crn"3 and 1.10 g.cm"3 have been identified as PM, GA and ER on the basis of their characteristic sedimentation properties and enrichment of the marker enzymes for PM (glucan synthase II), for GA (inosine diphosphatase, ID-Pase, and glucan synthase I) and for ER (mannosy 1transfer ase, MTase), in the respective fractions. The PM, GA and ER thus obtained were enriched 12.8, 7 and 8 folds in their specific marker enzymes relative to the crude microsomal fraction (12,000-105,000 xg pellet) and were free of cross contamination as judged by the presence/absence of marker enzymes specific for different endomembranes. About 10 percent of the total acid phosphatase, APase, (E.C.3.1, 3.2) activity in the 18h imbibed pea cotyledons was associated with the microsomes which were almost completely devoid of 5'-nucleotidases and hexose phosphatases. Of the total microsomal APase activity, about 35, 4.5 and 6.4 percent was associated with the PM, GA and ER respectively. ii p-nitrophenyl phosphate (pNPP) was the best substrate for the membrane-bound enzymes from PM, GA and ER followed by ATP which was hydrolysed to the extent of 53, 61 and 74 percent relative to pNPP, by PM-, GA-, and ER-APase respectively. Phosphorylated sugars and nucleoside monophosphates were only slightly hydrolysed by the membrane-bound APase particularly the PM-bound. The apparent Km values of PM-, GA-, and ER- associated enzymes using pNPP as substrate were 500 jjM, 500 ;jM and 310^uM respectively. Their Vmax values were 66.7, 28.6 and 33.3 /UM per min per mg protein, respectively. The PM-, GA-, and ERassociated APases exhibited identical optimum pH range of 5.25 - 5.75, above and below which the activity of the enzymes declined sharply. The PM-, GA- and ER-APases were activated 25, 15 and 18 percent by EDTA. Citrate activated the PM-, GA-, and ERassociated enzymes to 426, 140 and 233 percent respectively whereas tartarate stimulated these enzymes to the extent of 375, 126 and 220 percent in order. Na+ and K+ were without effect. Most of the divalent metal ions tested (Mg2+, Ca2 +, Mn2 +, Zn2 +, Hg2 +, Cu2 +, Ni2 +, lOmM) were inhibitory to the PM-, GA-, and ERAPases, although the potency of inhibition varied markedly. Zn2 +, Mn2 +, Hg2+ and Cu2+ were highly potent inhibitors. Ni2 +, Ca^ and Mn^ had differential inhibitory effect on these membrane-bound enzymes. While there was little inhibition of the PM-APase by a Ca2+, the GA- and ER-APase were inhibited by 43 and 67 percent respectively. Ni2+ inhibited the PM-, GA-, and ERAPase by 83, 43 and 63.5 percent respectively, whereas Mg2+ inhibited these enzymes by 36, 55 and 58, in order. Pi, F and Mo70^7 were strong inhibitors of the three membrane-bound APases. The PM-bound APase was stable for one month at 0-4 C whereas the GA-, and ER-APases were rendered totally inactive in one week. However, about 80 percent of activity was retained upto 3 days storage at the same conditions. Apart from their difference in their stabilities, the membrane-bound enzymes were remarkably similar with respect to pH optimum, metal ion effect and response to inhibitors and activators. A major acid phosphatase was purified to homogeneity from the PM of pea cotyledons by selective solubilization of the enzyme with 1 percent CHAPS at a protein-to-detergent ratio of 2:3 in the presence of 5mM EDTA, followed by ion exchange chromatography on DEAE-Sephadex, acid precipitation at pH 5.0 and CM-Sephadex column chromatography. Both native and SDS-PAGE of the enzyme revealed the presence of a single polypeptide chain of around 68 kD molecular weight, though molecular weight by gel filtratioon was found to be 69,000 - 70,000 daltons. The purified enzyme was highly unstable losing total activity in 3 days at 0 - 4°C and after one week at - 20°C. The purified PM-APase exhibited maximal activity between 5.2 to 5.6 pH range and mximal stability over a pH range of 4.8 to 6.4. The Km and Vmax values for pNPP as substrate were 3.1 x 10~4 M and 2 mM per min per mg protein, respectively. Inorganic phosphate (Pi) and fluoride inhibited the enzyme in a competitive and noncompetitive manner, respectively. The Ki value for Pi was found to be 0.4 mM. Besides pNPP, the PM-APase also hydrolysed nucleoside di and nucleoside triphosphates but with only 30-40 percent efficiency. Nucleoside monophophates and phosphorylated sugars were not hydrolysed. The enzyme was unaffected by citrate or tartrate, contrary to the PM-bound enzyme to which they were strong activators. Metal ions were not required for activity. Mg2+ and Ca2+ showed only slight inhibition while Mn2+ and Ni2+ inhibited the enzyme to about 33 and 60%, respectively. Zn2+, Hg2+, Cu2+, F" and molybdate were strong inhibitors, inhibiting the enzyme activity almost totally. Na+ and K+ were without effect. The PM-APase was found to be a glycoprotein with 21.1% carbohydrate. Digest ion of the enzyme with endo-N-acety1- g-Dglucosaminidas- e H (endo-H) released about 70 percent of the total carbohydrate content of the enzyme indicating that the oligosaccharide moiety was asparagine-linked high mannose type. Both periodate treatment of the enzyme and deg lycosy1 at ion by endo-H resulted in loss of enzyme activity indicating the essential role of the carbohydrate moeity for the activity of enzyme. The Golgi apparatus acid phosphatase was also purified to homogeneity using essentially the same purification scheme used for the purification of the PM-APase except that gel filtration was introduced in the purification procedure in place of DEAESephadex . The molecular weight of the GA-APase was 65,500 daltons by gel filtration and 61,100 daltons by SDS-PAGE. The pH optimum of the enzyme was in the range of pH 5.2 to 5.6. Km and Vmax values were 3.6 x 10"4 M and 0.87 mM/min/mg protein, respectively. The enzyme was most active towards pNPP followed by nucleoside triphosphates (ca 35%). Nucleoside diphosphates were hydrolysed only slightly (10-15 percent) whereas 5'-mononucleotides and phosphorylated sugars were not hydrolysed at all. The enzyme did not require metal ions for activity. Ca2, Mg2+, and Mn2+ inhibited the enzyme from 20 to 30 percent. Hg2+, Cu2+, Zn2+ and Ni2+ were potent inhibitors, inhibiting 100, 80, 62 and 82 percent of the enzyme activity, respectively. F", PO| and Mo7°24 were also strong inhibitors. The mode of inhibition by fluoride and orthophosphate was noncompetitive and competitive, respectively. The enzyme activity was not stimulated by citrate and tartarate. The enzyme was a glycoprotein containing about 19.1 percent carbohydrate. Endo-H treatment released about 50 percent of the carbohydrate in 20 h indicating that the enzyme was linked to the asparagine residue of the peptide chain through N-glycosidic linkage and was of a high mannose type. The GAAPase was also senistive to endo-H and periodate treatments, although the sensitivity was relatively smaller than the PMAPase. However, the results indicated the presence of Nglycosidically linked oligosaccharides and the requirement of the carbohydrate for the enzyme activity of the GA-APase. Based on the close resemblance between the PM-APase and GA-APase, it is suggested that the GA-APase may be a precursor of PM-APase in pea cotyledon cells and that it may be used as a model glycoprotein enzyme for studying the intracellular transport of proteins that are destined to become PM associated proteins in pea cotyledon cells.
URI: http://hdl.handle.net/123456789/531
Other Identifiers: Ph.D
Appears in Collections:DOCTORAL THESES (Bio.)

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