Please use this identifier to cite or link to this item: http://hdl.handle.net/123456789/527
Title: STUDIES ON SOME PLANT MEMBRANE GLYCOPROTEIN ENZYMES : PURIFICATION AND PROPERTIES OF 5' - NUCLEOTIDASE AND ACID PHOSPHATASE FROM THE MEMBRANES OF ARACHIS HYPOGAEA COTYLEDONS
Authors: Mittal, Richa
Keywords: GLYCOPROTEIN ENZYMES
NUCLEOTIDASE
ACID PHOSPHATASE
ARACHIS HYPOGAEA COTYLEDONS
Issue Date: 1986
Abstract: The germinating peanut .(Arachis hypoqaea) cotyledon, plasma membrane (PM) , Golgi-apparatus (GA) and endoplasmic reticulum (ER) fractions have been shown to contain a glycoprotein enzyme, i 5 -nucleotidase. The enzyme was purified from PM and GA fractions by the selective solubilization of the enzyme in a highly active and stable form with 0.5% octylglucoside in 50 mM Tris-HCl, pH 7.2, at a protein to detergent ratio of 2:3 in the presence of Mg + and EDTA followed by ion exchange chromatography on DEAEcellulose column. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) both PM-nucleotidase and the GA-nucleotidase showed a single protein band corresponding to the molecular weights of 55 K and 53.7 K daltons, respectively. The PM-enzyme had a broad _3 pH optimum of 5.0 - 6.0. The K^ and Vmax values were 1.0 x 10 M i and 8.5 pmol/min/mg protein with adenosine 5 -monophosphate (AMP) as substrate, respectively. The enzyme was found to be highly specific for 5*-AMP. Other nucleotides (GMP, UMP, CMP, ADP, GDP, UDP, ATP, GTP and CTP) as well as phosphorylated sugars were not hydrolyzed. p-Nitrophenyl phosphate was hydrolyzed at relatively much lower rate (15%) and the substrate affinity (l/fO was only t one-tenth that of AMP. Accordingly, the 5 -nucleotidase was referred to as AMPase. —3 The enzyme was competitively inhibited by ADP (K- = 2.4x10 M) and was also inhibited by NaF in a non-competitive manner with a K- value of 35 x 10"3M. Divalent cations, Ca2+, Mg2+, Hg2+, Zn2+, Ni + and the monovalent cations, K+, Li+ and Na+ had no effect on the enzyme activity. The purified AMPase was highly unstable losing its total activity within 24 h at -20°C or 4°C. While under these condit ions the crude solubilized enzyme (unpurified octylglucoside extract) was stable for several weeks indicating that some sta bilizing factors, most likely phospholipids, were lost during the enzyme purification. The plasma membrane AMPase was found to be a glycoprotein enzyme with 42.7% carbohydrate, composed of mainly D-mannose, N-acetyl-D-glucosamine and D-glucose. Evidence is presented that part of the carbohydrate moiety may be linked to the protein (apoenzyme) through the N-glycosidic linkage. The purified Golgi-apparatus AMPase resembles closely with the PM-AMPase with only very minor differences, implicating the transport of AMPase from GA to the PM. The microsomal membrane fraction (12,000 - 105,000 x g pellet) , obtained from 2-days old germinating peanut cotyledons, was surprizingly devoid of AMPase activity but contained suffi ciently high activity of acid phosphatase. Extraction of membrane fraction with 0.5% octylglucoside at a protein to detergent ratio of 1:2 solubilized about 65% of the acid phosphatase activity from the microsomal membranes. DEAE-cellulose column chromato graphy yielded three acid phosphatase containing protein peaks, eluting at 50 mM (enzyme I), 100 mM (enzyme II) and 200 mM (enzyme III) NaCl gradients. The folds of purifications of the enzyme I, II and III were 69.4, 26.5 and 16.1 with 58.03, 11.9 and 7.16 per cent yields, respectively. The isoenzyme I was found to be relatively pure by polyacrylamide gel electrophoresis as in addition to a major protein band, with electrophoretic mobility of 0.23 (relative to bromophenol blue) two minor bands were also present. On SDS-gel electrophoresis, the major band splitted into a major and a minor protein bands corresponding to molecular weights of 46.7 K and 50.1 K, respectively, indi cating that the enzyme may be composed of two or more subunits. Finally, the acid phosphatase isoenzyme I was obtained in pure form by preparative polyacrylamide gel electrophoresis. Like AMPase it was found to be a glycoprotein with 40% carbohydrate. The optimum pH, K^ and Vmax values for the hydrolysis of p-nitrophenyl phosphate were found to be 4.75, 10 mM and 5.5 umol/min, respectively. Orthophosphate inhibited the enzyme non-competitively with Ki of 34.5 mM. In this regard the enzyme differs from AMPase which was not inhibited by P. . Thermo dynamic parameters for the enzyme-p-nitrophenyl phosphate inter action were determined.
URI: http://hdl.handle.net/123456789/527
Other Identifiers: Ph.D
Appears in Collections:DOCTORAL THESES (Bio.)



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