Increase in β- N -Acetylglucosaminidase Activity during Germination of Cotton Seeds

Abstract

MATERIALS AND METHODS A marked increase in ,B-acetylglucosaminidase (2-acetamido-2-deoxy-fi-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) activity was observed in the germinating cotyledon of cotton seeds. The enzyme was isolated from cotton seedlings and purified to study its physiological function in the germination of cotton seeds. The purification procedure involves ammonium sulfate fractionation, ion-exchange chromatography, gel filtrations, and concanavalin A-Sepharose 4B chromatography, and the purified ,B-N-acetylglucosaminidase was shown to be homogeneous by disc electrophoresis. The molecular weight was estimated to be about 125,000 by gel ifitration. The enzyme hydrolyzed both p-nitrophenyl-N-acetyl-.8-D-glucosamine andp-nitrophenyl-N-acetyl-/8-D-galactosamine. Whenp-nitro-phenyl-N-acetyl-8-D-glucosamine was used as substrate, Km and V,,,,, were 0.625 nanomolar and 228 moles per minute per milligram, respectively, and optimum activity was at pH 5.6. The enzyme liberated a8-linked N-acetyl-glucosamine from chitin, ovalbumin, and pronase-digested wheat germ lectin. PLANT MATERIAL Seeds of Gossypium hirsutum L. Im 216, which had been submerged in distilled H20 for 4 h, were planted in sterile vermiculite in plastic pans. After planting, seedlings were grown with a daily supply of distilled H20 in a greenhouse with a 16-h photoperiod at 27 C and an 8-h dark period at 21 C. Hypocotyl tissue emerged 2 days after germination, whereas development ofthe cotyledons did not occur until day 3. The plant tissue was harvested at the time indicated by removing vermiculite and washing the seedlings with cold distilled H20. ENZYME EXTRACTION For the determination of 8i-acetylglucosaminidase activity, cot-yledon halves and hypocotyls from five plants were homogenized with a prechilled mortar and pestle. Grinding was accomplished by using 10 ml 50 mm Na-acetate buffer (pH 5.0)/g fresh weight. The resultant slurry was squeezed through four layers of cheese-cloth and centrifuged at l0,OOOg for 20 min. The supernatant was used for the enzyme assay and determination of protein content. For the purification of 8-N-acetylglucosaminidase, a similar extraction method was followed. Some proteins, such as phytolectin and cotyledonary reserve protein, are known to have N-acetylglucosamine as a constituent of the carbohydrate component. This amino sugar is released from reserve glycoprotein following proteolytic activities during pea seed germination (2). Meanwhile, N-acetylglucosamine also is incorporated into glycoprotein through the mediating lipid structure (6). These facts imply that f8-N-acetylglucosaminidase may play a role in liberating the amino sugars from the reserve glyco-protein which, in turn, maintains the supply of amino sugars for glycoprotein synthesis during germination. Recently, ,8-N-acetylglucosaminidase has been used extensively for the study of oligosaccharide structure of glycoprotein (5, 15) and purified from a wide variety of biological materials (1, 7, 13), along with other glycosidases. This enzyme cleaves terminal, Ii-(1-4)-linked N-acetylglucosamine and N,N'-diacetylchitobiose moiety of asparagine-linked oligosaccharides of various glycopro-teins (11). However, the physiological function in plant metabolism has not yet been determined. The study presented here was undertaken to determine the change in enzyme activity during germination and to characterize f8-N-acetylglucosaminidase in germinating cotton seed.