The Association of Acid Hydrolase Activity with the Microsomal Fraction from Pea Cotyledons (Pisum sativum L.)

A fraction enriched in microsomal membranes was prepared fromdeveloping pea cotyledons by differential centrifugation andfound to contain 5-10% of the total extractable -mannosidase,-and ß-galactosidase, hexosaminidase, ß-glucosidaseand p-nitrophenylphosphatase (PNPase). Further purificationof this microsomal fraction on linear sucrose density gradientswith or without EDTA confirmed the association of the majorityof the glycosidase activity with ER membranes whereas PNPasewas associated with a different unidentified membrane componentfound at a density of 1:19 g cm^–3. The microsomal-associatedglycosidases were divided into luminal and membrane-bound fractions,the ratio being different for each individual glycosidase. PNPasewas entirely membranebound. Neither the membrane-bound glycosidasesnor PNPase could be released from the membranes by ionic treatment,changes in pH or competition with monosaccharide solutions.Chromatofocusing of the glycosidases from the microsomal fractionshowed that specific isozymes of -mannosidase and ß-galactosidasewere associated with the membranes and lumen respectively butthere was no consistent relations between these and the isozymespresent in the protein bodies. The significance of these observationswith regard to the intracellular targeting of newly synthesizedenzymes from their site of synthesis to specific organellesis discussed. Key words: Endoplasmic reticulum, Glycoproteins, Glycosidases, Lectin, Phosphatase, Protein transport