Processing and secretion of alpha-mannosidase forms by Dictyostelium discoideum.
We are studying the biosynthesis and processing of acid hydrolases from Dictyostelium discoideum. We prepared antibody to highly purified alpha-mannosidase from the spent medium of stationary phase cultures. It precipitated alpha-mannosidase but not beta-hexosaminidase, alpha-glucosidase, beta-glucosidase, or any of the major proteins in cell lysates or secretions. The antibody precipitated a 150,000- and an 80,000-dalton protein in addition to mature forms (56,000-62,000 daltons) of alpha-mannosidase subunits. The possibility that the 150,000 and 80,000 dalton bands were precursors of mature forms was evaluated by pulse-chase experiments. Following a 20-min pulse labeling period, only the 150,000-dalton protein was detected in the immunoprecipitate. Apparent conversion of this form into 80,000- and 60,000-dalton forms was observed following a 30-min chase. During the next 90 min continued accumulation of 60,000-dalton and appearance of 62,000-dalton forms was observed while the 80,000-dalton form disappeared. The fate of the 150,000-dalton precursor depended on nutritional conditions. In cells conditioned with fresh growth medium intracellular processing predominated. Less than 10% of either the precursor or mature forms was secreted in 8 hr. However, when cells were shifted from growth medium to starvation buffer, secretion of precursor soon predominated. After a 1-hr lag period, cells began secreting 150,000-dalton precursor into the medium. After 4 hr in starvation buffer, the rate of secretion of 150,000-dalton form increased by at least an order of magnitude while processing was markedly diminished. This may be a case where nutritional conditions control the sorting of an acid hydrolase precursor.
D000918:Antibody Specificity; D003470:Culture Media; D004023:Dictyostelium / Q000201:enzymology*; D004792:Enzyme Precursors / Q000378:metabolism; D007163:Immunosorbent Techniques; D008361:Mannosidases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D043323:alpha-Mannosidase
D003470:Culture Media; D004792:Enzyme Precursors; D008361:Mannosidases; D043323:alpha-Mannosidase