Developing cotyledons of P. vulgaris L. were labeled for 30 min with [3H] amino acids, homogenized and the proteins fractionated on sodium dodecylsulfate (SDS) polyacrylamide gels. Fluorographs of these gels showed that the polypeptides of phaseolin, the major reserve protein of P. vulgaris, were synthesized as precursors which could be distinguished from the polypeptides of mature phaseolin by their slightly lower mobility. When extracts of cotyledons labeled for 45 min with [3H] amino acids were fractionated on isopynic sucrose gradients, radioactive phaseolin banded at the same density (1.14 g cm-3) as the endoplasmic reticulum (ER)-marker enzyme NADH-cytochrome c reductase. Fractionation in the presence of 3 mM MgCl2 indicated that the newly-synthesized phaseolin was associated with the rough ER. Pulse-chase experiments showed that phaseolin was transiently associated with the ER and later accumulated in the protein bodies. Treatment of isolated ER with proteinase K showed that phaseolin polypeptides were degraded only if Triton X-100 was present, indicating that phaseolin was membrane-protected, probably enclosed within the vesicles. ER-associated phaseolin associated to an 18S form at pH 4.5 in the presence of 0.3 M NaCl and 100 mM sodium acetate. The polypeptides of ER-associated phaseolin had a slightly lower mobility on SDS-gels than polypeptides of protein body phaseolin. ER-associated phaseolin had a carbohydrate content of 6.8%, while protein body-derived phaseolin had a carbohydrate content of 6.2%. When cotyledons were labeled simultaneously with [14C] amino acids and [3H] glucosamine or with [14C] amino acids and [3H] mannose, the [3H]/[14C] ratio of ER-derived phaseolin was similar to that of protein body derived phaseolin, indicating that the faster mobility of SDS-gels was not due to the detachment of carbohydrate. Experiments in which the carbohydrate side chains were removed with endoglycosidase H, and the resulting polypeptides subjected to electrophoresis in SDS-gels showed that the differential mobility of the glycopolypeptides of phaseolin resided in their polypeptide chains.
A PRECURSOR OF THE RESERVE PROTEIN PHASEOLIN IS TRANSIENTLY ASSOCIATED WITH THE ENDOPLASMIC RETICULUM OF DEVELOPING PHASEOLUS-VULGARIS COTYLEDONS
BOLLINI R;
1982
Abstract
Developing cotyledons of P. vulgaris L. were labeled for 30 min with [3H] amino acids, homogenized and the proteins fractionated on sodium dodecylsulfate (SDS) polyacrylamide gels. Fluorographs of these gels showed that the polypeptides of phaseolin, the major reserve protein of P. vulgaris, were synthesized as precursors which could be distinguished from the polypeptides of mature phaseolin by their slightly lower mobility. When extracts of cotyledons labeled for 45 min with [3H] amino acids were fractionated on isopynic sucrose gradients, radioactive phaseolin banded at the same density (1.14 g cm-3) as the endoplasmic reticulum (ER)-marker enzyme NADH-cytochrome c reductase. Fractionation in the presence of 3 mM MgCl2 indicated that the newly-synthesized phaseolin was associated with the rough ER. Pulse-chase experiments showed that phaseolin was transiently associated with the ER and later accumulated in the protein bodies. Treatment of isolated ER with proteinase K showed that phaseolin polypeptides were degraded only if Triton X-100 was present, indicating that phaseolin was membrane-protected, probably enclosed within the vesicles. ER-associated phaseolin associated to an 18S form at pH 4.5 in the presence of 0.3 M NaCl and 100 mM sodium acetate. The polypeptides of ER-associated phaseolin had a slightly lower mobility on SDS-gels than polypeptides of protein body phaseolin. ER-associated phaseolin had a carbohydrate content of 6.8%, while protein body-derived phaseolin had a carbohydrate content of 6.2%. When cotyledons were labeled simultaneously with [14C] amino acids and [3H] glucosamine or with [14C] amino acids and [3H] mannose, the [3H]/[14C] ratio of ER-derived phaseolin was similar to that of protein body derived phaseolin, indicating that the faster mobility of SDS-gels was not due to the detachment of carbohydrate. Experiments in which the carbohydrate side chains were removed with endoglycosidase H, and the resulting polypeptides subjected to electrophoresis in SDS-gels showed that the differential mobility of the glycopolypeptides of phaseolin resided in their polypeptide chains.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
