Abstract
We have obtained evidence in vivo for the intracellular site of sialylation of glycoproteins and have determined the kinetics of intramembranous transport of newly synthesized sialoglycoproteins. Radioactivity of sialoglycoproteins was determined at different times in homogenate, smooth and rough endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions of liver either from slices which had been incubated with radioactive N-acetylneuraminic acid or from mice which had been injected with radioactive sugar. At short times after labeling, the Golgi apparatus contained the highest specific activity of radioactive sialoglycoproteins and over half of the total cellular radioactive sialoglycoproteins. By 15 to 25 min, most of the radioactivity in the Golgi apparatus had decreased with a concomitant increase of radioactivity in the plasma membrane. Radioactivity in the smooth and rough endoplasmic reticulum accounted for less than 20% of the total cellular radioactivity throughout. Control experiments indicated that leakage of soluble, labeled sialoglycoproteins from membrane vesicles and adsorption of them onto vesicles was minimal. In contrast to the above studies, labeling for short times with radioactive mannose, showed, as expected from studies in vitro, most of the radioactivity in the rough endoplasmic reticulum. At longer times, the radioactivity in the rough endoplasmic reticulum decreased, with an increase in the plasma membrane. Throughout, the radioactivity in the smooth endoplasmic reticulum was low.
Highlights
The tissue slices were preincubated in medium containing the radiolabeled sugar at 0°C for 10 min, and the incubaother sugars and the isolation of CMP-NeuAc as aninterme- tion was started by placing the slices into a 37°C water bath in an diate in the above conversion suggested that this procedure may be useful to study the intracellular site of sialylation of glycoproteins and to determine the kinetics of intramembranous transport of newly synthesized sialoglycoproteins
Determination of Radioactiuity-To determine the amount of radioactivity incorporated into glycoproteins, aliquots of isolated membrane fractions or cell homogenates were precipitated with phosphotungstic acid (1%in 0.5 N HC1).We have shown previously [3] that labeling with [’HINeuAc is very specific, and that up to24 h, all the radioactivity in the phosphotungstic acid-insoluble fraction is in C3H]NeuAc
This suggests that theincorporation of NeuAc into liver glycoproteins occurs in the Golgi apparatus and that the majority of the labeled proteins are transported to the plasma membrane with a lag time of approximately 15min
Summary
Dioactivity in the roughendoplasmicreticulum de- Radiolabeled S~gars-[9-~H]NeuAcwas prepared enzymatically creased, with an increase in the plasma membrane. .by the method of Warrenand Glick [11] from N-acetyl-~-[6-. The absence of any detectable metabolism of NeuAcl to preparation and incubation of mouse liver slices were performed as described previously [3]. Pulse-labeling of liver slices was performed as described by Jamieson and Palade(l2) for pulse-labeling of pancreas slices. The tissue slices were preincubated in medium containing the radiolabeled sugar at 0°C for 10 min, and the incubaother sugars and the isolation of CMP-NeuAc as aninterme- tion was started by placing the slices into a 37°C water bath in an diate in the above conversion suggested that this procedure may be useful to study the intracellular site of sialylation of glycoproteins and to determine the kinetics of intramembranous transport of newly synthesized sialoglycoproteins.
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