1. The fractionation of intracellular albumin labelled with radioactive l-leucine was studied in rat liver by means of isoelectric focusing. 2. Isoelectric fractionation was compared with ion-exchange chromatography for purification of radioactive intracellular albumin obtained by antibody precipitation. Similar results were obtained with both methods of separation. Purified albumin contains only a minor amount of the radioactivity. The remainder is associated with albumin-like protein(s). 3. The albumin-like protein has the properties of a precursor of plasma albumin. 4. The distribution and turnover of radioactive albumin in rough and smooth microsomal fractions and in a Golgi-rich fraction were studied. 5. It is concluded that newly synthesized albumin, as such, appears only momentarily if at all in any intracellular structure before its appearance in the plasma. 6. It is also concluded that the rate-limiting step in the secretion of plasma albumin is the conversion of precursor(s) into albumin. We can find no evidence to suggest that there is any significant transport of albumin, as such, during the course of secretion.
1. The labelling of intracellular and extracellular serum albumin was studied in liver slices and in whole rats by using new methods for the purification of the protein. 2. The results suggest that a polypeptide precursor is formed that is converted relatively slowly into serum albumin. 3. The effect of liver cell K(+) has been examined by a double-label method and it is shown that K(+) accelerates the rate of conversion of ;precursor' into albumin. The rate of transit of albumin across the cell membrane appears to be unrelated to the concentration of K(+) within the cell. 4. The time-course of incorporation of radioactive amino acid into albumin follows a sigmoidal mode. There is a pronounced time-lag before label starts to appear in intracellular albumin, and a further time-lag before it appears in extracellular albumin. 5. In slices the sum of intra- and extra-cellular label rises steadily from 30min after the start of labelling with a pulse of labelled leucine or valine and continues to rise for at least another 60min. This occurs whether labelling is stopped by addition of excess of carrier amino acid or with cycloheximide (100mum) or both. 6. The intracellular albumin content remains constant whether slices are maintained with low or normal intracellular K(+) concentrations. 7. Specific radioactivities of intracellular albumin (and fractions thereof) and of extracellular albumin were determined in vitro and in vivo. The results show that the intracellular albumin cannot be a precursor of extracellular albumin, unless a very small compartment is turning over much more rapidly than the bulk of the liver albumin or even of the microsomal albumin.
1. Antibody precipitation of serum albumin from rat liver extracts yields impure preparations of the protein. 2. When rat liver is labelled with l-[1-(14)C]leucine, antibody precipitation of albumin leads to material that is contaminated with a protein or proteins of very high specific radioactivity. Only 10-25% of the radioactivity of the antibody precipitate is associated with serum albumin. 3. A chromatographic procedure is described that can be used to separate radiochemically pure serum albumin from antibody precipitates obtained from extracts of rat liver. 4. Extracellular albumin secreted by liver slices yields a precipitate with antibody which contains much less radioactive impurity. About 70-90% of the radioactivity is associated with serum albumin. Serum albumin separated by antibody precipitation from rat serum labelled in vivo was not contaminated with the radiochemical impurities associated with intracellular albumin. 5. A simple method is described of obtaining the content of serum albumin in rat liver extracts by the technique of isotope dilution and ion-exchange chromatography.
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