Uptake and intracellular transport in rat liver of formaldehyde-treated bovine serum albumin labelled with 125I-tyramine-cellobiose.
1. Endocytosis of formaldehyde-treated bovine serum albumin by rat liver sinusoidal cells has been followed by injecting rats with the protein labelled with 125I-tyramine cellobiose (125I-TCfBSA). 125I-TCfBSA is quickly taken up by the liver; the radioactivity present in the organ reaches a plateau 5-10 min after injection and is maintained for up to at least 180 min. During the first 5 min most of radioactivity remains acid-precipitable. After which, labelled acid-soluble components are produced at a constant rate for up to 30-40 min. 2. Differential centrifugation shows that radioactivity is first recovered mainly in the microsomal fraction. Within a few minutes it exhibits a distribution pattern similar to that of lysosomal enzymes, being chiefly located in the mitochondrial fractions. 3. Isopycnic centrifugation in a sucrose gradient of the microsomal fraction isolated 1 min after injection indicates a similar distribution for radioactivity and alkaline phosphodiesterase. Later, the microsomal radioactivity distribution curve is shifted towards higher densities and becomes distinct from that of the plasma-membrane enzyme. After isopycnic centrifugation in a sucrose gradient of the total mitochondrial fraction a considerable overlapping of acid-precipitable and acid-soluble radioactivity distributions is observed without significant changes with time. The same is observed in a Percoll gradient except that after a relatively long time (greater than 30 min) of injection a marked shift of radioactivity distribution towards higher densities occurs. 4. A pretreatment of rats with Triton WR 1339, a density perturbant of liver lysosomes, causes a striking shift of acid-soluble radioactivity distribution in a sucrose gradient towards lower densities while having markedly less influence on the acid-precipitable distribution. As a result, a distinction between the distribution of both kinds of radioactivity becomes clearly apparent. A preinjection of yeast invertase, modifies the acid-soluble distribution without having a significant effect on the acid-precipitable distribution up to 30 min after 125I-TCfBSA injection. 5. Glycyl-1-phenylalanine-2-naphthylamide largely releases acid-soluble radioactivity associated with the mitochondrial fraction, whatever the time after 125I-TCfBSA injection. On the other hand the proportion of acid-precipitable radioactivity present in the fraction that can be released is almost zero at 10 min after injection, and it later increases. 6. The results presented here are best explained by supposing that, after being trapped in small pinocytic vesicles, 125I-TCfBSA is quickly delivered to the endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)
Misquith
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Laboratoire de Chimie Physiologique, Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium.
Wattiaux-De Coninck
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S
Wattiaux
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R
D000818:Animals; D002458:Cell Fractionation; D002475:Cellobiose / Q000378:metabolism; D002500:Centrifugation, Isopycnic; D004151:Dipeptides / Q000494:pharmacology; D004705:Endocytosis; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012709:Serum Albumin / Q000378:metabolism*; D014439:Tyramine / Q000378:metabolism
D004151:Dipeptides; D012709:Serum Albumin; C038394:tyramine-cellobiose adduct; D002475:Cellobiose; C041865:glycylphenylalanine 2-naphthylamide; D014439:Tyramine