By means of the magnetic float method, the partial specific volumes of edestin and gelatin have been redetermined. The new values fit the general pattern of agreement between observed and calculated partial specific volumes of proteins. Since this correspondence depends on neglecting electrostriction, measurements were made at different values of pH, and also in urea solution, on gelatin, ovalbumin and serum albumins. The findings are consistent with the hypothesis that compact protein molecules contain an "excluded volume," which may alter in size under different conditions.Accurate measurements of the partial specific volume v of a protein have hitherto been difficult to make.3 They are important in the calculation of molecular weight M from ultracentrifuge data, because an error of 1% in v results in one of at least 3% in M. In the absence of complicating factors, it should be possible to calculate v from the amino acid composition of a protein.4•5 When this was done, calculated and observed values were the same6 with a few exceptions, notably those for edestin and gelatin. However, it was necessary to ignore electrostriction to reach this agreement.The present study was designed to take advantage of the high accuracy to which density determinations may be made by the float method in exploring the relation between v and amino acid composition. The effect of environment (¿>H and solvent composition) was examined, and particular attention given to those proteins for which the calculated values did not agree with experimental ones available in the literature. MaterialsTwo samples of high quality gelatin were used, both prepared by Eastman Kodak Co. One was derived from calfskin, the other from pigskin. The ash content was found to be less than 0.04% in both cases.A stock solution was prepared by dissolving gelatin in warm water, pouring the solution into a dialysis bag, and dialyzing for some days at 5°against a large volume of water.About 24 hours before measurements were begun, the flask was transferred to a room at 30°where subsequent weighing and adding operations were carried out.Three, preparations of edestin were made. Ultracentrifugal analysis of the first, I, which was made from hemp seed by the method of Bailey,6 showed a main peak with two smaller peaks. Preparation II, made by a procedure similar to that of Goring and Johnson,7 *gave an ultracentrifuge pattern containing one major and one minor component. A sample of edestin from Nutritional Biochemicals Corp.(lot 1341) was purified by similar methods until the minor heavy component was almost eliminated (preparation III).Lysozyme was obtained from Armour and Co. (lot 003 LI), and from Dr. L. R. Wetter (lot L.R.W.). It was dissolved in and dialyzed against phosphate buffer (pH 6.8, ionic strength 0.1), the small amount of insoluble material being filtered off before dialysis. Since some loss of lysozyme occurs through ordinary membranes, and only 300 mg. of lot L.R.W. was available, dialysis of this particular solution was restricted to 24 hours at ro...
After the injection of a small dose (1 jtg/100 g of body weight) of _151-labeled human asialotransferrin type 3 in rats, the radioactivity became rapidly associated with the liver. However, during the ensuing 12 hr a significant fraction of the dose returned to the circulation as protein-bound S5I. The protein released by the liver was indistinguishable by gel filtration from the original preparation and was precipitable by an antiserum to human transferrin. Nevertheless, it no longer bound to the immobilized Gal/GalN-specific lectin from rabbit liver. However, binding could be restored to a large extent by treatment with neuraminidase, indicating that the loss of binding was due to resialylation. Changes in the electrophoretic mobility of asialotransferrin released by the liver showed that resialylation was partial-i.e., it involved the attachment oftwo or three sialyl residues. From analysis by deconvolution of the plasma curve of partially resialylated asialotransferrin it was calculated that the liver "repaired" this way approximately one asialotransferrin molecule out of four. Plasma clearance of partially resialylated asialotransferrin was similar to that of nondesialylated transferrin.A minor portion (15-17%) of human transferrin phenotype C consists of molecules which, after desialylation, bind avidly (1) to the Gal/GalN-specific lectin (2) of the rat liver. We designated this fraction human asialotransferrin type 3 (HAsTf-3; ref.3). Suspended rat hepatocytes internalize HAsTf-3 via the above lectin (4). However, only a small fraction of the intracellular HAsTf-3 undergoes catabolism, whereas the rest is released showing no signs of proteolytic digestion. Cell-associated radioactivity decreases considerably more slowly in the suspension than is the time required for "2I-labeled HAsTf-3 ('"IHAsTf-3) to emerge from the hepatocytes, implying that the ligand is being repeatedly endo-and exocytosed. We termed this movement of HAsTf-3 the diacytic pathway (4) in contradistinction to the well-known lysosomal pathway for other asialoglycoproteins (5). During diacytosis, HAsTf-3 is entrapped in a subcellular particle that is ofa lesser equilibrium density than the vesicle that transports internalized asialoorosomucoid (6).The unusual handling of HAsTf-3 by the hepatocyte raises the question of how the liver ofthe intact rat ultimately disposes of this asialoglycoprotein. Because our earlier experiments in vivo were too short to provide an answer (3), we have now conducted studies lasting up to 12 hr. These showed that rat liver processed small doses of HAsTf-3 slowly in (at least) two ways-namely, catabolism and partial resialylation. Here we report our findings relating to resialylation. MATERIALS AND METHODSMaterials. Na125I, Na1311, 59FeC13, and N-acetyl-D-[6-3H(N)]mannosamine (19 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) were obtained from New England Nuclear. Neuraminidase from Vibrio cholerae was from GIBCO and neuraminidase from Diplococcus pneumoniae was a gift from M. Lowe and G. Ashwell (National ...
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