Isolation and partial characterization of the rat liver ligandosome fraction

Uptake of 125I-insulin by the liver of intact rats is followed by rapid translocation of label to low-density vesicles. Subcellular-fractionation studies indicate that, although the 125I associated with these vesicles is predominantly trichloroacetic acid-precipitable, there is an acid-soluble component arising from processing of the hormone in vivo. H.p.l.c. analysis indicates that the acid-precipitable 125I is not intact iodoinsulin, but may correspond to 'clipped insulin'. Isolated low-density vesicles degrade the acid-precipitable iodopeptide intravesicularly when incubated at 37 degrees C in iso-osmotic medium at pH7. The rate constant for intravesicular degradation is consistent with the rate of insulin clearance by the liver in vivo. Pretreatment of the rats with chloroquine resulted in a decrease in proteolysis of the iodopeptide within the isolated vesicles.

Section: Discussionsupporting

confidence: 57%

“…Preparative discontinuous-density-gradient centrifugation Liver homogenate (2.5 ml) was layered on to a discontinuous gradient of 5 ml of sucrose (p= 1.09g-cm-3) and lOml of sucrose (p = 1.14g.cm-3), both solutions containing 22mM-ethanol and 1 mM-Na,EDTA, pH7.2 (Pease et al, 1984).…”

Section: Analytical Fractionationmentioning

confidence: 99%

Degradation of endocytosed insulin in rat liver is mediated by low-density vesicles

Pease¹,

Smith²,

Peters³

1985

“…8, after 20 min post-perfusion, with the addition of unlabelled transferrin to the post-perfusion solution (fine line). The results obtained after 20 min post-perfusion in the absence of added transferrin were intermediate between those shown in the Figure. derived from the Golgi complex (Pease et al, 1984). The fractions enriched with '251-transferrin contained the Golgi-membrane marker enzyme galactosyltransferase, but could be partly differentiated from that enzyme marker by treatment with digitonin.…”

Section: Discussionmentioning

confidence: 99%

“…Portions of the liver from the experiment described above were homogenized in the standard manner and a low-density-vesicle fraction was prepared by centrifugation in a discontinuous sucrose density gradient as described previously (Pease et al, 1984). The vesicle fraction was suspended in 0.25 M-sucrose/Hepes, pH 7.4, and was divided into four aliquots.…”

Section: Release Of Transferrin From the Low-density-vesicle Fractionmentioning

confidence: 99%

Uptake and subcellular processing of 59Fe-125I-labelled transferrin by rat liver

Morgan¹,

Smith²,

Peters³

1986

The uptake of transferrin and iron by the rat liver was studied after intravenous injection or perfusion in vitro with diferric rat transferrin labelled with 125I and 59Fe. It was shown by subcellular fractionation on sucrose density gradients that 125I-transferrin was predominantly associated with a low-density membrane fraction, of similar density to the Golgi-membrane marker galactosyltransferase. Electron-microscope autoradiography demonstrated that most of the 125I-transferrin was located in hepatocytes. The 59Fe had a bimodal distribution, with a larger peak at a similar low density to that of labelled transferrin and a smaller peak at higher density coincident with the mitochondrial enzyme succinate dehydrogenase. Approx. 50% of the 59Fe in the low-density peak was precipitated with anti-(rat ferritin) serum. Uptake of transferrin into the low-density fraction was rapid, reaching a maximal level after 5-10 min. When livers were perfused with various concentrations of transferrin the total uptakes of both iron and transferrin and incorporation into their subcellular fractions were curvilinear, increasing with transferrin concentrations up to at least 10 microM. Analysis of the transferrin-uptake data indicated the presence of specific transferrin receptors with an association constant of approx. 5 X 10(6) M-1, with some non-specific binding. Neither rat nor bovine serum albumin was taken up into the low-density fractions of the liver. Chase experiments with the perfused liver showed that most of the 125I-transferrin was rapidly released from the liver, predominantly in an undegraded form, as indicated by precipitation with trichloroacetic acid. Approx. 40% of the 59Fe was also released. It is concluded that the uptake of transferrin-bound iron by the liver of the rat results from endocytosis by hepatocytes of the iron-transferrin complex into low-density vesicles followed by release of iron from the transferrin and recycling of the transferrin to the extracellular medium. The iron is rapidly incorporated into mitochondria and cytosolic ferritin.

“…After homogenization in 0.25 M sucrose\Hepes buffer (containing 10 mM Hepes buffer, pH 7.2, 1 mg\ml bacitracin and 2.5 mM NEM), an endosomal fraction was prepared by discontinuous sucrose gradient centrifugation [23]. This endosomal preparation contains 60 % of the recovered trichloroacetic acid-precipitable radiolabel, but less than 4 % of lysosomal enzymic activities [24]. The isolated endosomal population was subsequently washed free of cytosol by a 1 : 10 dilution in 10 mM Hepes buffer, pH 7.4, containing 0.1 M KCl and 2.5 mM NEM, and pelleted by centrifugation (100 000 g at 4 mC for 30 min).…”

Section: Preparation Of Endosomes Containing Radiolabelled Insulinmentioning

confidence: 99%

The characterization of endosomal insulin degradation intermediates and their sequence of production

Seabright

Smith

1996

Insulin degradation within isolated rat liver endosomes was studied in vitro with the aid of three 125I-insulin isomers specifically labelled at tyrosine (A14, B16 and B26). Chloroquine and 1,10-phenanthroline were used to minimize insulin proteolysis during endosome preparation, whereas the manipulation of endosomal processing of insulin in vitro by Co2+ ions (to activate) and 1,10-phenanthroline (to inhibit) permitted the study of degradation intermediates and their time-dependent production. Structural and kinetic analysis of intermediates isolated from both intra- and extra-endosomal compartments allowed the determination of major cleavage sites and the probable sequence of proteolytic events. It was found that 125I-tyrosine is the ultimate labelled degradation product of all iodo-insulin isomers, suggesting that endosomal proteases are able to degrade insulin to the level of its constituent amino acids. 125I-tyrosine was also the only radiolabelled product able to cross the endosomal membrane. Intra-endosomal insulin degradation proceeds via two inter-related cleavage routes after metalloendoprotease cleavage of the B-chain. One pathway results from an initial cleavage in the centre region of the B-chain (B7-19), probably at B14-15, whereas the major route results from a cleavage at B24-25. B24-25 cleavage removes the B-chain C-terminal hexapeptide (B25-30), which is subsequently cleaved by an aminopeptidase activity to produce first the pentapeptide B26-30 and then 125I-tyrosine. The isolation of intact radiolabelled A-chain from the degradation of 125I-[A14]-insulin suggests that further degradation of proteolytic intermediates containing cleaved B-chain proceeds via interchain disulphide reduction. The A-chain is then processed by several cleavages, one of which occurs at A13-14.