The distribution of galactosyl transferase was studied using trans and cis Golgi fractions isolated by a modification of the Ehrenreich et al . procedure (1973. J. Cell Biol. 59 :45-72) as well as an intact Golgi fraction isolated by a new one-step procedure. Two methods of assay were used . The first method analyzed the ability of Golgi fractions to transfer galactose (from uridine diphosphogalactose [UDP-gal] substrate) to the defined exogenous acceptor ovomucoid . The second method assessed the transfer of galactose from UDP-gal substrate to endogenous acceptors (endogenous glycosylation) . The trans Golgi fraction (Golgi light) was highly active by the first method but revealed only low activity by the second method . Golgi fractions enriched in central and cis elements (the Golgi intermediate, heavy and especially the intact Golgi fraction) were highly active in both methods of assay . The endogenous glycosylation approach was validated by gel fluorography of the endogenous acceptors. For all Golgi fractions, transfer of galactose was revealed to secretory glycopeptides. It is concluded that galactosyl transferase activity in vivo occurs primarily in central and cis Golgi elements but not trans Golgi vesicles .The discovery by Leblond and colleagues (26, 35) that i. v. injected [3H]galactose was incorporated within the Golgi apparatus directly led to the use of galactosyl transferase as a marker enzyme for purified Golgi fractions (e .g . 4,6, 11,12,14,15,[22][23][24][25]32). This enzyme activity is usually assayed in the presence of detergent and measures the transfer of galactose (from UDP-gal)' to a defined exogenous acceptor (either G1cNAc or free G1cNAc residues on specific glycopeptides) . In such assays little account has been taken of the subcellular distribution of endogenous peptide acceptors.In vivo, galactose transfer is expected to occur where there is both enzyme and endogenous acceptor. We have therefore attempted to localize the site of transfer of galactose to endogenous acceptors by assessing endogenous galactosyl transferase activity of a variety of Golgi fractions which differ in their morphological characteristics.'Abbreviations used in this paper are: G1cNAc, N-acetylglucosamine; hGH, human growth hormone, SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis ; STKM, 0.25 M sucrose, 50 mm Tris HCl pH 7.4, 25 mM KCI, 5 MM MgCIZ; UDP-gal, uridine diphosphategalactose; VLDL, very low density lipoprotein. MATERIALS AND METHODS Isolation of Golgi FractionsGolgi fractions from microsomal pellets were isolated exactly as described previously (4)
Electron microscope radioautography has been used to study hormone-receptor interaction. At intervals of 3, 10, and 20 min after the injection of l~I-insulin, free hormone was separated from bound hormone by whole body perfusion with modified Ringer's solution. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined.At 3 min, lmI-insulin has been shown to be exclusively localized to the hepatocyte plasmalemma (Bergeron et al., 1977, Proc. Natl. Acad. Sci. U. S. A., 74:5051-5055). In the present study, quantitation indicated that 10 ~ receptors were present per cell and distributed equally along the sinusoidal and lateral segments of the hepatocyte plasmalemma. At later times, label was found in the Golgi region. At 10 min, both secretory elements of the Golgi apparatus and lysosome-like vacuoles were labeled, and at 20 min the label was especially concentrated over the latter vacuoles. Acid phosphatase cytochemistry showed that the vacuoles did not react and therefore were presumed not to be lysosomal. These Golgi vacuoles may constitute a compartment involved in the initial degradation and/or site of action of the hormone. Control experiments were carded out at all time intervals and consisted of parallel injections of radiolabeled insulin with excess unlabeled hormone. At all times in controls, label was diminished over hepatocytes and was found primarily over endothelial cells and within the macropinocytotic vesicles and dense bodies of these cells. Kupffer cells and lipocytes were unlabeled after the injection of l~zI-insulin with or without excess unlabeled insulin. KEY WORDS insulin receptor 9 Golgi apparatus lysosomesThe initial interaction of l~I-radiolabeled insulin with the hepatocyte plasmalemma was recently demonstrated in vivo by electron microscope radioautography (4). Little is known about the fate of labeled polypeptide hormones after binding to the surface of their target cells, In the present study, we have approached the problem by examining the distribution of label at different times after l~zI-insulin administration. We have demon-J. CELL BIOLOGY 9 The Rockefeller University Press
A tissue-screening survey employing quantitative radioautography was carried out at 2 min after the intravascular injection of 125I-insulin into laboratory rats. The results revealed a substantial binding of insulin to cells forming the proximal convoluted tubule in kidney, hepatocytes of liver, acinar cells of the pancreas, parenchymal cells of the adrenal cortex and medulla, and epithelial cells of the gastrointestinal tract. Control experiments indicated that this binding was due to a specific interaction with the insulin receptor, except in the case of kidney where the binding was shown to be nonspecific. Although the major target for insulin action (liver) clearly demonstrated specific insulin binding, several other classical targets (adipocytes, skeletal, cardiac, and smooth muscle cells) showed no specific 125I-insulin binding and therefore indicated the limits of sensitivity of the in vivo radioautographic method. Nevertheless, the working hypothesis of a direct correlation of insulin receptor density with insulin action points to the hitherto unemphasized targets of pancreas, adrenal gland, and gastrointestinal tract as major sites of insulin action in the body.
The rationale of the specific-binding assay was applied to the detection of the liver insulin rece tor in vivo.Quantitative electron microscope radioautogap~y indicated that, 3 min after an intraportal injection,'12I-insulin was exclusively located to the hepatocyte plasmalemma. The specific-binding assay is routinely used in biochemical studies to assess quantitatively the recognition of 125I-labeled insulin (125I-insulin) by its receptor in vitro (e.g., refs. 1-3). Specific binding is based on a consideration of the law of mass action, which states that high concentrations of unlabeled insulin will compete with '25I-insulin for binding to receptor sites. Thus, receptor preparations (experimental samples) are incubated in vitro with saturating or subsaturating (physiologic) levels of 125I-labeled hormone and an identical control preparation incubated with the same amount of l25-Ilabeled hormone but with an "excess" of unlabeled hormone. Specific binding is defined as the difference in bound hormone between the experimental and control samples (1).We have previously applied this rationale to enable the visualization by electron microscope radioautography of polypeptide hormone receptors to purified subcellular fractions derived from the plasmalemma and Golgi apparatus of liver homogenates.t In the present in vivo investigation the approach of the specific-binding assay has enabled us to screen (by light microscope radioautography) several fixation and perfusion methods and has led to the selection of the fixation procedure that most clearly showed specific insulin binding while still maintaining adequate morphologic preservation of the liver tissue. The resulting electron microscope radioautography hence directly visualizes the 125I-insulin and thereby marks the location of the insulin receptor. MATERIALS AND METHODS 125I-Insulin. Porcine insulin (24.4 units/mg; ConnaughtLaboratories, Toronto) was iodinated by chloramine T (4, 5). The 125I-insulin was freshly prepared immediately before each experiment and the integrity of the hormone was assessed by specific binding to microsomes derived from human placenta (4). The specific activity of the 125I-insulin was 160,tCi/,ug. (7). Following dehydration, the blocks were embedded in Epon.The control animals were treated identically except the portal vein injection (0.1 ml) contained 142.5 X 106 dpm of '25I-insulin plus 50 jig of unlabeled insulin.Light and Electron Microscope Radioautography. For light microscope radioautography, semithin (0.5-Mm) sections were prestained in iron/hematoxylin and coated with Kodak NTB2 emulsion (8). Following various times of exposure, the radioautographs were developed with freshly prepared .For electron microscope radioautography, thin sections (silver-grey interference color) were cut on an LKB-Huxley ultramicrotome and a monolayer of Ilford L-4 emulsion was applied (9). Following exposure periods, the radioautographs were developed with . The sections were poststained with uranyl acetate (11) and lead citrate (12) and viewe...
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