A quantitative model of traffic between plasma membrane and secondary lysosomes: evaluation of inflow, lateral diffusion, and degradation.

We present here a mathematical model that accounts for the various proportions of plasma membrane constituents occurring in the lysosomal membrane of rat fibroblasts (Draye, J.-P., J. Quintart, P. J. Courtoy, and P. Baudhuin. 1987. Eur. J. Biochem. 170: 395-403; Draye, J.-P., P. J. Courtoy, J. Quintart, and P. Baudhuin. 1987. Eur. J. Biochem. 170:405-411). It is based on contents of plasma membrane markers in purified lysosomal preparations, evaluations of their half-life in lysosomes and measurements of areas of lysosomal and plasma membranes by morphometry. In rat fibroblasts, structures labeled by a 2-h uptake of horseradish peroxidase followed by a 16-h chase (i.e., lysosomes) occupy 3% of the cellular volume and their total membrane area corresponds to 30% of the pericellular membrane area. Based on the latter values, the model predicts the rate of inflow and outflow of plasma membrane constituents into lysosomal membrane, provided their rate of degradation is known. Of the bulk of polypeptides iodinated at the cell surface, only 4% reach the lysosomes every hour, where the major part (integral of 83%) is degraded with a half-life in lysosomes of integral to 0.8 h. For specific plasma membrane constituents, this model can further account for differences in the association to the lysosomal membrane by variations in the rate either of lysosomal degradation, of inflow along the pathway from the pericellular membrane to the lysosomes, or of lateral diffusion.

Degradation of proteins microinjected into IMR-90 human diploid fibroblasts.

Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte-mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination.

Most iodinatable fibroblast surface proteins accompany the cytoplast membrane during cytochalasin B-mediated enucleation of chick embryo fibroblasts.

Six different proteins are found to be reproducibly exposed on the cell surface of chicken embryo fibroblasts (CEF) by the criterion of lactoperoxidase-catalyzed iodination (250,000, 185,000, 130,000, 100,000, 87,000, and 75,000 daltons). We wondered whether cell enucleation might lead to a differential partition of these surface proteins with the karyoplast or cytoplast membrane. We found that there is a marked enrichment of most iodinatable cell surface proteins in the cytoplast after cytochalasin-mediated enucleation of cell monolayers. Nearly all the iodinatable fibronectin remains with the cytoplast. Of the six labeled proteins, the karyoplast membrane contains a small amount of the 130 kdalton protein as well as trace levels of the 100-, 85-, and 75-kdalton proteins. Proteolysis or selective shedding of membrane proteins were not significant factors in the relative exclusion of iodinatable membrane proteins from the karyoplast. The cytoplast could replace some exposed membrane proteins after removal by trypsinization; however, fibronectin was not detectable within 10 h. That the karyoplast was not capable of membrane protein synthesis and/or insertion was suggested by the lack of any change in the labeling pattern of karyoplasts up to 8-h incubation after enucleation. A variety of control studies indicated that the surface proteins identified in this report were cell-derived and not adsorbed serum components. That some of the iodinatable proteins are intrinsic membrane proteins was suggested by their resistance to removal by conditions thought to extract extrinsic membrane proteins (i.e., low salt, high salt, and NaOH washes). lack of effect of cytoskeletal disrupting agents (preliminary evidence) suggests the nonrandom partition of membrane proteins may depend on anchoring of membrane proteins by a system(s) in the cytoplast other than intact microtubules and microfilaments.

Studies on mono- and diiodohistidine. I. The identification of iodohistidines from thyroidal iodoproteins and their peripheral metabolism in the normal man and rat.

The problem as to whether iodohistidines are normally biosynthetized in thyroglobulin and thyralbumin has been examined both in man and the rat. Evidence has been obtained for the first time that diiodohistidine (DIH) is present in both species in these two iodoproteins. The biosynthesis of monoiodohistidine (MIH) in the thyroglobulin of the normal rat has been confirmed and extended to rat thyralbumin and to human thyroid iodoproteins. THE IODOHISTIDINE IDENTIFICATION IS BASED ON FIVE ORIGINAL METHODS INCLUDING: (a) the preparation of stable and radioiodine-labeled iodohistidines; (b) the protection of the labile iodohistidines during the iodoprotein enzymatic hydrolysis; (c) the isolation of iodohistidines by ion-exchange resin chromatography; (d) their separation from each other and from iodinated cationic butanol-insoluble compounds by Sephadex G-10 chromatography; and (e) their purification by successive crystallizations to a constant specific activity. Iodohistidine levels (in percent of protein radioactivity from iodide given in vivo) were found comparable in man and the rat. However, the values (mean +/-SE) for thyroglobulin (MIH, 0.61+/-0.10%; DIH, 0.050+/-0.015%) and for thyralbumin (MIH, 2.61+/-0.57%; DIH, 0.28+/-0.09%) differ significantly (P < 0.05). Iodohistidines are stable during in vitro exposure to iodotyrosine dehalogenase preparations. In contrast to iodotyrosines the iodohistidines when given in vivo to man either orally or intravenously were in large part recovered in 24-h urines.

Interaction of concanavalin A with rabbit thymocyte plasma membranes. Distinction between low affinity assoication and positively cooperative binding mediated by a specific glycoprotein.

1. We have analyzed the interaction of the mitogenic lectin, concanavalin A, with purified plasma membranes isolated from rabbit thymocytes. 2. Scatchard analyses show that in native membranes binding is positively cooperative at low concanavalin A concentrations and non-interacting at high lectin levels. 3. In contrast, membranes treated with 0.0064 M glutaraldehyde exhibit diphasic Scatchard plots, indicating the presence of high- and low-affinity binding sites. The high-affinity zone corresponds to the region of positive cooperativity in native membranes. 4. The number of high-affinity binding sites per cell-equivalent corresponds approximately to the number of glycoprotein (mol. wt. 55000) molecules (1-10(6)/cell), but account for less than 25% of the total lectin binding. 5. Treatment of membranes with 0.0064 M glutaraldehyde selectively crosslinks the glycoprotein (mol. wt. 55000) and its multimers, correlating directly with the modifications of concanavalin A-binding. 6. We conclude that high-affinity binding of concanavalin A to thymocyte membranes is a cooperative process mediated by the glycoprotein (mol. wt. 55000). We further conclude that the bulk of concanavalin A binding is through low-affinity associations, not involving specific membrane macromolecules.

Studies on the iodinated surface membrane proteins and concanavalin A agglutination of transformed Syrian hamster cells.

Chemically transformed Syrian hamster cells exhibit marked agglutination in the presence of the plant lectin, concanavalin A. In this report, we describe conditions which can alter this concanavalin A agglutinability, and compare the surface proteins from transformed cells which express different degrees of agglutinability. Lactoperoxidase-catalyzed iodination of tertiary Syrian hamster cells reveals the major iodinatable protein to be approximately 220 000 daltons. The transformed Syrian hamster cells do not contain this protein in an iodinatable form. Analyses of the transformed cells grown under conditions which decrease the concanavalin A agglutinability do not demonstrate any iodination of the 220 000 mol. wt. protein. These results depict the effects of growth and dibutyryl cyclic AMP on the iodinatable cell surface proteins of transformed cells and indicate that the absence of the I-220 000 mol. wt. protein is probably not a major determinant of concanavalin A agglutination.

Change of the protein composition of the thyroid colloid during treatment with propylthiouracil and thyroxine: a microgel electrophoretic study of single rat thyroid follicles.

Prophylthiouracil and thyroxine were given daily to rats for 4 weeks. Samples of colloid were collected in vivo from the superficial thyroid follicles during this period and their protein composition was analysed by gel electrophoresis. It was observed that the aggregates of thyroglobulin, i.e., the 27-S thyroid iodoprotein and the heavier fractions, were reduced to 50% after 1 week and were almost absent after 2 weeks. A faster migrating thyroglobulin fraction was observed in the samples of colloid and in the homogenate of the whole gland after 48 h of treatment. During the following period of treatment there was an increase in the relative amount of the faster migrating thyroglobulin fraction compared to 19-S thyroglobulin in the colloid, the former comprising approx. 75% of the globulins after 4 weeks, It can be concluded that propylthiouracil inhibits the formation of the 27-S iodoprotein and that a structurally altered and iodine-poor thyroglobulin fraction is accumulated in the follicle lumen.

Receptor binding of selectively labeled (Tyr-10) and (Tyr-13)-mono-125I-glucagons and competition by homologous 127I-labeled isomers.

Two monoiodinated derivatives of glucagon were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The purified (Tyr-10) and (Tyr-13)-mono-125I-labeled glucagon isomers were characterized and studied with respect to their binding to the receptors of isolated intact rat hepatocytes. The extent of steady-state binding to cellular receptor sites differed for the two labeled glucagon tracers at 37 degrees C as well as at 15 degrees C with (Tyr-10)-mono-125I-glucagon displaying higher receptor binding. The apparent equilibrium constants, Kd,app at 37 degrees C are 3.6 +/- 0.4 nM (mean +/- S.E. of three independent experiments) for the tyrosine-13-labeled tracer and 5.9 +/- 0.6 nM for the tyrosine-10-labeled glucagon with native glucagon as competitor. Since the observed Kd in the competition assay is a function of the true Kd values of the monoiodinated radioactive glucagon isomers and native glucagon, the dissociation constants were also measured with chemically identical tracer and competitor. Under these conditions, we obtained Kd values of 1.3 +/- 0.2 nM for the tyrosine-10-labeled analog and 2.0 +/- 0.2 nM for the tyrosine-13-labeled glucagon isomers confirming the higher receptor binding affinity of (Try-10)-mono-125I-glucagon. All competition curves fit the mathematical expression for a model of non-cooperative binding to a single class of receptors.

Evidence for a secretion of thyroxine by isolated hog thyroid cells.

Isolated thyroid cells prepared from hog thyroid glands by tryptic dispersion were incubated with 131I- for 1--6 h. Free [131I]thyroxine was identified in the incubation medium by three chromatographic methods. Neither [131I]iodotyrosines nor [131I]triiodothyronine were detected. The [131I]thyroxine released in the medium by 100 mul of cells (packed cell volume) after a 6-h incubation period amounted to 1.16% (S.E. = +/- 0.39) of the total radioactivity. The medium [131I]thyroxine represented 15--25% of the total [131I]thyroxine synthesized during the 6 h of incubation. Thyrotropin, 1--60 munits/ml, increased the medium [131I]thyroxine content 2-4 fold. Dibutyryl cyclic AMP mimicked the effect of thyrotropin. The amount of medium [131]thyroxine was strictly related to the amount of incubated cells but was independent of the volume of the incubation medium. When prelabeled cells were incubated in the presence of methimazole the increase in medium [131I]thyroxine was quantitatively related to a decrease in the intracellular [131I]thyroxine. Addition of dinitrotyrosine, an inhibitor of the deiodinase activity, induced the release of iodotyrosines in the incubation medium. That the incubation supernatant of isolated thyroid cells did contain free thyroxine but not iodotyrosines suggests that the normal mechanisms of proteolysis of thyroglobulin and deiodination of iodotyrosines inside the cells are preserved. From these data, it was concluded that the thyroxine release by isolated cells represents a real secretion.

Identification of an intracellular pathway of thyroxine synthesis by dispersed thyroid cells.

This study deals with the identification of the biochemical events involved in the metabolic sequence leading from the synthesis to the release of thyroxine in the dispersed thyroid cell system. (1) Using an experimental model allowing the differentiation between intracellular and extracellular sites of iodination, it is shown that thyroxine is synthesized inside the cells by an iodinating system sensitive to thyrotropin stimulation. (2) The secretion of thyroxine synthesized inside the cells is not mediated by an exocytotic-endocytotic phenomenon. Colchicine, vinblastine, fluoride, propanolol and chlorpromazine, at concentrations equal to or 10--100-times higher than those required to inhibit hormone release in follicular-organized thyroid tissue have no effect on thyrotropin-stimulated thyroxine secretion. (3) The secretion involves the intracellular proteolysis of hormone-containing iodoprotein(s) which, in addition to free thyroxine, generates free mono- and diiodotyrosines. Free thyroxine is released into the incubation medium and iodotyrosines are deiodinated under normal conditions and accumulate in the presence of an inhibitor of iodotyrosine deiodinase: 3,5-dinitrotyrosine. This proteolysis is inhibited by 5 mM chlorpromazine. These data indicate that the complete metabolic sequence leading from the uptake of iodide to the release of free thyroxine into the incubation medium can be described as an 'intracellular metabolic sequence for thyroxine synthesis'.

Metabolism of radioiodinated bovine parathyroid hormone in the rat.

Metabolism of bovine 125I-labeled parathyroid hormone was studied in the rat by gel filtration and by sequence analysis of the iodinated fragments. Analysis of the kinetics of hormone metabolism shows that iodinated intact hormone has a multiexponential disappearance curve with a rapid (3 min) initial and a slower (48 min) second component. Iodinated fragments, which rapidly increase during the first 12 min after injection of the intact hormone, subsequently disappear from the circulation with a t1/2 of no greater than 48 min. Plasma samples collected at various time-intervals after intravenous injection of bovine 125I-labeled parathyroid hormone were gel filtered on Bio-gel P-100. Four radioactive peaks were seen. The first and second peaks eluted, respectively, at the void volume of the column and at the position of intact hormone. The third peak consisted of iodinated fragments, and the last peak eluted at the salt volume of the column. Sequence analysis of the iodinated fragments in the third peak showed that it was heterogeneous, containing several different, but closely related, polypeptides. Before 48 min after injection, the most-abundant fragment is one whose amino-terminal amino acid is residue 34. The amino-terminal residue of the next most-common fragment is the amino acid at position 37. No fragments representing cleavages closer to the amino-terminus than residue 34 were seen. The results of these studies are virtually identical with those previously obtained in the dog. The similarities found in the sites of hormone proteolysis and in the kinetics of hormone metabolism in the rat and dog, coupled with the less direct evidence indicating that similar cleavages are also present in man and bovine, are consistent with the view that proteolysis of parathyroid hormone is peripheral tissues is specific, at least in mammalian species, and may be a critical step in controlling the availability of biologically active hormone.

Autoregulation of iodide transport in the rabbit: absence of autoregulation in fetal tissue and comparison of maternal and fetal thyroid iodination products.

Maternal and fetal rabbit thyroid glands were compared as to their ability to respond to excess iodide in vitro with a reduction in subsequent iodide transport activity. Preincubation of maternal thyroid tissue slices for 2 h with excess iodide (30 microM) resulted in a 31% reduction in the subsequently measured thyroid-medium radioiodide concentration ratio. In contrast, similar iodide pretreatment had no significant effect on fetal iodide transport. In all other respects, fetal iodide transport, although it was 10 times higher, did not differ significantly from maternal transport activity. Combined radiolabeled maternal (125I) and fetal (131I) rabbit thyroid tissue was eluted on Sephadex G-25 columns. Fractions were analyzed for both 125I and 131I activity, and the maternal to fetal ratios (125I/131I) were determined for each fraction. The majority of iodoproteins eluted with the void volume, and the 125I/131I ratio was constant in these fractions. Thereafter, two peaks of elevated 125I/131I activity could be observed. Peak A eluted below lysozyme (Mr = 14,300) and above insulin (Mr = 6,000), with an apparent mol wt of 8,000 to 10,000. A second peak, peak B, eluted from the column at a site similar to that of MIT or a protein of Mr of 2,000. Ascending paper chromatography of this latter peak of 125I/131I activity and MIT was carried out in two solvent systems. In both systems, peak B comigrated with MIT. These findings suggest that the failure of fetal thyroid tissue to exhibit autoregulation may be associated with the reduced iodination of a compound with an approximate Mr of 8,000 to 10,000. The role of this substance in iodide transport remains to be identified. The reason for the apparent increase in the labeling of MIT observed in maternal vs. fetal tissue is unknown.

Studies on the heterogeneity of labeled iodoprotein from iodine-replete and iodine-deficient rats as determined by susceptibility to proteolysis.

Thyroid iodoprotein from rats fed a high-iodine diet (HID) or a low-iodine diet (LID) were labeled with radioiodine in vivo for periods ranging from 4 hr to several days. Standardized aliquots of thyroid homogenate from rats with various treatments were digested for 4 hr with 1% pancreatin or with 0.003% pancreatin after 30 min pretreatment with beta-mercaptoethanol (ME-P). Four-hr labeled iodoprotein from both LID and HID rats was equally susceptible to digestion with 1% pancreatin; however, such iodoprotein from LID rats was more susceptible to digestion with ME-P than that from HID rats. With increasing intervals up to 7 days between administering radioiodine and removing the thyroids, there was a progressive rise in the resistance to digestion in iodoprotein from LID rats, but only a slight increase in resistance in iodoprotein from HID rats. If propylthiouracil was added to the diet beginning 24 hr after radioiodine administration, there was a marked increase in the rate of development of resistance of iodoprotein to digestion. Radioautographs showed that the radioiodine was localized primarily in the peripheral follicles after 2 days PTU. Similar differences in susceptibility to digestion were found in purified thyrogobulin prepared from HID and LID rats. No change in susceptibility to digestion of thyroid iodoprotein with time after labeling was seen in hypophysectomized LID rats in which thyroid secretion and thyroglobulin turnover is known to proceed at an extremely slow rate. The data indicate that there are at least two types of iodinated thyroglobulin in the rat thyroid. One is readily susceptible to digestion and has a rapid turnover in the thyroid. The other is more resistant to digestion, has a slow rate of turnover and is located primarily in the peripheral follicles.

Afrikander cattle congenital goiter: characteristics of its morphology and iodoprotein pattern.

The morphology and some properties of the complex iodoprotein pattern of the genetically determined congenital goiter in Afrikander cattle is described. The goiter contained irregularly shaped follicles which were devoid of colloid and the follicular epithelial cells were elongated, measuring about 20 micrometer in length compared to 10 micrometer for normal thyroid cells. The goiter cells contained apical clusters of larger and more numerous lysosomes than normal thyroid cells. Apical vesicles containing electron-dense material which were in contact with the plasma membrane could be seen in most normal thyroid cells, but were extremely scarce in the goiter. In 36 cell profiles studied none was found. The endoplasmic reticulum cisternae of the goiter differed significantly from normal thyroid cells. Fewer ribosomes were seen to be attached to the membranes of goiter cells. Furthermore, unlike normal thyroid cells, many free polysomes were seen in goiter cells. The characteristics of the unusual iodoprotein pattern of the goiter extract, resolved by gel chromatography and sucrose density gradient centrifugation, were qualitatively and quantitatively similar to that described previously (Endocrinology 91, 470, 1972). A relatively small amount of the total soluble protein was iodinated. Of these, only a 12S sedimenting species was precipitated by antithyroglobulin immunoglobulin. When separated on polyacrylamide gels containing sodium dodecyl sulfate and mercaptoethanol, this 12S species was resolved into at least 14 polypeptide components ranging in molecular weights from less than 66,000--330,000. Three of the bands, representing a small percentage of the total protein, seemed to comigrate with the major polypeptides of thyroglobulin and were also precipitated with rabbit antihyroglobulin immunoglobulin. The data indicate that glycosylation of iodoproteins was not affected although 19S thyroglobulin synthesis and subsequent storage were drastically impaired.

Euthyroidism via iodide supplementation in hereditary congenital goiter with thyroglobulin deficiency.

We have studied hereditary congenital goiter in an inbred strain of goats. On a normal diet, the goats were hypothyroid and iodide taken up by the gland was released rapidly in the form of iodinated macromolecular material. This resulted in a low thyroidal iodine pool. An extremely low amount of thyroglobulin-related antigens (12 microgram/g tissue; normal, 100 mg/g tissue) was detected in the goitrous gland by RIA. Addition of 1 mg I-/day to the diet made the goats euthyroid and the serum protein-bound iodine increased to high values. Even under these conditions, however, the amount of thyroglobulin-related antigens was not significantly elevated (19 microgram/g tissue). On sucrose gradient, these antigens sedimented in the 7S region. No thyroglobulin-related antigens sedimented at 12S or 19S. Besides iodoalbumin, a heterogeneous group of abnormal iodoproteins containing T3 and T4 was observed with this high iodide intake and may have accounted for the hormone production. The abnormal iodoproteins had a molecular weight of 300,000-500,000 and a sedimentation value of about 7S. From these findings, we conclude that these goats were unable to synthesize thyroglobulin but with excess iodide, there was sufficient formation of T3 and T4 in the abnormal iodoproteins to make the animals euthyroid.

Thyroidal iodine metabolism during the development of the chick embryo.

Stable iodine was measured in the thyroid gland of the chick embryo from day 9 to day 20 of incubation in order to evaluate quantitatively the functional development of the gland. Total iodine content increased progressively during incubation. From day 9 to day 17 of incubation, this increase resulted from the increases of pellet-bound iodine and of soluble iodine. Afterwards, it essentially paralleled the increase of the soluble thyroglobulin-bound iodine which reflected the increase in both thyroglobulin content and the degree of iodination of the thyroglobulin. The total iodine, thyroglobulin-bound iodine and thyroglobulin (TG) content, increased as power functions of time during incubation, with critical times on days 11 and 15. Their concentrations also increased during the whole incubation period, while the iodide concentration remained roughly constant (25 ng/mg) from day 13 to day 19. Only one iodoprotein, 19.5 S TG, was found, and its heterogeneity of iodination was demonstrated during the whole period of incubation studied (from day 11 to day 20). The degree of dissociation with sodium dodecyl sulfate (SDS) of the TG into 12 S subunits decreased as the degree of iodination of the TG increased. Throughout embryonic development, iodine was bound more and more to TG molecules, which were resistant to dissociation with SDS. While the average iodine content of the TG increased, no appreciable changes were found in iodotyrosine and iodothyronine percentages of TG-bound iodine: monoiodotyrosine, 26%; diiodotyrosine, 43%; thyroxine 12%; 3,5,3'-triiodothyronine, 2.5%. As a consequence, a linear relationship existed for each iodoamino acid between the number of its residues per mole of TG and the iodine content of TG (127I atoms per mole)-- about 30 atoms of iodine was required to form 1 mole of T4. The low efficiency of the TG of the chick embryo as a thyroidal hormone-forming protein was compensated for by its high degree of iodination.

Thyrotropin binding to cultured lymphocytes and thyroid cells.

The TSH-binding properties of human lymphocytes in continuous culture were studied and compared to those of bovine and human thyroid cells in primary culture. Both lymphocytes and thyroid cells had maximal TSH-binding capacity at pH 5.2. At pH 7.4, thyroid cells bound 15% but lymphocytes bound only 3% of the amount bound at pH 5.2. At 37 C, maximal binding of [125I]iodo-TSH to lymphocytes was reached within 60--90 min and maximal binding to thyroid cells was reached within 15--20 min. TSH binding to lymphocytes was salt sensitive, being inhibited to 50% by 0.2 mM MgCl and 0.4 mM CaCl2 and by 20 mM Kl, KCl, and NaCl. The saturable binding of bovine TSH (bTSH) to thyroid cells at pHs 5.2 and 7.4 was above 90% of the total binding. Saturable binding of bovine TSH (bTSH) to thyroid cells at pHs 5.2 and 7.4 was above 90% of the total binding. Saturable binding to lymphocytes at pH 5.2 was also above 90%, but at pH 7.4 was 75% of total. At pH 5.2, both cell types displayed identical displacement curves of [125I]iodo-bTSH by unlabeled bTSH. Pure hCG, human placental lactogen, human GH, and insulin cross-reacted to less than 1% with [125I]iodo-bTSH binding to lymphocytes at pH 5.2, whereas a crude preparation of hCG and human FSH plus human LH showed a strong cross-reaction. Nonhormone glycoproteins, including mucin, normal human gamma-globulin, and bovine thyroglobulin showed intermediate cross-reactivity. At pH 7.4, the cross-reactivity of normal human gamma-globulin, bovine thyroglobulin, and pure hCG with bTSH binding to both lymphocytes and thyroid cells was below 1%. The TSH-binding properties of lymphocytes and thyroid cells show many similarities but differ in kinetics and the relative binding capacity at neutral pH. Although the physiological significance of these differences is not yet clear, cultured cells provide a convenient system for studies of TSH-receptor interaction.

Proteolytic processing of thyroglobulin by extracts of thyroid lysosomes.

The release of T4 and T3 from the prohormone thyroglobulin (Tg) occurs in thyroid lysosomes. To examine the role of cathepsin-B, -D, and -L, the three major endopeptidases in this process, we incubated rabbit [125I]Tg, labeled in vivo, with lysosomal extracts from human thyroids. Iodopeptide formation was evaluated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after short term incubations (20-45 min), while iodoamino acid release was assessed by paper chromatography after long term incubations (8 and 24 h). Using pepstatin to inhibit cathepsin D, Z-Phe-Ala-CHN2 to inhibit both cathepsin B and L, and Z-Phe-Phe-CHN2 to selectively inhibit cathepsin L, we obtained the following results: 1) blocking of all three endopeptidases reduced both iodopeptide formation in short term experiments and iodoamino acid release in long term experiments by 80-90%; 2) iodopeptide formation was reduced by 85% with Z-Phe-Ala-CHN2, by 56% with Z-Phe-Phe-CHN2, and by 26% with pepstatin; 3) iodoamino acid release was reduced by 60-80% with Z-Phe-Ala-CHN2 and by 40-50% with either Z-Phe-Phe-CHN2 or pepstatin at 8 h, but by less than 20% at 24 h; pepstatin and Z-Phe-Phe-CHN2 together reduced iodoamino acid release by 80% and 60% at 8 and 24 h, respectively. Limited hydrolysis of Tg by lysosomal enzymes produced at least eight peptide fragments of less than 100,000 mol wt. Three of these, together representing 32% of the 125I released, resulted from cleavages in the C-terminal region of Tg corresponding to residues 2487, 2393, and 2390 of cDNA-derived human Tg. Several other peptides, together containing 38% of the 125I released, included the N-terminus of Tg. These C-terminal and N-terminal fragments contained three of Tg's four major hormonogenic sites, but none of the cleavage sites fell close to the hormone sites themselves. We conclude that 1) the formation of discrete iodopeptides precedes the release of iodothyronines and iodotyrosines from Tg; 2) the cysteine proteinases are more important than cathepsin D in this process; and 3) these endopeptidases selectively cleave Tg to favor the production of hormone-containing intermediates for subsequent processing by exopeptidases.

Specific binding of iodinated growth hormone to rat liver in vivo.

The specific uptake by rat liver of human (hGH) and bovine (bGH) GHs labeled with 125I was studied by an in vivo procedure. A significant reduction of the uptake was observed when labeled hormones were injected together with different amounts of the corresponding native GH. This reduction was dose dependent, and the concentration of native hormone that prevents 50% of the liver uptake of the labeled hormone was close to 12 microgram/100 g BW. In normal rats, only native hGH or bGH significantly decreased the liver uptake of [125I]iodo-bGH, while bovine PRL (oPRL) or heat-denatured bGH were inactive. The highest inhibition of the uptake of [125I]iodo-hGH by rat liver was obtained when this labeled hormone was injected either together with hGH or with bGH plus oPRL while partial displacement was observed with bGH or oPRL. These data suggest that hGH binds to both somatotropic and lactogenic sites in the liver of normal rats. In hypophysectomized animals, only the somatogenic binding sites could be detected.

Thyroid hormone regulation of extrathyroidal iodoproteins.

The effect of thyroid status on plasma and tissue levels of labeled nonextractable iodine (NEI) derived from the metabolism of radioiodothyronines was examined in the rat. Concentrations of radioiodoprotein were substantially elevated in plasma, kidney, and liver in thyroidectomized animals 72 h postinjection of [125I]triiodothytonine ([125I]T3). Similarly, total rat concentrations of radioactive NEI were increased (52%) 72 h after injection of [125I]T3. NE125I concentrations from [125I]T3 in plasma, kidney, and liver were diminished progressively in thyroidectomized animals maintained on increasing doses of thyroxine replacement, demonstrating that iodoprotein levels were inversely related to thyroid state. The plasma disappearance rate of radioiodoprotein from [125I]T3 was markedly slowed in hypothyroid animals and accelerated in intact controls rendered hyperthyroid with daily injections of T4, 8 mug/100 g BW. Propylthiouracil (PTU) treatment of thyroidectomized rats maintained on T4, 2 mug/100 g BW per day resulted in increased NE125I from [125E]T3 in plasma, kidney, and liver. The results of the foregoing investigations suggest that thyroid hormone regulates levels of iodothyronine-derived iodoproteins by influencing the rate of degradation of iodoproteins. Moreover, the observed elevation of iodoprotein levels in T4-maintained thyroidectomized animals after PTU administration appears consistent with the modification of thyroid status due to the peripheral antithyroxine effect of PTU.