Incorporation Of 125I Research Articles

Characterization and Purification of Iodinated Bovine Thyrotropin by High Performance Liquid Chromatography*

Reversed-phase (RP) HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography have been used to study the incorporation of 125I into bovine (b) TSH by the lactoperoxidase-catalyzed radioiodination procedure. Two preparations of [125I]bTSH were studied, being freshly iodinated bTSH and tracer purified from this preparation by the method of receptor adsorption. It is demonstrated with these methods that both the alpha- and beta-subunits of bTSH are labeled with 125I, and that the tracer purified by receptor adsorption retains this incorporation pattern. However, the implied theoretical specific activity of (at least) 2 I atoms per TSH molecule (or approximately 140 muCi/micrograms) suggested by this result was not achieved, with observed tracer specific activity being 30-60 muCi/micrograms, indicating that hormone molecules with varying extents of labeling must exist. Evidence to support this was provided by comparison of the MIT/DIT ratios for the 2 tracer preparations. Receptor adsorption decreased the MIT/DIT ratio from 75:25 in the freshly iodinated bTSH to 93:7, indicating the selection of particular iodinated species. Tryptic mapping by RP-HPLC was used to study both tracer preparations, and it is shown that at least 14 iodine-containing tryptic peptides may be resolved for each preparation, which is greater than the theoretical maximum of 13 peptides if every tyrosine was labeled and tryptic cleavage occurred at all possible lysine and arginine residues. Tracer heterogeneity was also studied by purification using RP-HPLC. Selection of peak fractions demonstrated that intact [125I]bTSH may be recovered from RP-HPLC which in TSH radioreceptor assay exhibit increased assay sensitivity, increased saturable binding, and decreased nonsaturable binding.

A detailed examination of the iodination of β-galactosidase: stoichiometric inactivation by nonspecific iodination

The incorporation of 125I, using lactoperoxidase, and the subsequent inactivation of beta-galactosidase in the period when incorporation and inactivation were stoichiometric were investigated in detail. The high pressure liquid chromatographic (HPLC) radioactive profiles of the tryptic peptides of samples taken in the stoichiometric period showed that, although two labelled peptides predominated, there were other labelled peptides. The predominating peptides were shown to be the mono- and di-iodinated forms of the peptide containing Tyr-253. This confirmed the result of an earlier study, but quantitation showed that this iodination accounted for only 15-18% of the total. To show that the other labelled peptides in the HPLC profiles were not merely oxidized or partially digested forms of the peptide containing Tyr-253, two experiments were carried out. In one of the experiments, two of the other labelled peptides were isolated and identified as iodinated forms of the peptide containing Tyr-285 (5-7% of the incorporation). In the other experiment, four additional labelled fractions from the HPLC eluate were treated further with trypsin. No further digestion was observed and thus these peptides did not result from incomplete digestion of the sequence containing Tyr-253. Overall, these results show that, although the incorporation of 125I was stoichiometric with inactivation, no single Tyr was responsible for the inactivation as was tentatively suggested previously. The competitive inhibitor isopropyl-beta-D-thiogalactopyranoside (IPTG) was effective in reducing the rates of inactivation of the enzyme and incorporation of 125I, but the same peptides were labelled in the presence of IPTG as in its absence.

Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition-particle-mediated translational arrest by glucose.

The biosynthesis of insulin in the islets of Langerhans is strongly controlled at the translational level by glucose. We have used a variety of experimental approaches in efforts to dissect the mechanisms underlying the stimulatory effect of glucose. To assess its effects on rates of peptide-chain elongation, isolated rat islets were labelled with [3H]leucine at different glucose concentrations in the presence or absence of low concentrations of cycloheximide. Under these conditions, at glucose concentrations up to 5.6 mM, endogenous insulin mRNA did not become rate-limiting for the synthesis of insulin, whereas stimulation of non-insulin protein synthesis was abolished by cycloheximide at all glucose concentrations, indicating either that insulin synthesis is selectively regulated at the level of elongation at glucose concentrations up to 5.6 mM, or that at these concentrations inactive insulin mRNA is transferred to an actively translating pool. Glucose-induced changes in the intracellular distribution of insulin mRNA in cultured islets were assessed by subcellular fractionation and blot-hybridization using insulin cDNA probes. At glucose concentrations above 3.3 mM, cytoplasmic insulin mRNA was increasingly transferred to fractions co-sedimenting with ribosomes, and relatively more of the ribosome-associated insulin mRNA became membrane-associated, consistent with effects of glucose above 3.3 mM on both the initiation of insulin mRNA and SRP (signal recognition particle)-mediated transfer of cytosolic nascent preproinsulin to the endoplasmic reticulum. When freshly isolated islets were homogenized and incubated with 125I-Tyr-tRNA, run-off incorporation of 125I into preproinsulin was increased by prior incubation of the islets at 16.7 mM-glucose. The addition of purified SRP receptor increased the run-off incorporation of [125I]iodotyrosine into preproinsulin, especially when the islets had been preincubated at 16.7 mM-glucose. These findings taken together suggest that glucose may stimulate elongation rates of nascent preproinsulin at concentrations up to 5.6 mM, stimulates initiation of protein synthesis involving both insulin and non-insulin mRNA at concentrations above 3.3 mM, and increases the transfer of initiated insulin mRNA molecules from the cytoplasm to microsomal membranes by an SRP-mediated mechanism that involves the modification of interactions between SRP and its receptor.

Purification of bile acid-binding proteins from rat hepatic cytosol. Use of a photoaffinity label to detect novel Y′ binders

A 125I-labelled photolabile derivative of cholic acid has been used to investigate the organic anion-binding Y′ fraction from rat liver, prepared by the method of Sugiyama, Y., Yamada, T. and Kaplowitz, N. (1982) Biochimica Biophysica Acta, 709, 342–352. The use of this photoaffinity probe led to the discovery of previously undescribed bile acid-binding proteins. A comprehensive purification scheme for the Y′ proteins which allows the isolation of these novel binding species is described. Electrophoretic analysis shows that the Y′ binders can be divided into two groups. The proteins in group 1 are dimeric and the 5B, 6E and 7F binding species consist of subunits with approximate molecular masses of 19.6, 15.6 and 14.9 kDa, respectively. The group 2 binding proteins, 5C, 5D and 8C, are monomeric and have molecular masses of approximately 36.2, 36.2 and 33 kDa, respectively. Calculation of the incorporation of 125I by these proteins showed that the group 1 proteins displayed a significantly greater specific incorporation of radioactivity than group 2. The specificity of 125I-labelled 3β-azidocholylhistamine is further demonstrated by analysis of tryptic digests of photoaffinity labelled Y′ binders and glutathione S-transferases AA, A, D and F by reverse-phase high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The majority of the radioactivity was shown to be incorporated into a single component, which was not coincident with the free photoaffinity label.

Synthesis of peptide labelled with p-iodophenylalanine which corresponds to the first amphipathic region of apolipoprotein C-I.

The amino terminal fragment (1-15) of apolipoprotein C-1, H-Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe14-Gly was prepared by the solid phase method. However, the phenylalanine residue at position 14 was replaced with p-iodophenylalanine in the chemical synthesis. The preparation of t-butyloxy-carbonyl-p-iodophenylalanine is described. After completion of the synthesis, the product was deprotected and cleaved from the resin with liquid HF. The peptide was purified by gel filtration on Sephadex G-25 and preparative high performance liquid chromatography. The purified material was shown to be homogeneous by amino acid analytical data and by chromatography in three different analytical reversed phase HPLC systems. The peptide was then labelled by the chloroamine-T procedure and good incorporation of 125I was obtained. After purification of the product by gel filtration on Biogel P-2, the labelled pentadecapeptide was tested for ability to bind to Very Low Density Lipoproteins (VLDL) in the following manner: VLDL was isolated from normolipemic serum by ultracentrifugal flotation and 100-microliter samples were incubated with labelled material dissolved in 200 microliter of 0.5 M NaCl, 0.02 M sodium phosphate buffer, pH 7.4 at 37 degrees for 30 min. The VLDL fraction was again isolated by ultracentrifugal flotation and the incorporation of radioactivity into the lipoprotein was measured. Under these conditions, a sample of [3H]-native apolipoprotein C-I was incorporated to an extent of 83% of the total sample, while the [125I]-pentadecapeptide exhibited an incorporation of 8.7%.

Photoaffinity labeling of the DDT1 MF-2 cell ?1-adrenergic receptor

In this study, we have used an alpha 1-adrenergic receptor photoaffinity ligand, 2-[4-(4-azido-3-iodo-benzoyl)-piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (125I-APD), to label covalently the alpha 1-adrenergic receptor in a smooth muscle cell line. Our results indicate that in the absence of light, (125I)APD binds reversibly to a site in the DDT1 MF-2 cell membranes having pharmacological characteristics of an alpha 1-adrenergic receptor. Following incorporation of (125I)APD into partially purified membranes a single labeled band of protein with a Mr of 81 000 was visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incorporation of (125I)-APD into this band was affected by adrenergic agonists and antagonists in a manner consistent with an alpha 1-adrenergic interaction. Prazosin (alpha 1-selective) blocked incorporation of the label into the Mr = 81 000 protein while yohimbine (alpha 2-selective) did not. Of the adrenergic agonists, (-)-epinephrine and (-)-norepinephrine but not (-)-isoproterenol blocked labeling of the Mr = 81 000 protein. We conclude that the ligand binding site of the DDT1 MF-2 cell alpha 1-adrenergic receptor resides in a Mr = 81 000 protein.

Inhomogeneity of the Nucleus to 125 IUdR Cytotoxicity

Synchronized suspension cultures of Chinese hamster ovary (CHO) cells were used to determine the lethal effects produced by the decay of 125I incorporated into different subfractions of the nuclear genome. Such a shift in nuclear incorporation pattern was achieved by using the drug aphidicolin, which inhibits 95% of all nuclear DNA synthesis, is nontoxic to cells in a colony-forming assay, and does not modify the radiation response of CHO cells to X irradiation. In addition to shifting incorporation of 125I to only 5% of the nuclear genome, both nuclease digestions to characterize the molecular location of 125I and electron microscope autoradiography show an inhomogeneous distribution of sites of 125I incorporation in the presence of 5 micrograms/ml aphidicolin. These data in combination with survival curves of CHO cells labeled with 125I-iododeoxyuridine (125IUdR) either with or without aphidicolin showed a dramatic change in the survival response (DO: 30 decays/cell and 96 decays/cell, respectively). It is concluded, therefore, that the nucleus is not a homogeneous target for radiation-induced cell death because when subfractions of the nuclear genome are labeled, radically different levels in cell survival are obtained.

The relative importance of thrombin inhibition and factor Xa inhibition to the antithrombotic effects of heparin

The relative importance of antithrombin and anti-factor Xa activities of heparin fractions required to achieve optimal antithrombotic effects is unknown. To study this, we measured the effects of standard heparin, an octasaccharide heparin fraction (anti-factor Xa activity only), and dermatan sulfate (antithrombin activity only) on the prevention of thrombosis and related this to their anticoagulant effects in vivo in rabbits. Thrombosis was measured as the incorporation of 125I- fibrinogen into tissue thromboplastin-induced thrombi using a Wessler- type model. Ex vivo changes in thrombin clotting time (TCT) were used as an index of antithrombin activity, and a chromogenic anti-factor Xa assay was used to measure anti-factor Xa activity. In addition, the ability of the three sulfated polysaccharides to simultaneously inhibit the generation of thrombin activity and to enhance the inactivation of the factor Xa added to initiate thrombin generation in plasma was determined. Standard heparin, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 90%, prolonged the TCT by two seconds, and resulted in an anti-factor Xa level of 0.32 U/mL. The octasaccharide heparin fraction, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 41%, had no effect on the TCT, and resulted in an anti-factor Xa level of 0.28 U/mL. Higher doses of the octasaccharide resulted in a further increase in the anti-factor Xa levels but had no further effect on thrombus formation. Dermatan sulfate, in a dose of 500 micrograms/kg, inhibited thrombus formation by 95%, but had no affect on the TCT. These results indicate that the antithrombotic effect achieved by inhibiting factor Xa is limited and that better antithrombotic effects are achieved by heparin or heparin- like substances capable of influencing the inactivation and/or the generation of thrombin.

Biosynthesis and regulation of iodolipids in calf thyroid.

The incorporation of 125I into iodolipids was investigated in calf thyroid slices. Time course studies showed that the iodination of lipid reaches a plateau after 30 min of incubation. A highly significant correlation was found between iodination of lipid and of protein (r = 0.906), suggesting that both reactions may be related. Addition of PTU or MMI caused a significant inhibition of lipid iodination, indicating that a peroxidase could be involved in this reaction. The iodinated lipids are not attached to protein, since they migrated in BEA chromatography and pancreatin digestion of the samples did not modify the percentage radioactivity. The iodolipid fraction observed in BEA chromatography was eluted and analysed by TLC. Iodinated free fatty acids and neutral lipids comprised most of the radioactivity. Although iodolipids are present in every subcellular fraction, the 20 000 X g pellet had the greatest proportion of iodinated fatty acids and neutral lipids.

125I-labeled crosslinking reagent that is hydrophilic, photoactivatable, and cleavable through an azo linkage.

A radioactive crosslinking reagent, N-[4-(p-azido-m-[125I]iodophenylazo)benzoyl]-3-aminopropyl-N' -oxysulfosuccinimide ester, has been synthesized. The reagent is photoactivatable, water-soluble, cleavable through an azo linkage, and labeled with 125I at the carrier-free specific activity of 2000 Ci/mmol. Any protein derivatized with the reagent is thus converted into an 125I-labeled photoaffinity probe. Crosslinks are formed following photolysis with 366-nm light, and cleavage by sodium dithionite results in the donation of radioactivity to the distal partner in crosslinked complexes. The newly labeled proteins are then analyzed by gel electrophoresis and autoradiography. The compound was prepared by iodination of N-[4-(p-aminophenylazo)benzoyl]-3-aminopropionic acid using carrier-free Na125I and chloramine-T, followed by azide formation and conversion to the water-soluble sulfosuccinimide ester. As a model system, protein A-Sepharose was derivatized with the reagent under subdued light. Each derivatized protein A molecule contained only one crosslinker. The derivatized protein A-Sepharose was then photolyzed in the presence of human serum and subsequently treated with sodium dithionite. Analysis of the serum by gel electrophoresis revealed that 1.1% of the radioactive label originally present on the protein A-Sepharose was transferred to the heavy chain of IgG, which was the most intensely labeled protein in the gel. The next most intensely labeled protein was IgG light chain, which incorporated radioactivity that was lower by a factor of 3.6 than that of the heavy chain. These results demonstrated the specificity of the derivatized protein A-Sepharose as a photoaffinity probe. Photolabeling of IgG was the result of nitrene-mediated reactions and was not due to the incorporation of free 125I.

Preparation and biological reactivity of polyiodinated human growth hormone.

Studies with iodinated human GH (hGH) were undertaken to characterize the iodinated product and to evaluate the possibility that iodination might distort the hGH molecule sufficiently to interfere preferentially with binding to one or more classes of receptors on adipocytes. hGH containing an average of 1.2 atoms of 125I/molecule was subjected to cleavage with cyanogen bromide, and the cleavage products were examined by autoradiography after electrophoresis of the lyophilized reaction mixture on polyacrylamide gel. 125I was found to be distributed in the fragments according to their content of tyrosine, suggesting that there is no single tyrosine residue that is iodinated preferentially. Digestion of the iodinated hormone with pronase followed by paper chromatography revealed that 7.6 times as much iodine appeared as MIT than as DIT, suggesting that the incorporation of 125I into a tyrosine residue had little influence on the incorporation of a second iodine atom. By increasing the molar ratios of iodide and chloramine-T to hGH in the reaction mixture, increasing amounts of iodine were incorporated into hGH in a highly predictable and reproducible manner until a product containing 11-12 atoms of iodine/molecule hGH was obtained. The ratio of MIT to DIT decreased progressively with increasing incorporation of iodine. The data suggest that only six of the eight tyrosine residues of hGH are accessible for iodination. Iodinated hGH retained full biological potency with respect to stimulation of glucose oxidation (insulin-like effect) and lipolysis even after incorporation of 4.6 atoms of I/molecule hGH. Two preparations containing 9.4 and 9.6 atoms of I/molecule hGH retained full biological potency, whereas another preparation containing 9.4 atoms/molecule had approximately one third the potency of uniodinated hGH with respect to both activities. Some biological potency, at least with respect to stimulation of lipolysis, was retained even in maximally iodinated preparations. We conclude that iodination occurs randomly among the six accessible tyrosine residues, and even after polyiodination, the interaction of hGH with its receptors in adipocytes is not completely compromised.

Effects of prolonged administration of thyrotrophin on serum concentration, release and synthesis of thyroid hormones in mice.

Mice were injected sc with TSH (0.5 U) at 12 h intervals for 5 days. Groups of mice were sacrificed daily to determine serum T4 and T3 concentrations, 4 h thyroidal 125I uptake, distribution of 125I among thyroidal iodoamino acids, and thyroidal content of T4 and T3. Serum T4 and T3 concentrations increased significantly after the initial injection of TSH and gradually decreased thereafter, reaching initial levels on the 3rd and 4th days, respectively. In contrast to serum hormone levels, thyroidal 125I uptake, incorporation of 125I into T4 and T3 increased significantly on the first day and remained elevated throughout the period of TSH-treatment. Thyroidal T4 content expressed as microgram/mg weight of tissue decreased significantly on the first day and thereafter remained constant. Thyroidal T3 content did not change significantly throughout the experimental period. The differences between thyroidal synthesis and thyroidal contents of T4 and T3 strongly suggest that thyroid hormone secretion is being continuously stimulated. Transient increases in serum T4 and T3 concentrations are probably due to a gradual increase in the rate of peripheral degradation of thyroid hormones. These results suggest that TSH-induced refractoriness in thyroidal iodine metabolism does not appear to exist, at least when TSH is given in vivo for 5 days.

Labeling of platelet surface proteins with 125I by the Iodogen method

A procedure for the 125I-iodination of platelet suspensions is described. The procedure utilizes Iodogen, a solid-phase oxidizing agent similar to chloramine-T. Platelets were labeled under a variety of conditions, including in the presence of 0.1% albumin, and showed between 7 and 28% incorporation of 125I. Best labeling results were obtained at low platelet concentrations (3–5 × 10 8 platelets/ml), short reaction times (15 min), and with 2-ml glass vials coated with 100 μg of Iodogen. Analysis of the labeled platelet proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed that the same major protein bands were labeled by this procedure as were labeled by the lactoperoxidase procedure. At low platelet concentrations, the Iodogen procedure gives twice the amount of iodine incorporation.

Preparation and properties of fluorescent polysaccharides

A new method for preparing fluorescein derivatives of polysaccharides is described. These derivatives are prepared by activation of the polysaccharide with cyanogen bromide and subsequent reaction with fluoresceinamine. The optimum conditions for coupling have been established in this report. Using this procedure, we have prepared fluorescein derivatives of a wide variety of polysaccharides. Degrees of substitution in the range of 3.0 × 10 −3 to 2.4 × 10 −2 mol of fluorescein per mole of monosaccharide equivalent were obtained. The fluorescent derivatives are stable: no free fluorescein was detected after incubation at 22°C for 48 h or at −10°C for 4 months. The fluorescein-derivatized polysaccharides were found to have the same potency in inhibiting lectin-mediated hemagglutination as the underivatized polysaccharide. In addition, these fluorescent polysaccharides can be radioiodinated to specific activities exceeding 10 6 dpm/μg due to incorporation of 125I into fluorescein. The cell binding properties of 125I-fucoidin and 125I-heparin are indistinguishable from the corresponding underivatized polysaccharides. This general approach for preparing fluorescent polysaccharides should produce useful reagents for localizing and quantifying cell surface carbohydrate-binding proteins (lectins).

Cellular adhesion to growth surfaces: a possible role for low molecular weight surface components.

Cell lines initiated from a fibrocystic hyperplasia and a carcinoma of the breast and an established human fetal lung fibroblast line, MRC5, were grown in culture. The surface proteins of the cells were iodinated in cell monolayer, and in suspension following harvesting from culture flasks using EDTA. Data are presented which show that cells labelled in suspension show reduced incorporation of 125I into surface proteins of high molecular weight (greater than 100,000 daltons) and considerably increased incorporation into proteins with molecular weight less than 100,000 daltons. These changes are discussed in relation to the possible release of surface material by the action of EDTA. It is also suggested, on the basis of the current findings, that the detachment of cells may expose low molecular weight components possible involved in the attachment of the cells to the substratum.

Studies on the bioactivity of radioiodinated highly purified bovine thyrotropin: analytical polyacrylamide gel electrophoresis.

Highly purified bovine TSH (stored in solution at -70 C) was radioiodinated by the stoichiometric chloroamine-T method. The iodinated material ws subjected to analytical polyacrylamide disc gel electrophoresis. TSH was eluted from gel slices (1 mm width) and was analyzed for radioactivity and bioactivity. The latter was determined using the cultured thyroid cell cAMP response assay. Radioactivity in the TSH preparation migrated separately from bioactivity, but concordant with the protein bands observed in gels run in parallel. Further studies performed on bovine TSH purified in our laboratory, as well as on a different TSH preparation of exceptionally high potency (both stored as lyophilized powder) revealed a different pattern, with TSH bioactivity and radioactivity eluting concurrently. Iodination of TSH did not alter its electrophoretic migration on disc gel electrophoresis. In all preparations polymorphism of TSH bioactivity was observed, with at least four separate protein bands containing TSH bioactivity being present in our preparation. The relationship between the degree of iodination and retention of TSH bioactivity was examined. Incorporation of 125I into TSH was greatly different at two different concentrations of chloramine-T. Despite this, however, the progressive loss of TSH bioactivity was similar at both concentrations, indicating that incorporation of iodine into the TSH molecule is not itself responsible for the decrease in bioactivity. These studies indicate variability among different TSH preparations in terms of their retention of bioactivity. Significant loss of TSH bioactivity appears to occur during storage in solution. The damage to the biological activity of TSH during the iodination procedure is more likely related to the oxidation process than to the incorporation of iodine.

Normal neutrophil function in human renal allograft recipients receiving Minnesota anti-lymphoblast globulin (MAG).

Equine anti-human lymphoblast globulin (MAG) used in combination with other immunosuppressive agents did not impair chemotaxis, phagocytosis, nitroblue tetrazolium (NBT) reduction or halogenation by peripheral blood neutrophils (PMN) from seven human recipients of cadaver renal allografts. MAG was given to the allograft recipients following transplantation, and the dosage was determined each day by quantitation of peripheral blood T and B lymphocytes. No deposition of immunoglobulin or complement on the recipient's PMN was detected by indirect immunofluorescence. Chemotaxis of PMN was determined in blind-well chambers, and phagocytosis of opsonized zymosan was quantitated by spectrophotometric measurement of solubilized formazan, and halogenation was measured by quantitating the incorporation of 125I into PMN that had phagocytosed opsonized zymosan. Chemotaxis, phagocytosis, NBT reduction, and halogenation by neutrophils from patients receiving MAG, prednisone and azathioprine were not impaired.

α-Halostilbenes related to diethylstilbestrol

Some new estrogens based on the diethylstilbestrol structure have been prepared for use as diagnostic, radiopharmaceuticals. By the use of structure activity relationships the optimal position for the introduction of iodine was determined to be the α position. The new α-halostilbestrols were prepared as their dimethoxy, monomethoxy and dihydroxy forms. These forms were successfully labelled using82Br,125I,128I and131I. A rationale for the mode of incorporation of125I and131I was developed which also explained the stereochemistry of incorporation. This incorporation required the use of the cuprous ion as a catalyst. It was found that in the absence of such a catalyst the incorporation of radioiodide proceeded very slowly and without stereospecificity whereas in the presence of cuprous ion it was rapid and the configuration of the substrate was maintained.

External membrane proteins of rabbit lung macrophages

Externally disposed polypeptides of rabbit lung macrophages were labeled using chloramine-T. Optimal conditions, chosen as those which maximized the incorporation of 125I without inhibiting phagocytosis of C3-opsonized lipopolysaccharide oil particles, were found to be dependent on concentrations of carrier iodide, chloramine-T, and the cells themselves. These macrophages inhibit the labeling reaction owing to an apparent abundance of surface sulfhydryl groups which preferentially become oxidized before labeling can occur. Analyzed on polyacrylamide gel electrophoresis, whole macrophages displayed major bands of radioactivity whose apparent molecular weights were: 317,000, 245,000, 186,000, 143,000, and 104,000 daltons. All bands were completely removed by trypsin treatment except a large band of 10,000–15,000 daltons which was removed by lipid solvent extraction and diminished by β-mercaptoethanol treatment of whole labeled cells. No label comigrated with actin at 42,000 daltons or with either of the two major proteins found in the lung lavage fluid. Very similar bands were found in podosomes, peripheral hyaline blebs of plasma membrane, prepared from whole labeled cells.

Lactoperoxidase-coupled iodination of bovine chromaffin granules.

Abstract— Chromaffin granules were iodinated with lactoperoxidase at either their external or internal membrane surfaces. When iodination of internal soluble granule proteins and membrane phospholipids was minimized, the majority of the membrane proteins, including the 83,000 component, were iodinated. Components with molecular weights 63,000, 61,000, 51,000, 44,000, 32,000, 26,000 and 19,000 had a higher 125I specific activity than did the other membrane components, suggesting they were more accessible at the outer membrane surface than were the other components. In the presence of detergent, the iodination of all membrane components was increased more than 10‐fold; the incorporation of 125I was now similar to their Coomassie Blue staining intensity in disc gels, indicating that all components were equally accessible to lactoperoxidase. In the presence of detergent, iodine incorporation into the MW 83,000 and 16,000 components was stimulated approx 100‐fold.The MW 83,000, 63,000, 61,000 and 37,000 components incorporated significant amounts of 125I when granule membranes were iodinated from their internal surface, suggesting these components have a portion of their polypeptide chain accessible at the inner membrane surface. Thus the MW 83,000 component, which we identified as dopamine β hydroxylase, and the MW 63,000/61,000 components, which are part of the membrane ATPase, can be iodinated from both membrane surfaces. This would suggest that these are transmembrane proteins. However, the major portion of all the proteins in this membrane were inaccessible to lactoperoxidase at either membrane surface.