O-(4-Diazo-3,5-di[125I]iodobenzoyl)sucrose, a novel radioactive label for determining organ sites of catabolism of plasma proteins.

A method is described for radiolabelling proteins with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose (DD125IBS). When proteins so labelled were degraded within lysosomes, the radioactive fragments were largely retained within the organelle. High specific radioactivities were obtained without changing the properties of the protein. The validity of the method was demonstrated in vivo in rats using the short-lived protein lactate dehydrogenase, isoenzyme M4, and the long-lived protein bovine serum albumin. Derivatization with DD125IBS did not alter the clearance of either protein. Uptake of DD125IBS-labelled lactate dehydrogenase, isoenzyme M4, by liver and spleen of rats was determined. Radioactivity in these tissues increased up to about 2 h after injection (at this time the protein has been almost completely cleared from the blood) and subsequently declined with a half-life of approx. 20 h. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. The results of these studies establish that DD125IBS covalently coupled to plasma proteins should be a useful radioactive tracer for identifying the tissue and cellular sites of catabolism of relatively long-lived circulating proteins.     

Application of O-(4-diazo-3-[125I]iodobenzoyl)sucrose for the detection of the catabolic sites of low density lipoprotein

The sites of degradation of human low density lipoprotein (LDL), are analyzed using the novel labelling compound O-(4-diazo-3-[125I]iodobenzoyl)sucrose (D125IBS). The decay from rat serum of D125IBS-labelled LDL is identical to the serum decay of conventionally iodinated (ICI method) LDL. The radioactivity derived from D125IBS-labelled LDL accumulates predominantly in the liver after intravenous injection and the hepatic radioactivity remains associated with the lysosomal compartment for an extended period of time, when compared to the radioactivity derived from conventionally iodinated LDL. It is concluded that the D125IBS labelling procedure is an interesting new tool to study the sites of catabolism of serum lipoproteins.     

Chemical modification of ribonucleosides with N-acetyl-3,5-di[125I]iodotyrosyl hydrazide

Several 3,5-diiodotryrosyl derivatives have been synthesized by both sodium iodideiodine and the sodium iodide-iodic acid methods. Conditions optimizing yield and purity of the product have been established for the latter reaction. Under those conditions, treatment of N-acetyl-tyrosyl ethyl ester with sodium [¹²⁵I]iodide and iodic acid gave N-acetyl-3,5-di[¹²⁵I]iodotyrosyl ethyl ester (ADITEE) with high specific activity. Hydrazination of [¹²⁵I]ADITEE produces N-acetyl-3,5-di[¹²⁵I]iodotyrosyl hydrazide. This hydrazide has been successfully used to modify four different ribonucleoside dialdehydes.     

Proteins of the kidney microvillar membrane. Asymmetric labelling of the membrane by lactoperoxidase-catalysed radioiodination and by photolysis of 3,5-di[125I]iodo-4-azidobenzenesulphonate.

Two methods were used to label pig kidney microvillar membrane proteins from the luminal and cytoplasmic surfaces of closed membrane vesicles. The first method was lactoperoxidase-catalysed radioiodination. The enzyme reagents, lactoperoxidase and glucose oxidase, were positioned inside the vesicles before sealing or externally after sealing, iodination being initiated by the subsequent addition of glucose and 125I-. After resolution of the labelled proteins by electrophoresis in the presence of dodecyl sulphate, asymmetric labelling patterns on radioautographs were observed. However, the major disadvantage of this method is the high degree of intramembrane labelling of the fatty acid chains of membrane lipids, a reaction that undermines any conclusions about the location of the label in that region of the protein supposedly exposed at the surface of the membrane. The second method overcame this disadvantage. A new hydrophilic photoreagent, 3,5-di[125I]iodo-4-azidobenzesulphonate, was synthesized via the intermediate, diazotized 3,5-di[125I]iodosulphanilic acid. It was transported by a Na+-dependent system into microvillar vesicles, thus permitting labelling from either side of the membrane when the vesicles were photolysed. The labelling of membrane lipids was less than with the first method and was essentially confined to the polar headgroups. The activity of several microvillar peptidases survived the labelling reaction and they could be identified in the immunoprecipitates after resolution of the detergent-solubilized membrane proteins by crossed-immunoelectrophoresis. Treatment with papain converted the detergent-solubilized form of susceptible enzymes into the proteinase-solubilized form, which lacked the intramembrane domain and any portion exposed at the cytoplasmic surface. Radioautography established that aminopeptidases M and A, dipeptidyl peptidase IV and neutral endopeptidase were transmembrane proteins. This novel approach to the investigation of membrane topology may be applicable to other complex membranes.     

Astatine-211 labeling of an antimelanoma antibody and its Fab fragment using N-succinimidyl p-astatobenzoate: comparisons in vivo with the p-[125I]iodobenzoyl conjugate

Astatine-211 labeling of an antimelanoma antibody, NR-ML-05, and its Fab fragment with N-succinimidyl p-[211At]astatobenzoate (2a) has been described. Preparation of the astatinated intermediate 2a was accomplished by distilling astatine-211 from an irradiated bismuth target directly into a reaction mixture containing an organometallic compound, N-succinimidyl p-(tri-n-butylstannyl)benzoate (1), and an oxidant, N-chlorosuccinimide, in 5% HOAc/MeOH. Trapping of distilled astatine as 2a was found to be efficient, resulting in 70-90% yields based on the amount of astatine-211 in the reaction mixture. The dry distillation technique employed gave recoveries of astatine-211 which ranged from 20% to 75%. Conjugation of 2a to NR-ML-05 and its Fab fragment was accomplished in 40-60% yields. The [211At]astatobenzoyl-conjugated antibodies were found to be stable in vitro when challenged by strong denaturants and nucleophilic reagents. Coinjected dual-labeled studies of the 2a astatinated antibodies and the same antibodies labeled with N-succinimidyl p-[125I]iodobenzoate (2b) in athymic mice bearing the human tumor xenograft A375 Met/Mix demonstrated that both radiolabeled antibodies had equivalent tumor localization. Data from the dual-labeled biodistribution of the intact antibody suggests that the astatine is stably attached. Data from the dual-labeled Fab fragment suggests that a portion of the astatine label is released as astatide, either from the astatinated Fab or from a catabolite.     

Adsorptive pinocytosis of 125I-labelled lactate dehydrogenase isoenzymes H4 and M4 by rat yolk sacs incubated in vitro.

The pinocytic uptake of 125I-labelled porcine lactate dehydrogenase isoenzymes H4 and M4 by 17.5-day rat visceral yolk sac incubated in vitro was saturable and binding obeyed Michaelis-Menten kinetics. The uptake characteristics of the two isoenzymes were very similar. For the H4 and the M4 isoenzymes, the dissociation constants of the protein-plasma-membrane complex were 0.62 microM and 0.84 microM respectively, and the maximum rates of uptake 0.13 and 0.26 nmol/mg of yolk-sac protein per h respectively. These findings contrast with those from studies in vivo, which show the M4 form is taken up by rat liver sinusoidal cells at a much higher rate than the H4 form, and point to different recognition systems for the adsorptive pinocytosis of simple non-conjugate proteins in yolk-sac epithelial cells and liver sinusoidal cells. Competition experiments indicate that binding of the H4 isoenzyme to the yolk-sac cells is restricted to hydrophobic interactions, whereas the binding of the M4 isoenzyme involves hydrophobic as well as positively charged sites on the protein molecules.     

Comparative affinities of adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) for [125I] AM and [125I] CGRP specific binding sites in porcine tissues.

We have investigated the binding characteristics of rat [125I] adrenomedullin (AM) and human [125I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [125I] rat AM and [125I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [125I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [125I] rat AM binding to various tissues were rat AM > or = human AM > or = human AM(22-52) > h alpha CGRP > or = h alpha CGRP(8-37) > sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20-300 fold more potent than hAM (22-52). When the same experiments were performed using [125I] h alpha CGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > h alpha CGRP > h alpha CGRP(8-37) in most of the tissues except in spleen and liver where h alpha CGRP was the most potent ligand. In lung, h alpha CGRP was almost as potent as rAM and hAM in displacing [125I] h alpha CGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.     

Preparation and in vivo evaluation of an N-(p-[125I]iodophenethyl)maleimide-antibody conjugate.

Protein sulfhydryl reactive N-(4-[125I]iodophenethyl)maleimide (IPEM, 5) was obtained from N-[4-(tri-n-butylstannyl)phenethyl]maleimide in 59-100% radiochemical yield. Conjugation of 5 to NR-ML-05 Fab, a murine anti-melanoma antibody Fab fragment that had been previously reduced with dithiothreitol (DTT), was effected in an average of 85% yield. Results from in vitro chemical challenges and serum stability studies on the IPEM conjugate of NR-ML-05 Fab (6) indicated a stable covalent attachment of the radioiodine. A biodistribution study of the IPEM conjugate in tumor-bearing athymic nude mice showed lack of significant accumulation of radioiodine in the thyroid and stomach which was an indication of in vivo stability. The observed uptake in tumor was consistent with that obtained for Chloramine-T- or p-iodobenzoate-labeled NR-ML-05 Fab conjugates.     

Kinetic mechanism of guinea-pig skeletal muscle lactate dehydrogenase (M4) with oxaloacetate-NADH and pyruvate-NADH as substrates

• 1.1. Guinea-pig skeletal muscle lactate dehydrogenase M₄ isoenzyme, with pyruvate and NADH as substrates, is adapted to an ordered bi-bi ternary complex mechanism at pH 7.0.
• 2.2. In the same conditions, the kinetic mechanism of the reaction, with oxaloacetate and NADH as substrates, is of the rapid equilibrium ordered bi-bi ternary complex type; NADH is the first substrate in the reaction sequence.

     

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors.

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.     

Fluid endocytosis by rat liver and spleen. Experiments with 125I-labelled poly(vinylpyrrolidone) in vivo.

1. Rates of fluid endocytosis of rat liver, spleen, hepatocytes and sinusoidal liver cells have been determined, by using 125I-labelled poly(vinylpyrrolidone) as marker. Poly(vinylpyrrolidone) was injected intravenously into rats, and plasma clearance and uptake by liver and spleen were estimated. From these data, rates of fluid endocytosis of 1.2 and 1.8 ml of plasma/g of protein per day were calculated for liver and spleen respectively. Essentially the same results were found in nephrectomized rats. 2. Hepatocytes and sinusoidal cells were separately isolated by the collagenase/Pronase method, and sinusoidal cells were further fractionated by centrifugal elutriation. Hepatocytes, sinusoidal cells, Kupffer cells and endothelial cells showed rates of fluid endocytosis of 0.96, 9.0, 19 and 13 ml of plasma/g of cell protein per day respectively. Total-body X-irradiation did not influence uptake of poly(vinylpyrrolidone) by spleen, indicating that spleen lymphocytes are not significantly involved in fluid endocytosis. 3. For liver a rate constant of exocytosis of 5% per day was found, whereas for spleen no significant loss of accumulated label could be demonstrated during a 21-day period. 4. Distribution of label over a great number of organs and tissues was measured 9 days after the injection. Liver, skin, bone and muscle together contained about 70% of the label present in the carcass; only spleen and lymph nodes contained more label per g fresh weight of tissue than liver.     

Dissociative binding of alpha- and beta-sulphoxides of biotinylamidoethyl-3-(4-hydroxy-3-[125I]iodophenyl)propionamide to avidin.

Previously we reported the dissociative binding of biotinylamidoethyl-3-(4-hydroxy-3-[125I]iodophenyl)propionamide to avidin [Garlick & Giese (1988) J. Biol. Chem. 263, 210-215]. In the present paper we report the corresponding binding of the alpha- and beta-sulphoxides of this parent compound to avidin. The 1:1 complex (obtained with avidin in excess) of the alpha-sulphoxide derivative with avidin has a dissociation half-life (t1/2) of 25 days, only 1.6 times as fast as the parent compound (t1/2 41 days). However, the corresponding beta-sulphoxide dissociates 446 times faster (t1/2 0.092 day) than the parent compound, this apparently being due to a steric effect. The alpha-sulphoxide is attractive as a tracer reagent to facilitate studies and applications of the avidin-biotin system.     

Concentration of [16 alpha-125I]iodoestradiol in human ovarian tumors in vivo and correlation with estrogen receptor content

The gamma emitting estrogen [16 alpha-125I]iodoestradiol was administered to 11 patients with ovarian cancer and 1 patient with endometrial cancer. At specific times after the administration of the tracer, portions of the tumor and of control tissues, fat and muscle, were removed and counted. The amount of radioactivity in these tissues was compared to the cytosolic estrogen receptor content of the tumor, measured by Sephadex LH-20 gel filtration, in biopsy specimens taken before the injection of the tracer. There was a strong correlation (p less than 0.005) between the estrogen receptor concentration in the biopsied tumor and the amount of radioactivity in the tumor. There was no correlation between the isotope in the muscle and the tumor receptor, nor between the radioactivity in the tumor and that in fat or muscle. As would be expected for a steroid receptor mediated process, the bulk of the total tissue radioactivity was present in the nuclear compartment of the tumors. This pattern was not observed in the muscle. Furthermore, the nuclear radioactivity in the tumors was positively correlated with the cytosolic estrogen receptor content. These experiments demonstrate that under in vivo conditions this gamma emitting estrogen is concentrated in tumors in a manner that is dependent upon the estrogen receptor. It was also found that the concentrations of radioactivity in the blood were high, producing low tumor to blood ratios. The blood level of isotope was not due to the presence of the unmetabolized steroid, which disappeared from blood rapidly, but was caused by circulating metabolites of the injected steroid. Since the concentration of the isotope in the tumor was dependent upon the estrogen receptor level, it would appear from these experiments that it is theoretically possible to use such compounds to image and monitor tumors that contain estrogen receptors. However, rapid metabolism would seem to preclude the use of 16 alpha-iodoestradiol itself for this purpose. These studies point to the possibility that the synthesis of analogs of 16 alpha-iodoestradiol, sterically protected against inactivation by rapid metabolism, may lead to a radiopharmaceutical agent that would be useful for imaging and monitoring estrogen receptor containing tumors.     

Acetylcholine receptors in normal and denervated rat diaphragm muscle. I. Purification and interaction with [125I]-alpha-bungarotoxin.

Acetylcholine receptors have been purified from junctional regions of normal rat diaphragm muscle and from extrajunctional regions of denervated diaphragm. The reaction of purified receptors with [122I]-alpha-bungarotoxin has been investigated by kinetic methods. The toxin-receptor complexes dissociated in a biphasic manner at 35 degrees with a rapidly dissociating component (t1/2 = 4 hr) and a slowly dissociating component (t1/2 is greater than or equal to 100 hr). The association reaction between toxin and receptor did not obey simple second-order kinetics but could be analyzed in terms of two classes of binding sites corresponding to the two rates of dissociation. This treatment of the data allowed derivation of association rate constants for the two sites. Value obtained for the dissociation constants were 3.7 times 10(-10) and less than or equal to 0.4 times 10(-10) M for the junctional receptor and 1.7 times 10(-10) and is less than or equal to 0.2 times 10(-10) M for the extrajunctional receptor. In each case it is the more tightly binding component that associates and dissociates more slowly. Receptors present in crude preparations were comparable to purified receptors in their reaction with [125I-alpha-bungarotoxin. The validity of the two site model is discussed in relation to the kinetic studies.     

The pinocytosis of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by rat yolk sac cultured in vitro.

The rates of uptake of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by 17.5-day rat yolk sac cultured in vitro were studied. Over a 6.5h period each substrate was accumulated at a constant and reproducible rate of approx. 2microliter/h per mg of protein. After accumulation in vitro, the three substances were released from the tissue into substrate-free medium at low rates. Sucrose present in the medium at concentrations up to 10 mg/ml was without effect on the accumulation of either [14C]sucrose or 125I-labelled poly(vinylpyrrolidone), but at higher concentrations inhibited the uptake of both substrates. Some batches of colloidal [198Au]gold had a significantly higher Endocytic Index (up to 5 microliter/h per mg of protein). The Endocytic Index of such a batch decreased with increasing substrate concentration, but colloidal gold did not decrease the Endocytic Index of 125I-labelled poly(vinylpyrrolidone). It is concluded that the three substrates enter the yolk sac by pinocytosis in the liquid phase. Those batches of colloidal [198Au]gold with higher Endocytic Indices are considered to enter also by adsorption on membrane binding-sites.     

Double labeling with [3H]thymidine and [125I]iododeoxyuridine as a method for determining the fate of injected DNA and cells in vivo

Mice were injected intravenously and intraperitoneally with preparations of intestinal nucleoprotein, spleen nuclei, mouse thymus cells, or human kidney T cells whose DNA had been labeled with both [3H]thymidine (TdR) and [125I]-iododeoxyuridine (IUdR). Since free TdR is reutilized more efficiently than free IUdR produced by enzymic hydrolysis of the exogenous DNA, the ratio of [3H]TdR/[125I]IUdR in the DNA fraction of the tissues of the recipient mice provides a measure of the amount of intact exogenous DNA in the tissue. In most instances, the doubly labeled exogenous DNA was almost completely hydrolyzed within 1 day injection, but survival of the DNA from whole cells could be demonstrated in some cases.     

The in vitro dissociation kinetics of (R,R)-[125I]4IQNB is reflected in the in vivo washout of the radioligand from rat brain.

We have determined the kinetics of dissociation of (R)-3-Quinuclidinyl (R)-4-[125I]Iodobenzilate ((R,R)-[125I]4IQNB) from muscarinic acetylcholine receptor preparations from the cortex, hippocampus, caudate/putamen, thalamus, pons and colliculate bodies. The dissociation curves are well described by a biexponential function and are consistent with subtype selectivity favoring slow dissociation from the M1, M3, and M4 receptors with a 20-fold faster dissociation rate for the M2 receptor. Following intravenous injection, (R,R)-[125I]4IQNB binds to receptor in the rat brain in concentrations which reflect the receptor concentration present in a structure. We determined the extent of radioligand present at two times, 2 and 24 hrs, as an indication of the relative proportions of m-AChR which exhibits rapid vs. slow dissociation of (R,R)-[125I]4IQNB. A good correlation between in vitro and in vivo results suggests that the relative populations of receptor subtypes can be imaged using in vivo pharmacokinetics of (R,R)-[125I]4IQNB.     

Preparation of the N-(4'-hydroxy-[3'-125I]iodophenethyl)-6-(4-O-diethylstilbestryl)hexanam ide for diethylstilbestrol radioimmunoassays.

The synthesis of a radioiodinated diethylstilbestrol (DES) derivative is described. This derivative was prepared by coupling the previously synthesized active ester of 6-(4-O-diethylstilbestryl)hexanoic acid with mono-[125I]iodotyramine in dry tetrahydrofuran (20 to 22 C, 16 hours). The mono-[125I]iodotyramine was prepared using a chloramine-T method and purified by paper electrophoresis. The final product, N-(4'-hydroxy-[3'-125I]iodophenethyl)-6-(4-O- diethylstilbestryl)hexanamide, was separated by thin-layer chromatography (cyclohexane/ethanol/NH4OH 2.5 N/acetone; 40:50:5:20, v/v/v/v); it was stable for 2 months in ethanol at 4 C and had a specific activity higher than 540 Ci/mmol. The [125I]DES amide synthesized was found to retain the immunoreactivity of DES, since it competed with [3H]DES or DES in an in vitro radioimmunoassay system for the binding sites of a rabbit anti-DES antibody; thus, it seems to be capable of replacing the tritiated tracer used so far in DES radioimmunoassays.