[125I]diiodofluorescein isothiocyanate: Its synthesis and use as a reagent for labeling proteins and cells to high specific radioactivity

We have synthesized a radioactive derivative of fluorescein isothiocyanate (PITC) by lactoperoxidase-catalyzed iodination of fluorescein amine using ¹²⁵I. The iodinated amine was purified by thin-layer chromatography and converted to the isothiocyanate by reaction with thiophosgene. The product was inferred to be the diiodo derivative of FITC by comparing its absorbance and fluorescence emission spectra with those of known standards. This reagent, [¹²⁵I]diI-FITC, shares many of the useful features of its congener, FITC. Specifically, it may be used to label under mild conditions of temperature and pH, and it is chemically stable. When erythrocytes were labeled with [¹²⁵I]diI-FITC, radioactivity was found principally in a major exposed protein of the cell surface, and very little hemoglobin was labeled. [¹²⁵I]diI-FITC may prove generally useful as a means of labeling proteins and cell surfaces to high specific radioactivity.     

Comparative reactivities of 125I-secretin and 125I-minus6-tyrosyl secretin with guinea pig and rabbit anti-secretin sera.

Since secretin contains only an N-terminal histidyl and no tyrosyl residue, a synthetic secretin has been commercially prepared containing tyrosine in place of phenylalanine to facilitate the preparation of a radioiodine labeled tracer. We have found that although the rate of iodination of 6-Tyr-secretin is more rapid than that of secretin, the efficiency of iodination is not greatly increased and the shelf-life of the labeled product is not prolonged. The striking disadvantage of the use of ¹²⁵I-6-Tyr-secretin as a tracer in radioimmunoassay is its diminished immunoreactivity with several guinea pig and rabbit antisera compared to ¹²⁵I-secretin.     

Binding of vasoactive intestinal peptide and its stimulation of adenylate cyclase through two classes of receptors in rat liver membranes effects of 12 secretin analogues and 2 secretin fragments

1. Vasoactive intestinal peptide (VIP) receptors were identified in crude rat hepatic membranes by ¹²⁵I-labelled VIP binding and by the ability of VIP to stimulate adenylate cyclase activity. The specificity of these receptors was evaluated by the capacity of secretin, synthetic secretin analogues, and secretin fragments to inhibit ¹²⁵I-labelled VIP binding and to stimulate adenylate cyclase. 2. The results were compatible with the existence of two classes of VIP binding sites that could be distinguised according to their affinity for VIP and their specificity. High-affinity sites were more specific for VIP as secretin was 175 times less potent than VIP for recognition of these sites while being only 33 times less potent than VIP for recognition of low-affinity sites. 3. Secretin analogues, monosubstituted in position 2, 3, 4, or 6 were less potent than secretin for adenylate cyclase stimulation as well as for the recognition of the two classes of receptors. [Val⁵]Secretin was more potent than secretin and appeared definitely more VIP-like than secretin; [Ala⁴, Val⁵]secretin were equipotent to secretin. 4. The fragment secretin (7–27) was unable to recognize VIP receptors and to stimulate adenylate cyclase. The substituted fragment [Gln[⁹,Asn¹⁵]secretin (5–27) recognized these receptors with weak potency but could not activate the enzyme.     

Specificity of receptors to vasoactive intestinal peptide in guinea pig brain.

The specific binding of VIP to guinea pig brain membranes was tested by 1/ the ability of eight VIP and secretin analogs and fragments to inhibit the binding of ¹²⁵I-VIP and 2/ the capacity of the same peptides to influence basal and VIP-stimulated adenylate cyclase activities. Among all peptides tested, only VIP, secretin, [Val⁵] secretin, and [Gln⁹, Asn¹⁵] secretin (5–27) were able to inhibit ¹²⁵I-VIP binding. The adenylate cyclase activity was stimulated by VIP, secretin and [Val⁵] secretin. [Gln⁹, Asn¹⁵] secretin (5–27) although inactive per se was able to inhibit the VIP-stimulated adenylate cyclase activity competitively.     

Convenient methods to determine the specific radioactivity of (gamma-32P)ATP of high specific activity.

A satisfactory method for the determination of the specific activity of highly labeled [γ-³²P]ATP has not been reported previously. Yields of high specific activity ³²P labeled material usually are too small to be detected by ultraviolet spectrophotometry or phosphate analysis. Recent reports describing the assay of ATP by enzyme catalyzed phosphate transfer to ³H labeled glucose (1) or galactose (2) are not suitable for use with highly labeled ³²P material since the crossover into the ³H channel will greatly exceed the radioactivity of the ³H labeled phosphate acceptor. Recently Schendel and Wells reported the preparation of essentially carrier free [γ-³²P]ATP. They indicated, however, that the specific activity of the labeled product could not be determined by conventional methods (3). We have developed and now routinely use an expedient method for the determination of the specific activity of picomole quantities of highly labeled [γ-³²P]ATP. This procedure measures the phosphate transfer from [γ-³²P]ATP to oligothymidylic acid [dT(pT)₁₀] catalyzed by bacteriophage T4 induced polynucleotide kinase. The specific activity is determined by measuring the radioactivity present in d-³²pT(pT)₁₀, and can be verified by an isotope dilution method employing the same assay. Specific activities as high as 240 Ci/mmole have been determined.     

Identification of new collagen formation with 125I-labeled antibody in bovine pericardial tissue valves implanted in calves

Failure of bovine pericardial tissue valve used in young patients may be due to a slow rejection process. Polyclonal anticollagen (Type I) antibody (IgG) was made in rabbits and purified by protein A affinity column. Two milligrams of IgG was labeled with 2 mCi of ¹²⁵I by the Iodogen method. Free iodide was separated by G-10 column. Affinity of ¹²⁵I-IgG was checked by radioimmunoassay. Two hundred and fifty microcuries of ¹²⁵I-IgG was injected in calves immediately after tissue valve implantation, and the calves were killed 4 h post-injection. After harvesting the valve, each of the three leaflets was separated into four zones, and radioactivity in each section was mapped with a γ counter. The radioactivity in tissue valve section was compared to that of normal aortic valve. The sections of tissue valve retain five to ten times more ¹²⁵I-IgG than control aortic valve. Iodine-IgG thus provides a sensitive technique for determination of residual antigenicity in tissue valve.     

A method for measurement of the specific radioactivity of the choline moiety of choline phospholipids.

The specific radioactivity of a choline phospholipid has been determined by a double-isotope method. Purified phospholipid was hydrolyzed to release labeled choline, and choline kinase was employed to label the choline with ³²P from [γ-³²P]ATP. The double-labeled phosphorylcholine was purified by ion-exchange chromatography on QAE-Sephadex, and the specific radioactivity of the choline was calculated from the isotope ratio. The method is sensitive, requiring only 5 nmol of choline with a specific radioactivity of 1 μCl/μmol, and the chromatographic isolation of phosphorylcholine is simple and reproducible.     

Preparation and characterization of biologically active iodo- and [3H]secretin

Synthetic secretin has been iodinated at the N-terminal histidine, leading to an almost 100% yield of mono- and diiodo-secretin (“lodo-secretin”). The catalytic exchange of iodine against tritium results in the preparation of secretin labeled with tritium mainly at the histidine residue (7 Ci/mmol). Iodo-secretin and [³H]secretin have the same potency in stimulating pancreatic adenylate cyclase as secretin, but the apparent affinity of [³H]secretin for this enzyme is twice as high as for iodo-secretin. [³H]Secretin binds rapidly to pancreatic plasma membranes. Adding excess unlabeled secretin reduces the tracer binding by about 70%.     

Simultaneous radioimmunoassay of secretin and gastrin

Radioimmunoassay techniques for the measurement of gastrointestinal hormones have been modified to allow simultaneous assay of both secretin and gastrin in the same plasma samples. The system employs antibodies with high specificity for these two hormones and both ¹²⁵I-labeled secretin and ¹³¹I-labeled gastrin as markers. Separation of the free from the bound form of the hormones was achieved by plasma- and dextran-coated charcoal. The unique separation method has been found to be independent of the uniformity of plasma samples. The sensitivity and the reliability of the simultaneous assays were comparable to those of the single assays. The fasting levels of human plasma secretin and gastrin obtained by the simultaneous assay were 62.9 ± 3.6 and 43.5 ± 2.8 pg/ml respectively.     

Radioiodination of nonionic detergents of the alkyl-phenol class: Triton X-100

A method is described by which nonionic detergents of the alkyl-phenol class can be labeled with ¹²⁵I. The detergent is labeled to a high specific activity, which provides a sensitive method for the detection of detergent molecules bound to proteins, and, in addition, may provide a method by which proteins may be labeled indirectly.     

Interaction between monensin and lysosomotropic amines in the regulation of the processing of epidermal growth factor by BALB/c 3T3 cells

Monensin, like the lysosomotropic amines Chloroquine and methylamine, caused a large accumulation of ¹²⁵I-EGF in BALB/c-3T3 cells that was due to specific increases in the amount of intracellular intact hormone. However using a pulse-chase paradigm of ¹²⁵I-EGF accumulation, marked differences were observed between monensin and the amines. When EGF was accumulated in the presence of monensin, there was a gradual loss of cell-bound radioactivity during a chase in the absence of the drug, and the labeled material recovered in. the medium primarily consisted of degraded hormone. The continued presence of monensin in the chase medium substantively prevented the loss of cell bound material, and what little was recovered in the medium consisted of intact ¹²⁵I-EGF. In contrast, when ¹²⁵I-EGF was accumulated in the presence of methylamine, predominately intact peptide was lost from the cells at a relatively high rate during the chase whether or not methylamine remained in the medium. When monensin was present in the chase medium following accumulation in the presence of either Chloroquine or methylamine, the loss of intracellular ¹²⁵I-EGF was essentially blocked.     

CELL LOSS FROM SEVERAL TYPES OF SOLID MURTNE TUMOUR: COMPARISON OF 125I-IODODEOXYURIDINE AND TRITIATED THYMIDINE METHODS

The method of measuring tumour cell loss rates in situ following radioactivity loss after a single injection of ¹²⁵I-iododeoxyurudine (¹²⁵I-UdR) was tested for its accuracy in five different types of murine tumour. To achieve this the method was compared with two others: (1) using ¹²⁵I-UdR, but excising tumours before the radioactivity determinations, with or without extracting DNA; (2) using tritiated thymidine and autoradiography. A third method was used on three of the tumours, in which ¹²⁵I-UdR-labelled tumours were grown in unlabelled hosts, followed by whole body counting of the tumour-bearing mice. In two of the tumours an increase was observed in total tumour radioactivity with time after ¹²⁵I-UdR injection. This prevented the estimation of cell loss parameters in these tumours. Approximately half the increase was due to reutilization of ¹²⁵I-UdR supplied from tissues within the mouse; approximately a third to an influx of labelled inflammatory cells (probably in response to infection accompanying ulceration of overlying skin); and the remainder to an increase in non-DNA radioactivity. In these tumours cell loss rates could be obtained from the whole body counting technique in which influxes of labelled cells and reutilizable radioactivity were eliminated. A comparison of either ¹²⁵I-UdR technique with the ³H-TdR technique showed good agreement of the cell loss factors for the low cell loss tumours. However, for tumours with high cell loss factors the ¹²⁵I-UdR technique gave lower values for cell loss. This implied that reutilization of ¹²⁵I-UdR within the tumour (i.e. from internal, not external sources) occurred in the high cell loss tumours. It is concluded that equating radioactivity loss with cell loss after an injection of ¹²⁵I-UdR is reasonable for some tumours, but will result in significant underestimates in others. For high cell loss tumours the ³H-TdR technique will give the     

Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells.

The protein neurotoxin II from the venom of the scorpion $\text{Androctonus}\text{australis}$ Hector was labeled with ¹²⁵I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non intereacting binding sites of high affinity (K_D = 0.3 – 0.6 nM) and low capacity (4000 – 8000 sites/cell) to electrically excitable neuroblastoma cells. Relation of these sites to the action potential Na⁺ channel is derived from identical concentration dependence of scorpion toxin binding and increase in duration and amplitude of action potential. The protein neurotoxin II from the sea anemone $\text{Anemona sulcata}$ also affects the closing of the action potential Na⁺ ionophore in nerve axons. The unlabelled sea anemone toxin modifies ¹²⁵I-labeled scorpion toxin II binding to neuroblastoma cells by increasing the apparent K_D for labeled scorpion toxin without modification of the number of binding sites. It is concluded that both $\text{Androctonus}$ scorpion toxin II and $\text{Anemona}$ sea anemone toxin II interact competitively with a regulatory component of the action potential Na⁺ channel.     

CELL LOSS AND INFLUX OF LABELED HOST CELLS IN THREE TRANSPLANTABLE MOUSE TUMORS USING [125I]UdR RELEASE

The [¹²⁵I]UdR loss technique was used to estimate cell loss from RIF-1, EMT6 and KHJJ tumors in order to determine the length of the delay between labeling and the beginning of the loss of labeled cells, and also to calculate a value for ø, the cell loss factor. To determine the importance of reutilization of label released from the gut and/or the influx of labeled host cells, the blood flow to some tumors was occluded during and for 30 min after injection of the label. Relatively small amounts of radioactivity entered occluded RIF-1 tumors during 9 days after injection of [¹²⁵I]UdR, indicating that reutilization of systemic label and influx of labeled host cells are not significant in this system. In contrast, substantial amounts of radioactivity entered occluded EMT6 and KHJJ tumors, reaching 40% of the total activity in non-occluded tumors during 6 days following injection. After corrections were made for this influx of label, the [¹²⁵I]UdR loss curves from RIF-1 and EMT6 tumors were essentially exponential from the first day following injection of label. This was interpreted as indicating the loss of proliferating as well as non-proliferating cells from both tumors. The cell loss factor derived from the [¹²⁵I]UdR loss curves corrected for influx appeared to agree well with published values derived from analysis of percent labeled mitoses curves. In contrast, the corrected [¹²⁵I]UdR loss curves from KHJJ tumors showed that loss of activity began three days after injection of label, indicating that primarily nonproliferating cells are lost from this tumor.     

Localization by whole-body autoradiography of intact and fragmented radiolabeled antibodies in a metastatic human colonic cancer model

In this report, we have employed macroautoradiography to compare the tumor targeting of ¹²⁵I-labeled anti-carcinoembryonic antigen (CEA) MAb (NP-4) to ¹²⁵I-labeled anti-colon-specific antigen-p (CSAp) MAb (Mu-9) and their labeled F(ab′)₂ and Fab′ fragments, in nude mice each bearing large dorsal human colonic tumor xenografts, and small nodular tumors in the liver and lungs. Using intact MAbs (NP-4 and Mu-9), clearance of background radioactivity was delayed to 3–7 days post-treatment. Treatment with F(ab′)₂ and Fab′ fragments of both NP-4 and Mu-9 MAbs, however, promoted clearance of background ¹²⁵I-radioactivity which was well advanced by 6–24 h and complete by 24–48 h after injection. Localization of ¹²⁵I-radioactivity in large and micrometastatic tumor perimeters was the most characteristic uptake pattern observed for both intact and fragmented MAbs. Qualitative analysis of macroautoradiographic images and quantitative densitometry indicated that the higher tumor-to-blood ratios achieved with labeled F(ab′)₂ and Fab′ fragments at early time points, compared to labeled whole immunoglobulin, appeared to be more a function of rapid plasma clearance, tumor mass, location of xenografts and specific tumor growth patterns than increased tumor penetrance by lower molecular weight univalent and bivalent immune fragments.     

Subcellular distribution of intraportally injected 125I-labeled insulin in rat liver

Time course studies revealed that at 30 s after intraportal injection of 200 μU of ¹²⁵I-labeled insulin per 100 g rat 47.9 ± 2.8% of the injected radioactivity was recovered from the liver homogenate by precipitation with trichloroacetic acid. Trichloroacetic acid precipitable radioactivity declined to very low levels during the next 30 min whereas trichloroacetic acid soluble radioactivity reached a peak value of 9.56 ± 1.9% at 5 min and declined gradually thereafter. At 30 s mean peak accumulations ±SE of 6.83 ± 0.42, 5.06 ± 0.27, 14.90 ± 1.85, and 3.58 ± 0.58% of injected radioactivity were recovered in trichloroacetic acid precipitates from the 700g (nuclei + debris), 10,000g (mitochondria + lysosome), 105,000g (microsomes), and supernatant (cytosol) subfractions, respectively. Mean peak values of 0.72 ± 0.08, 0.12 ± 0.02, and 1.11 ± 0.16% of injected radioactivity were recovered in the partially purified mitochondrial fraction, purified nuclei, and plasma membranes, respectively, as trichloroacetic acid precipitable material. Most of the trichloroacetic acid precipitable activities in the subfractions were immunoprecipitable. Trichloroacetic acid soluble radioactivity was found mainly in the cytosol and microsomal fractions. Peak specific activity (percentage of injected dose/mg protein × 10^?3) was highest in the microsomes, intermediate in the plasma membranes, and very low in the purified nuclei and partially purified mitochondrial fraction. The specific activity of the microsomes remained at or near peak levels for 5 min after ¹²⁵I-labeled insulin injection and then declined, whereas specific activity of the plasma membranes dropped precipitously to 25% of peak values at 5 min. Sephadex gel filtration of the radioactivity in the deoxycholate soluble fraction of microsomes at 5 min after ¹²⁵I-labeled insulin injection resulted in the elution of a major peak (Peak I) in the region of ¹²⁵I-labeled insulin and a minor peak (Peak II) in the region of the labeled A and B chains. Incubation of the fraction for 30 min at 37 °C with 3 mm reduced glutathione and 15 mm EDTA resulted in a reciprocal fall in Peak I and rise in Peak II. The data suggest that intraportally injected ¹²⁵I-labeled insulin is rapidly internalized and concentrated in the rat liver microsomes. The time courses of appearance and disappearance of trichloroacetic acid precipitable radioactivity in plasma membrane and microsomes further suggest, although do not prove, that insulin binds to plasma membranes before it is internalized. They also provide presumptive evidence suggesting that the sequential degradative pathway is operative in vivo.     

Internalization of I-human choriogonadotropin in bovine luteal slices : A biochemical study

Various intracellular organelles as well as outer cell membranes of bovine corpora lutea intrinsically contain gonadotropin receptors (Rao et al., J biol chem 256 (1981) 2628 [5]). In order to investigate whether exogenously added human Choriogonadotropin (hCG) can internalize and bind to the intracellular sites, bovine luteal slices that had been carefully checked with respect to structural and functional integrity were incubated with 0.1 nM ¹²⁵I-hCG. Following incubation, specific radioactivity was found to be associated with various intracellular organelles, but not with cytosol. The order of radioactivity uptake by subcellular organelles following a 2-h incubation was: Golgi medium > Golgi heavy > Golgi light > plasma MEMBRANES = rough endoplasmic reticulum > mitochondria-lysosomes> nuclei. The 5′-nucleotidase activity and electron microscopic examination of the fractions revealed that the presence of radioactivity in the intracellular organelles cannot be attributed solely to plasma membrane contamination.The internalization and intracellular binding of ¹²⁵I-hCG was time and temperature-dependent. Only excess unlabeled hCG and hLH (but not hCG subunits, FSH and PRL) competed with ¹²⁵I-hCG for internalization in luteal slices. Very little or no ¹²⁵I-hCG added was internalized in liver or kidney slices; luteal, liver and kidney slices accumulated neither ¹²⁵I-BSA nor ¹²⁵I.The radioactivity eluted from various luteal subcellular organelles was able to rebind to fresh corresponding organelles and came off Sepharose 6B columns in a position corresponding to native ¹²⁵I-hCG. The gel filtration profile of detergent-solubilized radioactivity revealed that ¹²⁵I-hCG was macromolecular bound. The degraded and altered ¹²⁵I-hCG was found in the incubation media.     

Lactoperoxidase-catalyzed iodination of surface membrane lipids

Lactoperoxidase-catalyzed iodination of intact cells, is known to label predominantly, if not exclusively, the exposed tyrosine residues of cell surface proteins. The present study demonstrates that during this iodination process surface membrane lipids are also iodinated through an enzyme-dependent step. Phosphatidylcholine, cholesterol-phosphatidylcholine liposomes and confluent secondary cultures of chick embryo cells were iodinated by the lactoperoxidase-glucose oxidase-glucose [¹²⁵I] procedure. Liposomes were efficiently labeled. In the cells, 20–30% of the radioactivity was found in proteins and 20–30% in the lipids. Both neutral and polar lipids were found to bind [¹²⁵I] covalently. Controls in which lactoperoxidase was omitted showed < 6% of the radioactivity found in liposomes or cells labeled with the enzyme.     

Analysis of lipid and protein components of ejaculated bull sperm surface and seminal plasma

Surface components of ejaculated bull sperm were radiolabeled by enzymatic iodination with lactoperoxidase and Na¹²⁵I. The sperm were lysed and the labeled components analyzed on SDS-7.5% polyacrylamide gels. Electrophoresis of solubilized radioactivity resolved six components with approximate molecular weights of 77, 61, 44, 36, 24, and 15 kilodaltons. To identify components that might be adsorbed to the bull sperm surface from seminal secretions, seminal plasma was labeled. Electrophoresis of labeled seminal plasma resolved four components with approximate molecular weights of 74, 33, 24, and 15 kilodaltons, each of which comigrated with a labeled sperm surface component. To identify the chemical composition of the radiolabeled components, labeled sperm surface and labeled seminal plasma were submitted to isopycnic density gradient centrifugation in cesium chloride. The ¹²⁵I incorporated into bull sperm surface separated into two discrete areas of radioactivity, one having a density characteristic of protein and the other, of lipid. Iodinated seminal plasma banded in one discrete area that had a density characteristic of protein. Electrophoretic analysis of each area of radioactivity recovered from the gradients demonstrated that five of the six sperm surface and all of the seminal plasma components were in the protein fractions. The 15-kilodalton sperm surface component banded as a lipid, whereas the 15-kilodalton seminal plasma componènt banded as a protein.