N-chloroacetyl-[125I]iodotyramine: an alkylating agent with high specific activity

The preparation of iodinated N-chloroacetyltyramine and its evaluation as a specific sulfhydryl reagent are described. N-Chloroacetyltyramine was synthesized by a carbodiimide-mediated condensation of chloro- or iodoacetic acid and tyramine·HCL, and the crystalline product was iodinated in a reaction with chloramine T to yield either a 3,5-[¹²⁵I]diiodotyramine derivative, or a trace-iodinated product when carrier-free ¹²⁵I was employed. These iodinated derivatives react specifically with sulfhydryl groups, as judged by their ability to label reduced but not unreduced ribonuclease A and immunoglobulin E. Specific activities of 1 Ci/mmole in ¹²⁵I or ¹³¹I can be readily achieved with both the diiodinated and trace-iodinated (carrier-free) derivatives, and the specific activity of the former can be used directly to quantitate sulfhydryl groups in subnanomolar quantities of protein. N-Chloroacetyl ¹²⁵I-labeled tyramine prepared by trace iodination with carrier-free ¹²⁵I is more useful when very high specific activities (100–1000 mCi/μmol) are required. The utility of these reagents is discussed.     

A functional hybrid ribosome binding site in tryptophan operon messenger RNA of Escherichia coli

We present evidence that repair of DNA damage induced by decay of incorporated ¹²⁵I after replication of the labeled duplex of Escherichia coli requires the recA⁺ gene function. Furthermore, only about half of the cells survive after label segregation even when that repair function is present. Our results support the possibility that repair of ¹²⁵I decay-induced lesions is asymmetric, being limited to damage initiated in only one of the two strands of the DNA duplex.     

Synthesis of iodine-125 labeled 3-quinuclidinyl 4′-iodobenzilate

Iodine-125 labeled 3-quinuclidinyl 4′-iodobenzilate has been prepared via the reaction of the corresponding boronic acid with sodium [¹²⁵I]iodide in the presence of a mild oxidant.     

The simultaneous determination of the products of estrogen 2- and 4-hydroxylase action; the use of high-performance liquid chromatography with electrochemical detection

Both trace-labeled and high-specific activity ¹²⁵I-labeled derivatives of hexadecapeptide gastrin (G) were prepared by reaction with the iodinated form of the imidoester, methyl p-hydroxybenzimidate. Reaction conditions for preparation of trace-labeled iodinated imidoester gastrin (IIE-G) were: excess imidoester to G (IIE:G, 20:1), pH 9.2, and a reaction time of 24 h. Following purification by gel filtration and silica gel chromatography, an IIE-G component was isolated which appeared homogeneous on thin-layer chromatography and retained the same biological and immunological properties as unmodified G. Somewhat different conditions were necessary to prepare high specific activity iodinated imidoester gastrin (IIE¹-G). These included reducing the volume (20 μl) and pH (7.5) at which the imidoester was iodinated and adjusting the concentrations of reactants to the same molar amounts as 5 mCi of carrier-free ¹²⁵I. Sufficient amounts of IIE¹-G were obtained by reversing the ratio of G and IIE¹ and reacting with a G excess (GIIE¹, 10:1). The purified IIE¹-G had a specific activity exceeding 1500 μCi/nmol and was used to establish a specific and sensitive radioimmunoassay for gastrin.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Estimation of DNA, RNA and 125 I- and 3 H-labelled DNA in the same sample

Estimation of DNA, RNA, and the specific activity of DNA after labelling with [³H]thymidine and/or [¹²⁵I]iodeoxyuridine has been accomplished using a recently developed procedure for the estimation of DNA with p-nitrophenylhydrazine (pNPH). Samples of the pNPH reaction mixture are used for RNA estimation by th orcinol procedure and for ¹²⁵I and tritium measurement. Correction for ¹²⁵I contribution to the tritium measurement in double labelling experiments is accomplished either by use of a simple calibration curve (for high ${}^3\text{H}{}^{125}\text{I}$ ratios) or by removal of ¹²⁵I by hydrolysis and precipitation as AgI; in the latter procedure the efficiency of removal of ¹²⁵I was greater than 99%.     

m-[125I]iodoaniline: a useful reagent for radiolabeling biotin

Biotinyl-m-[¹²⁵I]iodoanilide (BIA) was synthesized by coupling biotin to m-[¹²⁵I]iodoaniline via a mixed anhydride reaction. m-[¹²⁵I]Iodoaniline was produced from the tin precursor, which was prepared using a palladium catalyzed reaction of hexabutylditin with m-bromoaniline. The radioiodinated BIA derivative is characterized by a stable amide and/or intact ureido group on the biotin molecule; it may thus be a useful carrier for targeting radionuclides to avidin-conjugated antibodies previously localized on tumors.     

[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.     

Some quantitative aspects of the labelling of proteins with 125I by the iodine monochloride method

The labelling of proteins by the iodine monochloride method was studied by using a mathematical model. The equations used were primarily derived from the mass law equation of the isotopic exchange reaction between [¹²⁵I]iodide and iodine monochloride. For convenient application, all equations were programmed into a computing desk-top calculator. To support the validity of the theoretical model, a series of iodinations of insulin were performed under various labelling conditions. The results of these experiments compare well with the theoretically derived values. Deviations from the theoretical values occurring at molar ratios of [¹²⁵I]iodide to iodine monochloride < 0.1 and > 4.0 are explained and suggestions made about how to prevent them. The mathematical model was used to simulate the isotopic exchange, and the iodination reaction under various conditions, to study (a) the influence of the amount of [¹²⁵I]iodide on the amount of [¹²⁵I]iodine monochloride formed, (b) the influence of the specific radioactivity of [¹²⁵I]iodide on the amount of [¹²⁵I]iodine monochloride formed, and (c) the influence of the specific radioactivity of [¹²⁵I]iodide on the number of millicuries needed for labelling to a desired extent.     

Changes in the colchicine susceptibility of microtubules associated with neurite outgrowth: studies with nerve growth factor-responsive PC12 pheochromocytoma cells

The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [125I] bovine PTH(1-34). Binding was initiated by adding [125I]-bPTH(1-34) to bisected calvariae at 30 degrees C. Steady state binding was achieved at 90 min at which time 10 mg drg wt of calvaria specifically bound 17% of the added [125I]bPTH(1-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was less than 2%. Insulin, glucagon, and calcitonin (1 microgram/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(1-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(1-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [125I]bPTH(1-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.     

The canine renal parathyroid hormone receptor is a glycoprotein: characterization and partial purification

Covalent labeling of the canine renal parathyroid hormone receptor with [125I]bPTH(1-34) reveals several major binding components that display characteristics consistent with a physiologically relevant adenylate cyclase linked receptor. Through the use of the specific glycosidases neuraminidase and endoglycosidase F and affinity chromatography on lectin-agarose gels, we show here that the receptor is a glycoprotein that contains several complex N-linked carbohydrate chains consisting of terminal sialic acid and penultimate galactose in a beta 1,4 linkage to N-acetyl-D-glucosamine. No high mannose chains or O-linked glycans appear to be present. The peptide molecular weight of the deglycosylated labeled receptor is 62,000 [or 58,000 if the mass of bPTH(1-34) is excluded]. The binding of [125I]bPTH(1-34) to the receptor is inhibited in a dose-dependent fashion by wheat-germ agglutinin, but not by either succinylated wheat-germ agglutinin or Ricinus communis lectin, suggesting that terminal sialic acid may be involved in agonist binding. A combination of lectin affinity chromatography and immunoaffinity chromatography affords a 200-fold purification of the covalently labeled receptor.     

H-NMR of G-U-C and G-U-C-C in D2O: assignment of nonexchangeable protons and analysis of solution conformation

The ribonucleotide oligomers G-U-C and G-U-C-C have been synthesized enzymatically. These oligomers are cognates of the m⁷G⁴⁶-U⁴⁷-C⁴⁸-m⁵C⁴⁹ sequence found in the variable loop of t-RNA^phe. The ¹H-NMR chemical shifts of the base and ribose Hl; protons as well as the couplings. J_1'–2' of the ribose protons have been examined as a function of temperature. Assignments for these resonances have been completed, and used in the analysis of solution conformation for these oligomers. The results are consistent with the basic features of the A-RNA structure and suggest the absence of alternative ordered solution structures.     

Stimulation of ornithine decarboxylase synthesis in the rat thyroid

High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme·monospecific antibody complex as the immunizing antigen. This new antiserum preparation was successfully labeled with ¹²⁵I and was found to retain its specific immune properties. Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone and methyl xanthine in rat thyroids $\text{in vitro}$. ¹²⁵I-labeled antibody incorporation into the enzyme antibody complex after induction $\text{in vitro}$ showed an increase which paralleled the increase in enzymatic activity and thus suggested $\text{de novo}$ synthesis of thyroid enzyme protein.     

Comparative Binding of 125I-and 99mTc-Labeled Native and Glycated Low-Density Lipoprotein to Human Microvascular Endothelial Cells-Potential for Atherosclerosis Imaging?

Native (n), glycated (g), and glycoxidated (go) low-density lipoproteins (LDL) were labeled with ¹²⁵I or ^99mTc, and the labeling efficiency and binding were assessed for potential use of these LDL compounds in imaging analysis of atherosclerotic lesions (PPAR-γ receptors) by determining the number of specific receptors for nLDL, gLDL or goLDL on human microvascular endothelial cells as well as the K_D s using either ¹²⁵I-or ^99mTc-labeled LDLs. The specific activity of labeled gLDL and goLDL was much higher (for goLDL 20 times higher) than that of nLDL. Gel filtration of labeled LDLs revealed, however, that ^99mTc–g/goLDL is significantly degraded by the labeling reaction. No fragmentation was observed for ^99mTc-nLDL and all the ¹²⁵I-labeled LDL forms. Binding studies using both ¹²⁵I-and ^99mTc-nLDL indicated a weak binding affinity (K_D 10^{? 7}mol/L) to human microvascular endothelial cells. The binding affinity of ¹²⁵I-g/goLDL to these cells was significantly higher (K_D 10^{? 9}mol/L) and could be increased further by preactivation of the endothelial cells using TNFα. Incubation with ^99mTc-goLDL, however, did not result in specific binding of the ligand, possibly as a consequence of the fragmentation of the lipoprotein during the labeling. Scatchard transformation of the binding data with ^99mTc-gLDL revealed the presence of only a few binding sites. This was in contrast to the results obtained with ¹²⁵I-labeled gLDL, which revealed a much higher membrane density of scavenger receptors for this ligand. We conclude that for in vitro binding studies as well as for potential in vivo imaging, only ¹²⁵I-labeled goLDL should be used, whereas nLDL may be applied as ¹²⁵I-or ^99mTc-labeled ligand.     

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.     

Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

Circular (e.g. simian virus 40) and linear (e.g. λ phage) DNAs have been labeled to high specific radioactivities (>10⁸ cts/min per μg) in vitro using deoxynucleoside [α-³²P]triphosphates (100 to 250 Ci/mmol) as substrates and the nick translation activity of Escherichia coli DNA polymerase I. The reaction product yields single-stranded fragments about 400 nucleotides long following denaturation. Because restriction fragments derived from different regions of the nick-translated DNA have nearly the same specific radioactivity (cts/min per 10[su3] bases), we infer that nicks are introduced, and nick translation is initiated, with equal probability within all internal regions of the DNA. Such labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.     

Prolonged retention of high specific activity by 125I-labeled angiotensin II — a consequence of ‘decay catastrophe’

Angiotensin II labeled with a single atom of ¹²⁵I per moleculed had been shown to retain 80% of the original specific activity of the radioiodinated peptide during storage for up to 5 months. This phenomenon is attributable to destruction of the angiotensin II molecule during the radioactive decay process, so that the immunoreactive peptide effectively desappears at the same rate as the incorporated ¹²⁵I atom. For this reason, monoiodinated angiotensin II can be employed for prolonged periods in radioimmunoassay and binding studies, with retention of high specific activity in the absence of formation of free iodide or interfering peptide fragments.     

Influence of circulating antigen on blood pool activity of a radioiodinated monoclonal antibody

Athymic mice with and without circulating CA 125 antigen were injected with 0.1–100μg of ¹³¹I-labeled OC 125 F(ab′)₂ antibody fragment. Both the blood clearance of ¹³¹I activity and the change in serum CA 125 were monitored over 24 h. Influence of CA 125 on blood pool activity could be avoided only at the 100 βg dose. In patient studies, circulating CA 125 levels decreased for the first 2 h after injection of OC 125 F(ab′)₂ but generally returned to preinjection levels shortly thereafter. In vitro binding studies using the sera from patients injected with ¹³¹I-labeled OC 125 F(ab′)₂ suggest that circulating CA 125 could interfere with the tumor uptake of the labeled antibody.     

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/μmol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [¹²⁵I]ICYP-diazirine binds with high affinity (K_d = 60 pM) to β-receptors from turkey erythrocyte membranes. Upon irradiation, [¹²⁵I]ICYP-diazirine is covalently incorporated in a M_r 40 000 protein. Stereoselective inhibition of photolabeling by the (?)enantiomers of alprenolol and isoproterenol indicated that the M_r 40 000 protein contains a β-adrenergic binding site. The yield of specific labeling was up to 8.2% of total β-receptor binding sites. The M_r 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [¹²⁵I]ICYP-diazirine resulted in specific labeling of two proteins with M_r 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximate M_r 67 000) was labeled specifically.     

Purification and immunological characterization of human myocardial MB creatine kinase

Elevated plasma MB creatine kinase (CK) is considered the most sensitive and specific diagnostic indicator of myocardial infarction. However, attempts to purify human MB CK have been unsuccessful. The need for purified human MB CK was further enhanced with the development of a radioimmunoassay for CK isoenzymes which would provide more prompt and specific detection of myocardial infarction. The major protein contaminant of MB CK is albumin which has been difficult to separate due to their similar electrophoretic mobility. Human hearts were obtained within 2 h postmortem and the tissue homogenized in 50 mm Tris-HCl (pH 7.4), 2 mm mercaptoethanol. The CK was recovered from the supernatant (31,000g) by ethanol extraction (50–70%). The resuspended pellet was fractionated on DEAE Sephadex A-50 with a salt gradient (50–500 mm, pH 8.0). The MB fraction contained about 90% albumin. The preparation was bound to an Affigel blue column and contaminating proteins other than albumin were eluted with 50 mm Tris-HCl (pH 8.0), 2 mm mercaptoethanol. MB CK was eluted with 250 mm NaCl, but the albumin remained bound. The MB fraction with a specific activity of 453 IU/mg represented an 80-fold increase in purity and exhibited a single protein band on polyacrylamide gels. Purified MB CK labeled with ¹²⁵I exhibited no binding to human albumin antiserum, but bound to MB CK antiserum, and unlabeled MB CK competitively inhibited binding of ¹²⁵I-MB CK in the radioimmunoassay system exhibiting a sensitivity for detection of plasma MB CK at the nanogram level.