High-performance liquid chromatography of iodine-labeled insulin and glucagon derivatives with on-line γ-detection

Insulin and glucagon were labeled with iodine. The reaction products were analyzed by high-performance liquid chromatography. It is shown that the pH of the reaction medium has a large effect on the position and the degree of iodine substitution as well as on the oxidation of the Met-containing glucagon and, furthermore, that the molar ratio of iodine to polypeptide hormone used during the labeling procedure affects not only the amount of iodine incorporated but also the distribution of iodinated products. The results show that certain iodinated derivatives are separated from each other and from the respective unlabeled polypeptide and thus can be obtained in a pure state.     

Effects of iodination of tyrosyl residues on the binding and action of glucagon at its receptor.

The binding and action of glucagon at its receptor in hepatic plasma membranes have been compared, as a function of pH, with that of glucagon containing iodotyrosyl residues. Iodinated glucagon, at pH 7.0 and below, binds to the receptor and activates adenylate cyclase with an affinity about threefold higher than that of native glucagon. At pH 8.5, the affinity of the receptor for native glucagon is the same as that seen at pH 7.0. However, iodinated glucagon binds with a lowered affinity with increasing pH. The decreased affinity of the iodinated hormone correlates with ionization of the iodotyrosyl phenoxy groups, which has a pKa of 8.2. It is suggested that the decreased affinity is actually due to the inability of the ionized iodoglucagon to bind to the receptor. The relative potency of native and iodoglucagon will depend, therefore, on the concentrations of ionized and un-ionized species of iodoglucagon, which in turn depend on the pH of the medium. We conclude that incorporation of iodine atoms in the tyrosyl residues of glucagon has two major effects: (i) the iodine atom increases hydrophobic interaction of the hormone with the receptor and (ii) ionization of the phenoxy groups results in the loss of biological activity possibly as the result of loss of hydrogen bonding capability. Thus, the tyrosyl residues in glucagon are critically involved in the function of the hormone.     

Iodinated phospholipids and the iodination of proteins of dog thyroid gland in vitro

Slices of dog thyroid gland were incubated with liposomes consisting of (125)I-labelled phosphatidylcholine (the iodine was covalently linked to unsaturated fatty acyl chains). The (125)I label of (125)I-labelled liposomes was incorporated into thyroid protein and/or thyroglobulin at a higher rate than was the (131)I label of either Na(131)I or (131)I(2). The iodine was shown to be protein-bound by the co-migration of the labelled iodine with protein under conditions where free iodine, iodide and lipid-bound iodine were removed from protein. The uptake of iodine from the iodinated phospholipid was probably due to phospholipid exchange between the iodinated liposomes and the thyroid cell membrane, since (a) (14)C-labelled phospholipid was metabolized to (14)CO(2) and (b) many lipids in the tissue slice became (14)C-labelled. A very strong inhibition of iodide ;uptake' from Na(131)I, caused by thiosulphate, produced only a minor inhibition of the incorporation of (125)I from (125)I-labelled liposomes into thyroid protein and/or thyroglobulin. This implies that free iodide may not necessarily be formed from the iodinated phospholipids before their entrance or utilization in the cell. Synthetic polytyrosine polypeptide suspensions showed some iodination by (131)I-labelled liposomes. In tissues with low tyrosine contents, such as liver and kidney, only a trace uptake was observed. Salivary gland showed some uptake. Endoplasmic reticulum of thyroid gland showed a higher iodine uptake than that of the corresponding plasma membranes. These experiments, together with the demonstration of the diet-dependent presence of iodinated phospholipids in dog thyroid, leads us to suggest that iodination of the membrane phospholipids of thyroid cells may be directly or indirectly involved at some stage in the synthesis of thyroglobulin, or exists as a scavenger mechanism, to re-utilize and/or recover released iodine from unstable compounds inside the thyroid cell.     

Identification by nuclear magnetic resonance of iodinated lipids in the dog thyroid.

Thyroid glands were obtained from dogs on high, normal or low iodine intake. The lipids of the cells and of purified plasma membranes of these thyroids were examined by nuclear magnetic resonance spectroscopy. The presence of an iodinated methine proton peak at δ = 4.5 ppm, has been recently reported as a means for detection of iodination of 1,2-disubstituted ethylenes by Zanger and Rabinowitz (J. Org. Chem. $\text{40}$, 248, 1975). The presence of iodinated lipids in the plasma membrane, and in the cell total lipids could easily be shown in the thyroids from animals that were fed a high iodine diet. The thyroids of animals with normal iodine diets showed only trace amounts of these lipids. In the thyroids of dogs on a low iodine diet, these iodinated lipids could not be detected. Na¹³¹I fed dogs showed by thin layer chromatography the presence of iodinated phospholipids.     

Iodinated melatonin: preparation and characterization of the molecular structure by mass and 1H NMR spectroscopy

Synthetic melatonin was iodinated by treatment with potassium iodide in the presence of an oxidizing agent, Iodo-Gen. The iodination products of melatonin were extracted with chloroform and separated by HPLC. The fraction showing immunoreactivity with respect to melatonin antisera was characterized as iodomelatonin by mass spectrometry, so that the substitution of iodine had occurred at a ring carbon atom. 1H NMR spectra showed the iodine to be incorporated at the C-2 position of the indole moiety. The N-[2-(2-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide (2-iodomelatonin) reported here is more useful than [3H]melatonin as a tracer in melatonin radioimmunoassay. This method offers also the possibility of preparing iodinated serotonin and other indoleamines for biological studies.     

Immunocytochemical study of argyrophil (Sevier Munger) endocrine cells of adult human pancreas

Bouin-fixed tissues from non-diabetic adult human pancreata display an argyrophil reaction mainly in the periphery of the islets with the silver technique of Sevier-Munger. The nature of these argyrophil cells was examined after restaining by an indirect immunocytochemical method using antibodies against insulin, glucagon, somatostatin and pancreatic polypeptide. After this procedure the argyrophil cells were identified as glucagon (A-) cells and pancreatic polypeptide (PP-) cells, although the latter exhibited a weaker reaction. The insulin (B-) cells and somatostatin (D-) cells were unreactive. The results show that the Seiver-Munger stain is of equal value to the Grimelius silver nitrate stain in adult human pancreatic islets after fixation in Bouin's fluid.     

Direct iodination of specific residues in crystals of yeast formylatable methionine-accepting transfer ribonucleic acid.

Crystals of yeast formylatable methionine-accepting transfer ribonucleic acid (tRNAfMet) were iodinated by using a modification of Commerford's procedure [Commerford, S.L. (1971) Biochemistry 10, 1993]. Chromatographic analysis of nuclease digestion products showed that radioactive iodine binds covalently to the 5 position of three nucleotide residues: U8, C73, and C74. These three iodine substitutions were assigned to three peaks in a difference Fourier synthesis comparing the iodinated derivative with native tRNAfMet. In this way the positions of U8, C73, and C74 were marked in the crystal structure of yeast tRNAfMet, providing guidepoints for the interpretation of a 4.5-A electron density map.     

Immunocytochemical study of argyrophil (Sevier Munger) endocrine cells of adult human pancreas

Summary Bouin-fixed tissues from non-diabetic adult human pancreata display an argyrophil reaction mainly in the periphery of the islets with the silver technique of Sevier-Munger. The nature of these argyrophil cells was examined after restaining by an indirect immunocytochemical method using antibodies against insulin, glucagon, somatostatin and pancreatic polypeptide. After this procedure the argyrophil cells were identified as glucagon (A-) cells and pancreatic polypeptide (PP-) cells, although the latter exhibited a weaker reaction. The insulin (B-) cells and somatostatin (D-) cells were unreactive. The results show that the Sevier-Munger stain is of equal value to the Grimelius silver nitrate stain in adult human pancreatic islets after fixation in Bouin's fluid.Supported by grants from the Swedish Medical Research Council (Project No. 102)     

Iodinated derivatives of the hormone avian pancreatic polypeptide

Reaction of avian pancreatic polypeptide with an iodine monochloride reagent at both pH 4 and pH 7.5 results in the differential modification of the four tyrosine residues in this peptide hormone. A total of 19 distinct iodinated derivatives were isolated by reverse-phase high-performance liquid chromatography, and their sites of iodination were characterized by both tryptic mapping and leucine aminopeptidase techniques coupled with HPLC. The pH 4 reaction produced 16 derivatives which, overall, represented substantial iodination at each tyrosine residue, whereas the pH 7.5 reaction was more directed, producing only 7 derivatives. Iodination at the C-terminal tyrosineamide 36 predominated at both pH values, and diiodo-Tyr 36 was found in the majority of the pH 7.5 derivatives. The relative of the four tyrosine residues with ICl were as follows: at pH 7.5, Tyr 36 much greater than Tyr 21 much greater than Tyr 27 greater than Tyr 7; at pH 4, Tyr 36 greater than Tyr 27 greater than Tyr 7 greater than Tyr 21.     

Iodinated salicylate-based poly(anhydride-esters) as radiopaque biomaterials

Poly(anhydride-esters) based on iodinated versions of salicylic acid were synthesized via both melt-condensation and solution polymerization techniques to generate radiopaque biomaterials. The poly(anhydride-esters) from iodinated salicylates were highly X-ray opaque compared to poly(anhydride-esters) from salicylic acid. Molecular weight and Young's modulus of polymers prepared by melt-condensation were typically two-to-three times higher than polymers prepared by solution methods. The glass transition temperatures of the polymers were dependent on the iodine concentration; polymers containing more iodine had higher glass transition temperatures. Cytotoxicity studies using mouse fibroblasts indicated that iodinated salicylate-based poly(anhydride-esters) prepared by both polymerization methods are biocompatible with cells at low polymer concentrations (0.01 mg/mL).     

The development of ketogenesis at birth in the rat.

The manner in which human liver cathepsin B (EC 3.4.22.1) digests glucagon was determined. After reaction of the proteinase with the substrate for 24h, more than 15 products were formed. During the first 7 h of reaction, eight products were formed; seven of these were dipeptides that originated from the C-terminal portion of the glucagon molecule, whereas the eighth peptide was the remaining large fragment of the hormone, consisting of residues 1-19. Measurement of the rate of formation of the products showed that cathepsin B degraded glucagon by a sequential cleavage of dipeptides from the C-terminal end of the molecule. Cathepsin B from both rat liver and bovine spleen was shown to hydrolyse glucagon by the same mechanism.     

Chemical and enzymatic modification of proteins in the 30 S ribosome of Escherichia coli

Methods are described for the iodination of ribosomal proteins by iodine monochloride and potassium iodide and bovine lactoperoxidase. Ribosomes that were maximally iodinated did not synthesize polyphenylalanine. About one-half of the tyrosine residues could be iodinated with iodine monochloride in the intact ribosome with no change in the sedimentation properties of the particle. When proteins were extracted and dissolved in 5 m-urea, all of the tyrosine residues could be iodinated with iodine monoehloride.     

INACTIVATION OF PEPSIN BY IODINE AND THE ISOLATION OF DIIODO-TYROSINE FROM IODINATED PEPSIN

In the presence of iodine at pH 5.0–6.0 a solution of pepsin absorbs iodine and the specific proteolytic activity of the solution decreases. The activity is less than 1 per cent of the original activity when the number of iodine atoms per mol of pepsin is 35–40. If the pH is 4.5 or less, iodine reacts very slowly and there is a correspondingly slower loss in activity. Glycyl tyrosine reacts with iodine in a manner similar to pepsin. Experiments were performed to determine the extent to which oxidation of pepsin by iodine occurs during iodination, and if such oxidation were responsible for the loss in enzymatic activity. Although the results were not absolutely decisive, there seems to be no appreciable oxidation taking place during iodination and no relationship between the slight oxidation and loss in peptic activity. From a dialyzed preparation of completely iodinated pepsin which was inactive and contained 13.4 per cent bound iodine, 82 per cent of the iodine was obtained in a solution which analyzed as a solution of diiodo-tyrosine. Because of the presence of a material which contained no iodine and prevented quantitative crystallization, only 53 per cent of the iodine containing substance could be crystallized. This 53 per cent was, however, identified as diiodo-tyrosine. The part of the titration curve which in pepsin and most proteins represents the phenolic group of tyrosine was, in the curve for iodinated pepsin, shifted toward the acid region as expected. From these results, it appears that the loss in proteolytic activity of pepsin, when treated with iodine under the specified conditions, is due to the reaction of the iodine with the tyrosine in pepsin.     

The earliest site of iodination in thyroglobulin is residue number 5

In most highly structured native proteins, as well as in thyroglobulin, the reactivity in vitro of the various tyrosyl residues toward iodine is widely different. The present work demonstrates that of nearly 70 tyrosyl residues present in rat thyroglobulin, there is one, residue number 5 from the NH2-terminal end, which has in vivo the highest affinity toward iodine, being the first one to be iodinated. In fact, when 6-(n-propyl)-2-thiouracil (PTU)-treated, iodine-deficient animals were injected with 125I and killed shortly after, we isolated from thyroid glands poorly iodinated thyroglobulin (about 1 iodine atom/thyroglobulin molecule), nearly 90% of the radioactivity of which was found as monoiodotyrosine. Although CNBr cleavage of this protein gave several fragments after gel electrophoresis only one of these, with apparent mass 27,000 Da, contained 125I. This fragment was isolated and fully characterized. Twelve cycles of automated Edman degradation were performed; the sequence found, i.e. N-I-F-E-X-Q-V-X-A-Q-X-L, indicated that the 27,000-Da fragment is the NH2 terminus of thyroglobulin. This portion of the polypeptide chain contains several tyrosyl residues which may well all be potentially involved in the early iodination of the protein. The observation that the removal of seven amino acids from the NH2 terminus is accompanied (at the fifth step) by the total disappearance of radioactivity in the resulting shortened peptide suggested that the fifth residue was the only one iodinated under these conditions. A second, more quantitative experiment was performed on thyroglobulin obtained from 6-(n-propyl)-2-thiouracil-treated animals whose death was postponed 24 h after the injection of 125I. In this case the radioactivity was found not only in a single CNBr fragment (27,000 Da) but also in other discrete species of lower molecular mass. The mixture of these peptides was subjected to seven steps of manual Edman degradation. Fragments before and after partial degradation were run in parallel on a polyacrylamide gel and the distribution of 125I compared. Besides some change in the background, the two profiles were identical except for the absence of the 27,000-Da species. This proves that all the 125I present in the 27,000-Da species was localized at the fifth residue, the same site at which the hormone molecule is preferentially synthesized under normal conditions. This result is not unexpected and is in accord with the known properties of thyroglobulin which has a polypeptide chain designed for efficient synthesis of the hormone even at low levels of iodination.     

Inhibition of thyroid-restricted genes by follicular thyroglobulin involves iodinated degree

Follicular thyroglobulin (TG) reflects the storage of both iodine and thyroid hormone. This is because it is a macromolecular precursor of thyroid hormone and organic iodinated compound in follicular lumen. Thus, it may have an important feedback role in thyroid function. In this study, monolayer cells were cultured and follicles were reconstituted with primary pig thyroid cells in vitro. Reconstituted follicles were treated with iodine and methimazole (MMI), a drug that blocks iodine organification and reduces the degree of TG iodination in follicular lumen. The high degree of iodinated TG in follicular lumen was observed to inhibit thyroid-restricted gene expression. To confirm this finding, monolayer thyroid cells were treated with a different degree of TG iodination at the same concentration. These iodinated TG were extracted from reconstituted follicles of different groups. In this manner, this study provides firsthand evidence suggesting that follicular TG inhibits the expressions of thyroid-restricted genes NIS, TPO, TG, and TSHr.     

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.     

Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus.

The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.     

Influences of gastro-intestinal polypeptides and glucose on glucagon secretion induced by cholinergic stimulation

Glucagon secretion from the endocrine pancreas is known to be enhanced by cholinergic stimulation. It has previously been described that vasoactive intestinal polypeptide (VIP) is a potent potentiator of this cholinergically induced glucagon secretion. In the present study, the effects of several gastro-entero-pancreatic polypeptides and glucose on glucagon secretion induced by the cholinergic agonist carbachol were investigated in vivo in the mouse. Carbachol was injected i.v. and it stimulated glucagon secretion. The polypeptides neurotensin and gastric inhibitory polypeptide (GIP) were both found to potentiate the carbachol-induced glucagon secretion, whereas substance P, pancreatic polypeptide, and two different molecular variants of cholecystokinin, CCK-8 and CCK-39, were without effect on cholinergically induced glucagon secretion. Neither of these polypeptides had any influence on basal glucagon secretion when tested over a wide dose range. Somatostatin and glucose both markedly inhibited carbachol-induced glucagon secretion. In conclusion: carbachol is a potent stimulator of glucagon secretion. This cholinergically induced glucagon secretion can be modified by several gastro-entero-pancreatic hormones influencing the release process both in potentiating and inhibiting direction. The physiological relevance of these interactions remains to be further investigated.     

The radioactive labeling of proteins with an iodinated amidination reagent.

An iodinated derivative of the imidoester methyl p-hydroxybenzimidate HCl (MPHBIM) has been synthesized for the selective labeling of proteins to high specific activity with radioactive iodine. In the first step, MPHBIM is reacted with radioactive iodide in the presence of chloramine T, and the iodinated derivative is precipitated from acidified solution to achieve partial purification. In the second step, the iodinated imidoester is redissolved at slightly alkaline pH and reacted with protein amino groups, to which it couples by amidine linkage. The coupling reaction proceeds in the presence of sulfhydryl reagents used to protect proteins. The main advantage of this two-step labeling procedure is that it avoids direct contact of the protein with potentially deleterious materials such as chloramine T or contaminants of the radioactive iodide.     

INACTIVATION OF PEPSIN BY IODINE : II. ISOLATION OF CRYSTALLINEl-MONO-IODOTYROSINE FROM PARTIALLY IODINATED PEPSIN

1. Pepsin solutions were iodinated at pH 5.0–6.0 until 10–20 per cent of the activity was lost and 1/20 (0.7 per cent) of the saturating amount of iodine had been introduced into the protein molecule. After alkaline hydrolysis 65 per cent of the original iodine was accounted for as mono-iodotyrosine although only 42 per cent was isolated as a crystalline product. No evidence was obtained to support the possibility that any group other than tyrosine in pepsin was iodinated. 2. Some of the properties of the crystalline l-mono-iodotyrosine were determined and compared to those of di-iodotyrosine. 3. One iodinated pepsin preparation was crystallized. The crystal form was the same as that of the original pepsin. A solubility curve of the crystals demonstrated that it was very different from pepsin and had nearly constant solubility.