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PubMed2005

Role of androgen receptor in the progression of human prostate tumor cells to androgen independence and insensitivity.

BACKGROUND: Various studies have implicated the androgen receptor (AR) in the progression of androgen-dependent human prostate cancer cells to androgen-independent and androgen-insensitive phenotypes, but the exact role of AR in progression is unclear. METHODS: To mimic the clinical situation and test the role of AR in progression, we cultured androgen-dependent LNCaP 104-S prostate tumor cells in the presence of the antiandrogen Casodex (bicalutamide) to derive resistant (CDXR) clones. In a second step, we cultured CDXR cells in the presence of the androgen R1881, which generated androgen- and Casodex-insensitive (IS) cells. These cells were then characterized with regard to AR function and the effect of ectopic AR expression or AR knockdown on androgen sensitivity. RESULTS: CDXR cells showed increased AR expression and transcriptional activity. CDXR cell proliferation was unaffected by Casodex but was repressed by androgen in vitro and in vivo. IS cells, on the other hand, had greatly reduced AR expression and activity compared to CDXR cells. Knockdown of AR expression in CDXR cells produced cells that were insensitive to androgen. Conversely, re-expression of AR in IS cells regenerated cells that were repressed by androgen. Knockdown of AR expression in 104-S cells produced cells that remained stimulated by androgen, while overexpression of AR in 104-S cells generated an androgen-repressed phenotype but did not confer androgen-independent growth. CONCLUSIONS: Increased AR expression determines whether prostate cancer cells are repressed by androgen, but is not required for androgen independence. These results may have implications for anti-AR therapy for prostate cancer.

Androgen Antagonists↗
PubMed2005

Complete androgen ablation suppresses prostate stem cell antigen (PSCA) mRNA expression in human prostate carcinoma.

BACKGROUND: Prostate stem cell antigen (PSCA) is a recently identified glycosylphosphatidylinositol (GPI)-anchored cell surface protein belonging to the Thy-1/Ly-6 family of cell surface antigens. Prior data in prostate cancers indicated that PSCA is directly regulated by androgens and PSCA expression increases with high-tumor grade, advanced stage, extracapsular invasion, and androgen-independent progression. The effect of complete androgen ablation (CAA) on tumor PSCA mRNA expression has not been elucidated. The purpose of the present study was to investigate the variations in the expression levels of PSCA mRNA before and after CAA, and further evaluate the clinically prognostic value of PSCA in human prostate carcinoma. MATERIALS AND METHODS: PSCA in situ hybridization (ISH) was performed on the cancerous pretreatment biopsy or transurethral resection of prostate (TURP) tissue of 42 men with primarily organ-confined prostate cancer before CAA, and on their tumor tissue from radical retropubic prostatectomy after CAA with bicalutamide and goserelin acetate for 3 months prior to undergoing radical prostatectomy. Tumor cytoplasmic staining of PSCA mRNA was evaluated by two independent pathologists and the differences of PSCA mRNA expression levels between the samples before and after CAA were analyzed using the Student's t-test. Thirty-six to forty months follow-up studies after radical retropubic prostatectomy were performed and aimed at assessing the correlation of PSCA mRNA expression level with local recurrences or metastases from the cancer. RESULTS: The percent of cells positive for PSCA mRNA by ISH labeling declined from 67.3% (0-89%)+/-9.4% before CAA to 33.8% (0-92%)+/-7.7% after CAA (P<0.001). Before CAA, 40 of 42 cases (95.2%) were positive for PSCA mRNA labeling, however, after CAA the percentage of positive reactivity of PSCA mRNA was decreased to 27 of 40 cases (67.5%), in which none was found with local recurrences or distant metastases after radical prostatectomy on follow-up. This decline in PSCA mRNA labeling was dependent on the original tumor grade with Gleason score of or=8: 73.4%+/-13.8% (P<0.05, respectively). The rest 13 cases had the increased percentage of cells positive for PSCA mRNA after CAA, in which 3 cases were found with local recurrences and 4 cases with distant metastases from tumor on follow-up. CONCLUSIONS: Our data demonstrate that CAA for prostate cancer can suppress PSCA mRNA expression with a tumor grade dependence and the increased expression of PSCA mRNA after CAA may be a clinically adverse predictor for tumor recurrences or distant metastases.

Aged↗
PubMed2006

The androgen receptor pathway is by-passed in prostate cancer cells generated after prolonged treatment with bicalutamide.

BACKGROUND: Experimental work in various prostate cancer models revealed that the androgen receptor is frequently upregulated and implicated in tumor progression. However, little attention has been paid to the androgen receptor-signaling pathway in the development of therapy resistance in patients who receive chronic treatment with a non-steroidal anti-androgen. METHODS: We have generated a novel subline, LNCaP-Bic, after prolonged treatment with androgen and bicalutamide in vitro. Proliferation of LNCaP-Bic cells in the absence or presence of androgen, tocopherol succinate, and/or bicalutamide was assessed by cell counting. Androgen receptor expression was determined by Western blot. Luciferase activity was measured in cells transfected with an androgen-responsive reporter. RESULTS: In basal conditions, proliferation of LNCaP-Bic cells increased more than threefold over that of control LNCaP cells. Neither synthetic androgen R1881 nor bicalutamide showed any effect on LNCaP-Bic growth in vitro. Androgen receptor expression did not differ between the cell subline generated in the presence of bicalutamide and parental LNCaP cells. The ability of R1881 to induce reporter gene activity in LNCaP-Bic cells was reduced by 56%. Tocopherol succinate caused inhibition of proliferation only in the parental cell line although the androgen receptor and prostate-specific antigen were down regulated by the vitamin E derivative in both parental LNCaP and LNCaP-Bic cells. CONCLUSIONS: Androgen receptor-mediated signal transaction is not enhanced in cells selected in the presence of bicalutamide. Our data may suggest that a more differentiated approach in targeting the androgen receptor is needed in prostate cancers that become resistant to classic endocrine treatment.

Androgen Antagonists↗
PubMed2006

Conditional expression of PTEN alters the androgen responsiveness of prostate cancer cells.

BACKGROUND: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is lost as a function of prostate tumor androgen dependence. While the transcriptional activity of the androgen receptor (AR) is inhibited by PTEN in androgen sensitive prostate cancer (CaP), the role of PTEN in androgen disease is unclear. METHODS: We developed a system where PTEN can be conditionally re-expressed at physiologic levels into a PTEN null metastatic human CaP cell line, C4-2, and androgen responsiveness examined. RESULTS: PTEN induction reduces cell growth and blocks the growth effect of synthetic androgen R1881. The anti-androgen Casodex enhances the growth-inhibitory action of PTEN and this effect is independent of Akt phosphorylation. Combined PTEN induction and Casodex, result in a further decrease in prostate specific antigen promoter activity compared to PTEN but not Casodex alone. CONCLUSIONS: PTEN induction confers androgen independent CaP cells enhanced responsiveness to the anti-proliferative effects of anti-androgens and this action may involve non-AR mediated effects.

Androgen Antagonists↗
PubMed2007

Prosaposin upregulates AR and PSA expression and activity in prostate cancer cells (LNCaP).

BACKGROUND: Prosaposin overexpression and/or genomic amplification have been demonstrated in androgen-independent (AI) prostate cancer cell lines and tissues. Here, we explored the possibility for a functional relationship between prosaposin and androgen receptor (AR) in LNCaP cells. METHODS: The effect of prosaposin or its active molecular derivatives (e.g., saposin C) on expression and activity of androgen receptor (AR) and prostate-specific antigen (PSA) was examined by using immunoblotting, RT-PCR, transfection, and reporter gene assays, immunofluorescence staining, and inhibitors of signal transduction pathways. RESULTS: Prosaposin or saposin C, in an AI-manner, (a) increased AR mRNA and protein expression and nuclear AR content and its phosphorylation state; (b) increased PSA mRNA and protein expression; and (c) upregulated PSA- and an androgen-inducible probasin (PB)-reporter gene activity in LNCaP and AR-transfected PC-3 cells. Induction of PSA expression and reporter activity was substantially blocked or prevented with the antiandrogen bicalutamide, pertussis toxin, or inhibitors of MAPK- and PI3K/Akt-signaling pathways, indicating an androgen-agonistic effect for saposin C that involves AR and multiple signaling pathways. CONCLUSIONS: The results for the first time introduce prosaposin as an androgen-agonist in prostate cancer cells. This finding, together with the growth-promoting effect and overexpression of prosaposin, may support a growth advantage to AI prostate cancer cells.

Androgen Antagonists↗
PubMed2007

A role for neurotensin in bicalutamide resistant prostate cancer cells.

BACKGROUND: Anti-androgens are administered as a principal treatment for prostate cancer. Aggressive hormone refractory disease is characterized in some cases by the development of a neuroendocrine phenotype. However little attention has been paid to resistance pathways selected for by long-term treatment with non-steroidal anti-androgens. METHODS: Using a resistant sub-line, LNCaP-Bic, we performed a comparative gene expression profiling using cDNA microarrays and target validation by qRT-PCR. Targets were then explored using cell proliferation, cell cycle analysis and in vitro invasion assays using siRNA technology. RESULTS: Neurotensin/Neuromedin N (NTS) was upregulated in the LNCaP-Bic line at both the transcript and protein level. The resistant line was found to have an increased proliferation rate, more rapid cell cycle progression and increased invasiveness through Matrigel. Each phenotypic difference could be reduced using siRNA knockdown of NT. CONCLUSION: Increased expression of NT in bicalutamide resistant prostate cancer cells induces cell proliferation and invasion suggesting that this peptide may contribute to the development of bicalutamide resistant prostate cancer.

Androgen Antagonists↗
PubMed1990

Effects of androgen and antiandrogen treatment on canine prostatic arginine esterase.

A series of experiments were conducted to investigate the regulation of the primary secretory protein of the canine prostate, arginine esterase, by androgens and/or new antiandrogen under development. In the first experiment, castration decreased (P less than 0.05) prostatic arginine esterase levels relative to intact controls (0.26 +/- 0.1 and 17.0 +/- 0.1 mumole/min/mg protein, respectively). Treatment of castrate dogs with either 5, 10, or 20 silastic capsules (8 cm length) containing the androgen 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) plus 1 capsule containing estradiol-17 beta (E2) or the i.m. injection of 25 mg 3 alpha-diol and 0.25 mg E2 for 12 weeks resulted in a dose-dependent increase (P less than 0.05) in prostatic arginine esterase activity (6.8 +/- 1.7, 19.0 +/- 3.6, 21.3 +/- 0.9, and 14.2 +/- 0.7 mumole/min/mg protein, respectively). In the second experiment, steroid treatment (10 3 alpha-diol plus 1 E2 silastic capsules) of castrate dogs for 12 weeks resulted in prostatic arginine esterase activity of 17.8 +/- 2.3 mu mole/min/mg. Co-administration of the steroidal androgen receptor antagonist. Win 49,596 (WIN) at doses of 0.625, 2.5, 10, or 40 mg/kg/day p.o., dose-dependently inhibited (P less than 0.05) prostatic arginine esterase activity (14.9 +/- 1.1, 14.3 +/- 1.3, 3.4 +/- 1.9, and 0.21 +/- 0.1 mumole/min/mg, respectively) to levels similar to that observed in castrate controls (0.14 +/- 0.03 mumole/min/mg). Administration of the nonsteroidal androgen receptor antagonist flutamide at 10 mg/kg/day p.o. to steroid-induced dogs also inhibited (P less than 0.05) arginine esterase activity (0.07 +/- 0.02 mumole/min/mg). In the last experiment, treatment of intact dogs with WIN at 0.625, 2.5, 10, and 40 mg/kg/day for 16 weeks dose-dependently reduced (P less than 0.05) arginine esterase levels (17.0 +/- 1.0, 16.3 +/- 1.5, 10.2 +/- 1.2, and 3.9 +/- 2.5 mumole/min/mg, respectively) compared to intact controls (14.4 +/- 1.2 mumole/min/mg). Histomorphologic and ultrastructural evaluation of prostates from dogs indicated that antiandrogen treatment resulted in glandular epithelial atrophy as well as a reduction in the number of secretory granules. The results of these experiments support that canine prostatic arginine esterase activity is under androgenic control, can be inhibited by antiandrogen treatment and may serve as a functional marker of the androgenic state of the prostate. Whether the effects of androgen and antiandrogens on prostatic arginine esterase is direct or indirect due to a general inhibitory effect on secretory epithelial cell function requires additional study. Furthermore, subject to further evaluation, the steroidal androgen receptor antagonist.(ABSTRACT TRUNCATED AT 400 WORDS)

5-alpha Reductase Inhibitors↗
PubMed1992

Casodex: a pure non-steroidal anti-androgen used as monotherapy in advanced prostate cancer.

Casodex, a pure potent non-steroidal anti-androgen, has been shown in a phase II clinical trial program to be very well tolerated and to have fewer side-effects compared with other anti-androgens. The most commonly reported side-effects (prompted by direct questioning) were breast tenderness (63.4%), breast swelling (52.5%), and hot flashes (23.6%). Gastrointestinal disturbances, hepatic impairment, alcohol intolerance, and problems with light adaptation are not associated with Casodex treatment. In this study, Casodex (50 mg once daily) was evaluated in 267 patients, 130 of whom have received it for more than 1 year. On assessment of best objective response, 55.5% of patients showed partial regression, 15.6% stable disease, 17.1% progression, and 11.8% were not assessable. These results are comparable with other standard hormonal therapies for advanced prostate cancer.

Aged↗
PubMed1994

Androgen ablation induces tenascin expression in the rat prostate.

Tenascin is a glycoprotein of the extracellular matrix of mesenchymal derived tissue compartments. Although the DNA sequences that code for tenascin production are known and the protein structure is well characterized, little is known about regulation of tenascin expression. Therefore, we are interested in hormonal aspects of tenascin expression. In this study, we addressed the question if androgen deprivation and prostatic involution would influence tenascin expression in the prostate. Methodologically, in two series of experiments, intact and orchiectomized testosterone propionate substituted male rats were subjected to one of the following hormonal treatments: a) flutamide, b) the antiandrogen casodex, and c) cyproterone acetate (CPA). As controls in each series, we used untreated controls and orchiectomized rats. After a period of 14 days of treatment, prostates were removed. Tenascin immunostaining of the sectioned specimen from the control and the hormonally treated animals demonstrated the following: 1) Little, if any tenascin immunoreactivity was detectable in prostates of untreated animals. 2) Androgen deprivation with either treatment resulted in tenascin expression in the stroma of the prostates. 3) Tenascin expression appeared to be variable both semiquantitatively and in the staining pattern detectable except in prostates treated with CPA, in which we observed the most uniform and most widespread staining pattern. From these results, we conclude that androgen deprivation induces tenascin expression in the stroma of the involuting prostate. Remodelling of the extracellular matrix of the stroma, known to appear in the process of prostate involution by androgen ablation, represents a process which has not been discussed with tenascin expression so far.

Androgen Antagonists↗
PubMed1994

Current clinical studies with a new nonsteroidal antiandrogen, Casodex.

The efficacy of Casodex (ICI 176,334; Zeneca Pharmaceuticals, Macclesfield, UK), a nonsteroidal antiandrogen, in the treatment of advanced prostate cancer has been compared in two trials with castration, either surgical (bilateral orchitectomy) or medical, with the gonadotropin-releasing hormone agonist Zoladex (goserelin acetate; Zeneca Pharmaceuticals). The half-life of Casodex is 7 to 10 days, and allows once-daily dosing. The first trial was a prospective, randomized study involving 150 patients receiving Casodex, 50 mg once daily p.o., and 154 patients undergoing castration. There were no significant differences between the two groups in subjective responses, time to treatment failure, or time to objective evidence of disease progression. The other study involved higher doses, the dose being selected after patients had received one of two doses of Casodex, 100 mg/day or 150 mg/day, in a double-blind study. On statistical analysis, it was clear that the higher dose was more effective, and thus most patients taking 100 mg/day were switched to 150 mg/day. Again, Casodex treatment was compared with medical or surgical castration. The higher dose of Casodex is well tolerated with a similar adverse event profile as the lower dose of 50 mg/day. Follow-up is as yet too short for an analysis of efficacy.

Aged↗
PubMed1994

Role of prostate-specific antigen as a predictor of outcome in prostate cancer.

Prostate-specific antigen (PSA) has been shown over the past few years to be a useful and sensitive marker for prostate cancer. During Phase III studies of the nonsteroidal antiandrogen, Casodex, in which different doses were compared with castration (either surgical or medical), serum PSA was measured on a regular basis. Attention was focused on the change in serum PSA from baseline after 3 months Casodex treatment and also on the number of patients receiving Casodex whose PSA returned to the normal range. Data from trials comparing Casodex, 50 mg/day, with castration showed a clear shortfall for Casodex compared with castration, in terms of percentage fall in PSA at 3 months, and also in the number of patients whose PSA fell into the normal range after 3 months. Subsequent analysis showed, however, that the PSA level was related to outcome in terms of time to progression. These data allowed the use of PSA to determine dose selection in a subsequent trial comparing Casodex, 100 mg/day or 150 mg/day, with castration. At the time of dose selection, changes in PSA showed a statistically significant difference between Casodex, 100 mg/day, and castration, but no significant difference between Casodex, 150 mg/day, and castration, either for the change in PSA at 3 months or for the proportion of patients whose PSA had fallen into the normal range. The idea that serum PSA levels can predict outcome in prostate cancer and that they are correlated with other measures of outcome, such as time to progression, is supported by these data. A decrease in PSA is not a true surrogate endpoint in that it cannot predict the outcome for an individual patient with complete accuracy, but it does correlate well with other measures of outcome, such as time to progression, for patient populations.

Analysis of Variance↗
PubMed1994

Differential inactivation of rabbit and yeast triosephosphate isomerase: effect of oxidations produced by chloramine-T.

Triosephosphate isomerase from rabbit has 5 Cys and 2 Met, while triosephosphate isomerase from yeast has 2 Cys (present in the rabbit enzyme in equivalent positions) and no Met. Since chloramine-T oxidizes Cys and Met, we determined the effect it has on the activity and structure of both enzymes. The activity of triosephosphate isomerase from rabbit was more sensitive to chloramine-T than that of the yeast enzyme (under conditions where the rabbit isomerase was completely inactive, the yeast enzyme exhibited approximately 50% activity). An initial effect of chloramine-T on triosephosphate isomerase was the oxidation of Cys and the formation of catalytically active acidic isoforms. For the yeast isomerase, the two processes were slower. Our data suggest that oxidation of Cys 126, which is conserved in all of the studied species, does not abolish catalysis. Chloramine-T also oxidized the two Met of the rabbit enzyme. At ratios of 50 chloramine-T/monomer, circular dichroism studies showed that the rabbit enzyme, but not that from yeast, underwent extensive alterations of tertiary and secondary structures. This was accompanied by formation of stable dimers, whose cross-linking was not through disulfide bonds. Studies of dimer formation at various enzyme concentrations showed that cross-linking was between monomers of the same dimer. Under conditions that led to cross-linking, rabbit triosephosphate isomerase took up 2.7 mol of 3H from NaB3H4/mol dimer, and the yeast enzyme incorporated only 0.4 mol of 3H. Thus cross-linking was most likely via a Schiff base. The results revealed the points whose modification caused inactivation of the rabbit enzyme.

Animals↗
PubMed1994

Regulation of two rat liver microsomal carboxylesterase isozymes: species differences, tissue distribution, and the effects of age, sex, and xenobiotic treatment of rats.

The preceding paper described the purification of two rat liver microsomal carboxylesterases, designated hydrolases A and B, that have high affinity (Km approximately 25 microM) and low affinity (Km approximately 400 microM) for para-nitrophenylacetate, respectively. The present study describes the preparation and purification of polyclonal antibodies against these purified enzymes. Each antibody was subjected to immunoabsorption chromatography to remove antibodies against epitopes common to both hydrolases A and B. The resulting isozyme-specific antibodies were used to study the regulation of hydrolases A and B by Western immunoblotting and Ouchterlony immunodiffusion. Liver microsomes from mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and humans contained one or more proteins that were immunochemically related and similar in size (M(r) approximately 60 kDa) to hydrolase A and/or hydrolase B. These proteins were preferentially recognized by the antibody against hydrolase A, except for cat liver microsomal esterase, which was preferentially recognized by antibody against hydrolase B. In rats, the levels of hydrolases A and B in liver microsomes were coregulated as a function of age, sex, and xenobiotic treatment of rats. The levels of both enzymes were very low in 1- and 2-week-old rats, but increased abruptly at 3 weeks of age in both male and female rats. Treatment of mature male rats with 11 known microsomal enzyme inducers caused little (< 35%) or no induction of hydrolase A or B, whereas treatment of rats with beta-naphthoflavone, pregnenolone- 16 alpha-carbonitrile or dexamethasone suppressed the levels of both enzymes. The kinetic analysis of para-nitrophenylacetate hydrolysis described in the preceding paper identified a high-affinity esterase (Km 20-35 microM) in rat liver, testis, lung, prostate, and pancreas and identified a low-affinity enzyme (Km 300-800 microM) in liver, kidney, small intestine, lung, brain, spleen, and heart. Immunoblot analysis established that hydrolase A was present in liver, testis, lung, and prostrate at concentrations that accounted for the high-affinity esterase activity in these tissues. Hydrolase A was not detected in the pancreas, even though this tissue contained low levels of a high-affinity esterase. Hydrolase B was detected in liver and kidney at concentrations that accounted for the low-affinity esterase activity in these tissues. Hydrolase B was not detected in the other tissues examined, some of which (e.g., small intestine) contained high levels of a low-affinity esterase. These results indicate that hydrolases A and B are independently expressed in a wide variety of extrahepatic tissues in rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Age Factors↗
PubMed1999

The active site of phosphorylating glyceraldehyde-3-phosphate dehydrogenase is not designed to increase the nucleophilicity of a serine residue.

Changing a catalytic cysteine into a serine, and vice versa, generally leads to a dramatic decrease in enzymatic efficiency. Except a study done on thiol subtilisin, no extensive study was carried out for determining whether the decrease in activity is due to a low nucleophilicity of the introduced amino acid. In the present study, Cys149 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus was converted into a Ser residue. This leads to a drastic reduction of the kcat value. The rate-limiting step occurs before the hydride transfer step. Selective, but slow, inactivation is observed with specific, structurally different, inhibitors of serine protease. The esterolytic activity of serine mutant towards activated esters is also strongly decreased. The rate-limiting step of the esterase reaction also shifts from deacylation in the wild type to acylation in the mutant. Altogether, these results strongly suggest that the low catalytic efficiency of the Ser mutant is due to a poor nucleophilicity of the hydroxyl serine group within the active site of the enzyme. The fact that (1) the apo --> holo transition does not change esterolytic and inactivating efficiencies, and (2) Ser149 Asn176 double mutant exhibits the same chemical reactivity and esterolytic catalytic efficiency compared to the Ser149 single mutant indicates that the serine residue is not subject to His176 general base catalysis. A linear relationship between the catalytic dehydrogenase rate, the kcat/KM for esterolysis, and the concentration of OH- is observed, thus supporting the alcoholate entity as the attacking reactive species. Collectively this study shows that the active site environment of GAPDH is not adapted to increase the nucleophilicity of a serine residue. This is discussed in relation to what is known about Ser and Cys protease active sites.

Amino Acid Substitution↗
PubMed1995

Chloramine-T in radiolabeling techniques. II. A nondestructive method for radiolabeling biomolecules by halogenation.

Chloramine-T (CAT) is commonly used in the radiolabeling of bioactive molecules by halogenation. CAT may be used either as a solution or in an immobilized form (Iodobeads) to release radioactive elemental iodine or other halogens by oxidation of their salts. CAT has a very high chlorine potential, and it causes oxidative damage to sensitive substrate molecules, such as peptides and proteins. In some cases, the substrates are completely destroyed. To reduce the chlorine potential of CAT, morpholine was mixed with CAT prior to exposure to the substrates. This formed N-chloromorpholine, in situ, which readily reacted with KI to form I2. The kinetics of the formation of N-chloromorpholine from CAT and morpholine were studied spectrophotometrically by following the disappearance of CAT at 250 nm. The reaction was found to be rapid at all pH's from 5 to 11. 1-Aminocyclohexanecarboxylic acid (a model amino acid) decomposed rapidly in the presence of CAT, but there was no decomposition in the presence of N-chloromorpholine. N-Chloromorpholine was compared to CAT solution and Iodobeads for the iodination of L-tyrosine. The formation of mono- and diiodotyrosine were followed by HPLC. On an equimolar basis (0.55 microM), N-chloromorpholine produced a much greater yield of the mono- and diiodinated tyrosine than Iodobeads. Furthermore, decomposition products were observed when tyrosine was exposed to Iodobeads for 15 min. When a CAT solution was used at a higher concentration (5.5 microM), a substantial amount of decomposition occurred, and the yields of the two iodinated species were very small.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloramines↗
PubMed1997

Cysteine-specific radioiodination of proteins with fluorescein maleimide.

A protocol is described for coupling of carrier-free iodine to protein sulfhydryl groups via fluorescein maleimide. 125I is first coupled to fluorescein maleimide in the presence of chloramine T. Iodination is stopped with sodium thiosulfate, and the iodine-substituted fluorescein maleimide is reacted with free cysteines of the protein. Excess label is then removed by gel-permeation chromatography. The procedure avoids exposition of the protein to oxidative conditions and does not require purification of the labeled carrier reagent. Suitability of the method for a given protein can be evaluated spectrophotometrically without employing radioactivity. It can be applied under denaturing conditions and may be particularly useful with mutant proteins carrying engineered single cysteine residues at sites that are not functionally critical.

Bacterial Proteins↗
PubMed2000

Chloramine-T in radiolabeling techniques. III. Radioiodination of biomolecules containing thioether groups.

A previously reported method for iodination of the tyrosine moiety of oxidation-sensitive biomolecules was found to cause unacceptable damage to biomolecules containing thiols and thioether groups. This was due to the oxidation of the sulfur-containing residues by molecular iodine (I(2)). To selectively iodinate the tyrosine moiety with minimum oxidation to the sulfur functionality, studies of the kinetics of the reactions between I-(3) and various amino acids and small peptides at various pH values in phosphate buffer were undertaken. Within the pH range studied (5.5-8.2), the results showed that the iodination reaction is strongly catalyzed by hydroxide ions, whereas the oxidation of the sulfur group was insensitive to pH. The results also showed that both reactions are strongly catalyzed by HPO-(4) ion. In a complex molecule, such as methionine-enkephalin, oxidation of the methionine residue (undesirable reaction) proceeds in parallel with iodination of the tyrosine residue (desirable reaction). If such a molecule was iodinated in 0.01 M phosphate buffer at pH values above 7.5, the iodination reaction would proceed much more rapidly than the oxidation reaction, resulting in a high yield of iodinated substrate with little oxidative damage.

Chloramines↗