The mechanisms of methionine oxidation concomitant with hormone radioiodination comparative studies of various oxidants using a simple new method

The mechanism of oxidation of methionine concomitant with iodination was studied by ascending paper chromatography using $\text{l}\text{-[Me-}{}^{14}\text{C}\text{]}\text{methionine}$. The ability of the principal iodination reactants to oxidize free methionine was measured in order to predict in situ methionyl oxidation during radioiodination of peptides. Iodide, oxidant, and tyrosine were tested individually and in combination. Dilute chloramine-T and H₂O₂ effectively oxidized free methionine, whereas electric current at the low levels used for iodination did not. Whereas KI₃ did not cause significant methionine oxidation, iodine in statu nascendi was a potent oxidant. Oxidation caused by chemical oxidants was markedly reduced by the addition of iodide, whereas the opposite effect was seen with electric current. In our in vitro system, tyrosine inhibited the effect of the chemical oxidants to different degrees, in the absence of iodide. Under conditions simulating actual ‘mild’ radioiodination conditions, with added tyrosine, none of the oxidants studied caused methionine oxidation; the presence of tyrosine and iodide appeared to preclude the oxidation of methionine. Both excess chloramine-T and high electric current resulted in the formation of a new compound, possibly a sulphinic acid derivative of methionine. The important finding was that the release of free iodine and its uptake by tyrosine were the dominant factors to be considered in the prevention of methionine oxidation.Our new chromatographic technique for the estimation of methionine oxidation in peptides is based on the observed similarity between the reactivity of free methionine and that of accessible methionyl residues in peptides. The method is simple, sensitive and reproducible.     

A membrane-bound aminopeptidase isolated from monkey brain and its action on enkephalin

A membrane-bound aminopeptidase which cleaves the tyrosin-glycine bond of enkephalin was purified about 1600-fold from monkey brain. This aminopeptidase hydrolyzed Leu-enkephalin with a K_m value of 35 μM and also hydrolyzed basic, neutral and aromatic amino acid β-naphthylamides. An apparently homogeneous enzyme consisted of a single polypeptide chain with a molecular weight of approx. 100 000. The optimum pH was in the neutral region. From the analysis of the reaction products, only aminopeptidase activity was detected. The enzyme was inactivated by metal chelators, but the activity could be restored by the addition of divalent cations, such as Co²⁺, Mg²⁺ and Zn²⁺. Puromycin, bestatin and amastatin, which are aminopeptidase inhibitors derived from microorganism, showed strong competitive inhibition of the enzyme, the most potent being amastatin, with a K_i value of 0.02 μM.     

Higher blood selenium level is associated with lower risk of hyperhomocysteinemia in the elderly

Background and aimsEarlier studies have reported inconsistent association between selenium (Se) and homocysteine (Hcy) levels, while no evidence could be found from Chinese population. To fill this gap, we investigated the association between blood Se and hyperhomocysteinemia (HHcy) of rural elderly population in China.MethodsA cross-sectional study on 1823 participants aged 65 and older from four Chinese rural counties was carried out in this study. Whole blood Se and serum Hcy concentrations were measured in fasting blood samples. Analysis of covariance and restricted cubic spline models were used to examine the association between Se and Hcy levels. Logistic regression models were used to evaluate the risk of prevalent HHcy among four Se quartile groups after adjusting for covariates.ResultsFor this sample, the mean blood Se concentration was 156.34 (74.65) μg/L and the mean serum Hcy concentration was 17.25 (8.42) μmol/L. A significant non-linear relationship was found between blood Se and serum Hcy, the association was inverse when blood Se was less than 97.404 μg/L and greater than 156.919 μg/L. Participants in the top three blood Se quartile groups had significantly lower risk of prevalent HHcy compared with the lowest quartile group. When defined as Hcy> 10 μmol/L, the odds ratios and 95% confidence interval of HHcy were 0.600 (0.390, 0.924), 0.616 (0.398, 0.951) and 0.479 (0.314, 0.732) for Q2, Q3, and Q4 Se quartile groups compared with the Q1 group, respectively. When defined as Hcy≥ 15 μmol/L, the odds ratios and 95% confidence interval of HHcy were 0.833 (0.633, 1.098) and 0.827 (0.626, 1.092), 0.647 (0.489, 0.857) for Q2, Q3, and Q4 Se quartile groups compared with Q1 group.ConclusionsOur findings suggest that higher blood Se level could be a protective factor for HHcy in the elderly.     

Regional Distribution of Methionine Adenosyltransferase in Rat Brain as Measured by a Rapid Radiochemical Method

Abstract: The distribution of methionine adenosyltransferase (MAT) in the CNS of the rat was studied by use of a rapid, sensitive and specific radiochemical method. The S -adenosyl-[methyl-¹⁴C] l -methionine ([¹⁴C]SAM) generated by adenosyl transfer from ATP to [methyl-¹⁴C] l -methionine is quantitated by use of a SAM-consuming transmethylation reaction. Catechol O -methyltransferase (COMT), prepared from rat liver, transfers the methyl-¹⁴C group of SAM to 3,4-dihydroxybenzoic acid. The ¹⁴C-labelled methylation products, vanillic acid and isovanillic acid, are separated from unreacted methionine by solvent extraction and quantitated by liquid scintillation counting. Compared to other methods of MAT determination, which include separation of generated SAM from methionine by ion-exchange chromatography, the assay described exhibited the same high degree of specificity and sensitivity but proved to be less time consuming. MAT activity was found to be uniformly distributed between various brain regions and the pituitary gland of adult male rats. In the pineal gland the enzyme activity is about tenfold higher.     

Secretion of [Met]Enkephalyl-Arg6-Phe7-Related Peptides and Catecholamines from Bovine Adrenal Chromaffin Cells: Modification by Changes in Cyclic AMP and by Treatment with Reserpine

Investigations into the effects of culturing bovine adrenal chromaffin cells in the presence (72 h) of dibutyryl cyclic AMP, forskolin, and reserpine on the level and release of [Met]enkephalyl-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline are reported. The assay for [Met]enkephalyl-Arg6-Phe7 immunoreactivity recognises both peptide B, the 31-amino acid carboxy-terminal segment of proenkephalin, and its heptapeptide fragment, [Met]enkephalyl-Arg6-Phe7. Treatments that elevate cyclic AMP increase the amount of peptide immunoreactivity in these cells; this is predominantly peptide B-like immunoreactivity in both control cells and cyclic AMP-elevated cells. Treatment with reserpine gives no change in total immunoreactivity levels, but does not result in increased accumulation of the heptapeptide [Met]enkephalyl-Arg6-Phe7 at the expense of immunoreactivity that elutes with its immediate precursor, peptide B. Cyclic AMP treatment causes either no change or a decrease in levels of accumulated noradrenaline and adrenaline. However, the release of [Met]enkephalin-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline is increased by 72-h pretreatment with forskolin or dibutyryl cyclic AMP, whether release is stimulated by nicotine or elevated potassium. In each case the molecular form of [Met]enkephalyl-Arg6-Phe7 immunoreactivity that is released approximately reflects the cell content. Pretreatment with reserpine has no effect on the total [Met]enkephalyl-Arg6-Phe7 immunoreactivity released, but does result in an increased release of the heptapeptide and a decrease in release of peptide B-like immunoreactivity. The studies suggest that the levels of [Met]enkephalyl-Arg6-Phe7 and peptide B available for release are controlled both at the level of proenkephalin synthesis and at the level of double-basic residue proteolysis.     

Taurine chloramine-induced inactivation of cofilin protein through methionine oxidation

Cofilin regulates reorganization of actin filaments (F-actin) in eukaryotes. A recent finding has demonstrated that oxidation of cofilin by taurine chloramine (TnCl), a physiological oxidant derived from neutrophils, causes cofilin to translocate to the mitochondria inducing apoptosis (F. Klamt et al. Nat. Cell Biol. 11:1241–1246; 2009). Here we investigated the effect of TnCl on biological activities of cofilin in vitro. Our data show that TnCl-induced oxidation of recombinant human cofilin-1 inhibits its F-actin-binding and depolymerization activities. Native cofilin contains four free Cys and three Met residues. Incubation of oxidized cofilin with DTT does not lead to its reactivation. A double Cys to Ala mutation on the two C-terminal Cys shows similar biological activities as the wild type, but does not prevent the TnCl-induced inactivation. In contrast, incubation of oxidized cofilin with methionine sulfoxide reductases results in its reactivation. Phosphorylation is known to inhibit cofilin activities. We found that Met oxidation also prevents phosphorylation of cofilin, which is reversed by incubating oxidized cofilin with methionine sulfoxide reductases. Interestingly, intact protein mass spectrometry of the oxidized mutant indicated one major oxidation product with an additional mass of 16 Da, consistent with oxidation of one specific Met residue. This residue was identified as Met-115 by peptide mapping and tandem mass spectrometry. It is adjacent to Lys-114, a known residue on globular-actin-binding site, implying that oxidation of Met-115 disrupts the globular-actin-binding site of cofilin, which causes TnCl-induced inactivation. The findings identify Met-115 as a redox switch on cofilin that regulates its biological activity.     

Methionine sulfoxide reductase: Chemistry,substrate binding,recycling process and oxidase activity

Three classes of methionine sulfoxide reductases are known: MsrA and MsrB which are implicated stereo-selectively in the repair of protein oxidized on their methionine residues; and fRMsr, discovered more recently, which binds and reduces selectively free L-Met-R-O. It is now well established that the chemical mechanism of the reductase step passes through formation of a sulfenic acid intermediate. The oxidized catalytic cysteine can then be recycled by either Trx when a recycling cysteine is operative or a reductant like glutathione in the absence of recycling cysteine which is the case for 30% of the MsrBs. Recently, it was shown that a subclass of MsrAs with two recycling cysteines displays an oxidase activity. This reverse activity needs the accumulation of the sulfenic acid intermediate. The present review focuses on recent insights into the catalytic mechanism of action of the Msrs based on kinetic studies, theoretical chemistry investigations and new structural data. Major attention is placed on how the sulfenic acid intermediate can be formed and the oxidized catalytic cysteine returns back to its reduced form.     

Methionine and lysine metabolism in the rumen and the possible effects of their metabolites on the nutrition and physiology of ruminants

Summary This article briefly summarizes the metabolism of methionine and lysine in the rumen of ruminant animals and known and inferable effects of their metabolites on the nutrition and physiology of ruminant animals.     

Posttranslational Processing of Carboxypeptidase E, a Neuropeptide-Processing Enzyme, in AtT-20 Cells and Bovine Pituitary Secretory Granules

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, “proCPE.” To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [³⁵S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37°C but was unaffected when cells were chased at either 20 or 15°C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCI₂. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.