Presence Of Met Research Articles

MET Receptor Tyrosine Kinase Sequence Alterations in Differentiated Thyroid Carcinoma

Activating mutations affecting the MET receptor tyrosine kinase are present in several types of human cancer, particularly in papillary renal cell carcinoma. Papillary thyroid carcinomas commonly express high levels of MET mRNA and protein, suggesting that increased MET signaling may be of importance in the molecular pathogenesis of differentiated thyroid carcinoma. To evaluate the role of MET mutations in thyroid carcinoma, we screened MET exons 2 to 21 for mutations in sporadic papillary, follicular, medullary, and anaplastic thyroid carcinomas using denaturing high-performance chromatography. A missense MET sequence alteration T1010I, located in exon 14 encoding for the juxtamembrane domain of MET, was found in 6 (6%) of the 104 thyroid carcinomas examined, whereas all 92 goiter samples had wild-type exon 14 (P = 0.031). Three (6%) of the 53 papillary, 2 (10%) of the 21 follicular, 1 (8%) of the 13 medullary, and none of the 17 anaplastic carcinomas studied had MET(T1010I). Four of the 6 T1010I sequence alterations were present also in the germline. MET protein expression showed no apparent association with the presence of MET(T1010I), and the clinical features of the patients with cancer with MET(T1010I) were similar to those of patients whose cancer did not harbor MET(T1010I). We conclude that MET(T1010I) sequence alteration is relatively frequent in differentiated thyroid carcinoma. The clinical and the molecular pathologic significance of this MET sequence alteration is not known.

Eucaryotic initiation factor 4B controls eIF3-mediated ribosomal entry of viral reinitiation factor.

The cauliflower mosaic virus reinitiation factor TAV interacts with host translation initiation factor 3 (eIF3) and the 60S ribosomal subunit to accomplish translation of polycistronic mRNAs. Interaction between TAV and eIF3g is critical for the reinitiation process. Here, we show that eIF4B can preclude formation of the TAV/eIF3 complex via competition with TAV for eIF3g binding; indeed, the eIF4B- and TAV-binding sites on eIF3g overlap. Our data indicate that eIF4B interferes with TAV/eIF3/40S ribosome complex formation during the first initiation event. Consequently, overexpression of TAV in plant protoplasts affects only second initiation events. Transient overexpression of eIF4B in plant protoplasts specifically inhibits TAV-mediated reinitiation of a second ORF. These data suggest that TAV enters the host translation machinery at the eIF4B removal step to stabilize eIF3 on the translating ribosome, thereby allowing translation of polycistronic viral RNA.

Decay kinetics of autogenously regulated CGS1 mRNA that codes for cystathionine gamma-synthase in Arabidopsis thaliana.

Cystathionine gamma-synthase (CGS) catalyses the first committed step in methionine (Met) biosynthesis in higher plants. Stability of CGS1 mRNA encoding CGS in Arabidopsis thaliana is regulated by negative feedback in response to Met application and the amino acid sequence of CGS itself acts in cis in this regulation. It is proposed that the regulation occurs during translation when the nascent polypeptide of CGS and its mRNA are in close proximity. This model predicts that inhibition of translation abolishes the regulation. To test this, we analysed the effect of translation inhibitor cycloheximide on the CGS1 mRNA decay. The half-life of CGS1 mRNA after the addition of transcription inhibitor actinomycin D in the absence and presence of 1 mM Met was 154+/-11 min and 81+/-5 min, respectively. Simultaneous addition of actinomycin D and cycloheximide stabilized CGS1 mRNA both in the presence and absence of Met, as essentially no decrease of CGS1 mRNA was observed. Moreover, cycloheximide treatment inhibited production of the truncated CGS1 RNA species, a possible degradation intermediate. These results indicated that inhibition of translation abolishes the CGS1 mRNA-specific decay process. Kinetic analyses indicated that about half the CGS1 mRNA is destined to CGS1 mRNA-specific decay when 1 mM Met was applied.

Detection and characterization of methionine oxidation in peptides by collision-induced dissociation and electron capture dissociation

Electron capture dissociation (ECD) and collision-induced dissociation (CID), the two complementary fragmentation techniques, are demonstrated to be effective in the detection and localization of the methionine sulfoxide [Met(O)] residues in peptides using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The presence of Met(O) can be easily recognized in the low-energy CID spectrum showing the characteristic loss of methanesulfenic acid (CH 3SOH, 64 Da) from the side chain of Met(O). The position of Met(O) can then be localized by ECD which is capable of providing extensive peptide backbone fragmentation without detaching the labile Met(O) side chain. We studied CID and ECD of several Met(O)-containing peptides that included the 44-residue human growth hormone-releasing factor (GRF) and the human atrial natriuretic peptide (ANP). The distinction and complementarity of the two fragmentation techniques were particularly remarkable in their effects on ANP, a disulfide bond-containing peptide. While the predominant fragmentation pathway in CID of ANP was the loss of CH 3SOH (64 Da) from the molecular ion, ECD of ANP resulted in many sequence-informative products, including those from cleavages within the disulfide-bonded cyclic structure, to allow for the direct localization of Met(O) without the typical procedures for disulfide bond reduction followed by SH alkylation.

Brain energy metabolism is compromised by the metabolites accumulating in homocystinuria

Homocystinuria is an inborn error of metabolism caused by severe deficiency of cystathionine β-synthase activity. It is biochemically characterized by tissue accumulation of homocysteine (Hcy) and methionine (Met). Homocystinuric patients present a variable degree of neurological dysfunction whose pathophysiology is poorly understood. In the present study, we investigated the in vitro effect of Hcy and Met on some parameters of energy metabolism in hippocampus of rats. CO 2 production from [U- 14 C ] acetate, glucose uptake and lactate release were assessed by incubating hippocampus prisms from 28-day-old rats in Krebs–Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of Hcy (10–500 μM) or Met (0.2–2.0 mM). Hcy and Met decreased CO 2 production in a dose-dependent manner and increased lactate release. In contrast, glucose uptake was not altered by the metabolites. The effect of Hcy and Met on cytochrome c oxidase activity was also studied. It was observed that Met did not alter this enzyme activity, in contrast with Hcy, which significantly inhibited cytochrome c oxidase activity. It is suggested that impairment of brain energy metabolism caused by the metabolites accumulating in homocystinuria may be related to the neurological symptoms present in homocystinuric patients.

Redox properties of Met(35) in neurotoxic beta-amyloid peptide. A molecular modeling study.

The beta-amyloid peptide (betaAP) is the principal component of plaque associated with the pathology of Alzheimer's disease. Part of its neurotoxicity appears to correlate with the ability of the peptide to reduce Cu(II) and form free radicals. Both processes are dependent on the presence and oxidizability of Met(35) in the C-terminus of the peptide but no mechanistic details on the reactions leading to Met oxidation are known. On the basis of previous studies with model peptides, we hypothesize that a one-electron oxidation of Met(35) in betaAP is facilitated through a neighboring group effect. Complexed to Cu(II) and/or in a lipid-mimicking environment, the solution structure of betaAP includes a large alpha-helical part. The solution NMR structure of betaAP1-40 in aqueous SDS micelles reveals an alpha-helix between residues 27 and 36, containing Met(35). In this helical C-terminus of betaAP, the peptide bond C=O group C-terminal of Ile(31) is located very close to the Met(35) sulfur and could stabilize a Met(35) sulfide radical cation through formation of an (S-O) three-electron bond. In the present paper, we have computationally validated this hypothesis using Langevin dynamics methods to determine the collision frequency of the Met(35) thioether sulfur and the oxygen atoms of several peptide bonds in the betaAP sequence. Nanosecond time scale computations were carried out for four distinct betaAP congeners, betaAP26-40, betaAP26-36, betaAP26-40(Ile(31)Pro), betaAP40-26, and their respective Met(35)-sulfur-centered cation radicals. Here, betaAP26-40, betaAP26-40(Ile(31)Pro) and betaAP40-26 are representative fragments of the full length betaAP1-42 or betaAP42-1 sequence, respectively, whereas betaAP26-36 represents a unique betaAP sequence for which biological data are available. Initial structures of betaAP26-40, betaAP26-40(Ile(31)Pro), and betaAP26-36 were selected to be identical to that of the betaAP26-40 or betaAP26-36 sequence in full-length betaAP1-40. As the structures of betaAP40-26 and betaAP42-1 are not known, various initial conformations such as alpha-helix and antiparallel beta-sheet were selected for betaAP40-26. Our computational results show that betaAP26-40, representative for the same sequence in full-length betaAP1-42, has the highest tendency to form (S-O) bonds between Ile(31)C=O and Met(35)S. We conclude that native betaAP1-42 has a higher tendency to support Met(35) oxidation through (S-O) bond formation, consistent with the experimental observation that betaAP1-42 is more neurotoxic compared to the other investigated sequences.

Independent roles of methionine and O-acetyl-l-serine in the regulation of the β subunit gene of β-conglycinin

The gene encoding the β subunit of β-conglycinin, one of the major seed storage proteins of soybean (Glycine max [L.] Merr.) , is downregulated by methionine (Met) and upregulated by O-acetyl-l-serine (OAS). We examined the interaction between Met and OAS in the regulation of the expression of the β subunit gene in in vitro-cultured immature soybean cotyledons. Application of Met reduced the accumulation of the β subunit both in the presence and absence of OAS in the media: Application of OAS increased the accumulation of the β subunit both in the presence and absence of Met. We also studied the regulation of the β subunit gene promoter by sulfur in Arabidopsis thaliana (L.) Heynh. mutant mto1-1, that over-accumulates soluble Met, using the β-glucuronidase (GUS) gene as a reporter. When grown under sulfur-deficient conditions, the GUS activity in seeds of the transgenic plants carrying the mto1-1 mutation was lower than that of the plants which do not carry the mutation, suggesting that the β subunit gene promoter was downregulated by the mto1-1 mutation under sulfur deficiency. When the transgenic plants carrying the mto1-1 mutation were grown under control and sulfur-deficient conditions, the GUS activity was higher under sulfur-deficient conditions, suggesting that the β subunit gene promoter was upregulated by sulfur deficiency even in the mto1-1 mutant background. Furthermore, OAS contents in immature siliques were elevated both in the mto1-1 mutant and wild-type plants under sulfur deficiency. Met contents in immature siliques of the mto1-1 mutant plants increased even under sulfur deficiency. There was a strong positive correlation between the GUS activities in seeds and the endogenous [OAS]/[Met] ratio. These results suggest that OAS and Met regulate the β subunit accumulation through independent pathways.

Design and characterization of a highly selective peptide inhibitor of the small conductance calcium-activated K+ channel, SkCa2.

Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1-3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tskappa, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala(1), Phe(2), and Met(7). By replacing Met(7) in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab(7), a selective SKCa2 inhibitor (K(d) = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab(7) by replacing His(521) with the corresponding SKCa2 residue (Asn(367)). Intracerebroventricular injection of Lei-Dab(7) into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab(7) will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.

3‐[123I]IODO‐α‐methyl‐L‐tyrosine as a substrate of human L‐type amino acid transporter‐1

AbstractWe have clarified the membrane transport characteristics of 3‐[123I]Iodo‐α‐methyl‐L‐tyrosine by molecular biological methods with cDNAs of an amino acid transporter. Uptake and inhibition studies of this compound were performed with human L‐type amino acid transporter‐1 and a heavy chain of human 4F2 cell surface antigen co‐expressed Xenopus laevis oocytes. 3‐[125I]Iodo‐α‐methyl‐L‐tyrosine uptake was decreased by the presence of Ala, Ser, Thr, Cys, Leu, IIeu, Phe, Met, Tyr, His, Trp, Val, Asn, and Gln. The uptake was Na+‐independent, and was activated by a heavy chain of human 4F2 cell surface antigen.

Effects of methionine supplement on methionine incorporation in rat embryos cultured in vitro.

The effect of supplementary L-methionine (Met) on the incorporation of methionine was evaluated in 9.5-day rat conceptuses cultured in vitro. Parallel experiments with L-leucine (Leu) were performed for comparison. Conceptuses were cultured for 24 hr in the presence of 3H-labeled Met or Leu, and the incorporation of radiolabel into the embryo and visceral yolk sac was measured. Supplementary Met proportionately increased the incorporation of Met, but supplementary Leu did not have as great an effect on the incorporation of Leu. A hypothesis is presented to explain these findings. It is proposed that Met, but not Leu, is a rate-limiting nutrient for organogenesis-stage rat embryos cultured in rat serum. The results are also discussed with reference to the established efficacy of supplementary folic acid in decreasing the incidence of neural tube defects in human populations and to claims that Met reverses certain teratogenic phenomena, both in vitro and in vivo.

Functional interference between contacting amino acids of homeodomains

In a protein, the function of an amino acid at some position depends on the amino acids at other positions. Here we demonstrate a functional interference between base-contacting amino acids (at positions 50 and 54) of homeodomains. When, in the context of Antennapedia or Goosecoid homeodomains, Lys 50 is paired to Tyr 54 or Ala 54 and Gln 50 is paired to Met 54, the resulting proteins efficiently discriminate among different DNA sequences. In contrast, in the presence of the pair Lys 50-Met 54, both homeodomains show a reduced capability to discriminate among different DNA sequences. Sequence selection experiments performed in the context of the Goosecoid homeodomain suggest that the presence of Met 54 precludes the base-discriminating function of Lys 50. These results may explain why the pair Lys 50-Met 54 is never found in natural homeodomains.

Effects of ipriflavone and its metabolites on human articular chondrocytes cultivated in clusters

Ipriflavone (IP) is an isoflavone derivative that was suggested to have bone-sparing effects in post-menopausal and senile osteoporosis. A moderate stimulatory effect of IP and its metabolites on proliferation of osteoblastic cells was reported in rat osteoblastic osteosarcoma cell line. We investigated the effects of different concentrations (0, 1, 10 and 100 micrograms/ml) of IP and its metabolites (MET I, II, III and V) on the incorporation of [3H] thymidine and production of proteoglycans (PG) and type II collagen (COL II) by human articular chondrocytes during a 12-day period, in a three-dimensional chondrocyte culture model. [3H]thymidine uptake was measured in chondrocyte clusters, and specific PG and COL II radioimmunoassays were performed every 4 days on the culture medium and cell clusters. Incubation with IP or its metabolites did not affect [3H]thymidine uptake regardless of the dose. PG released into the culture medium and PG cluster content rose significantly (P < 0.025) in presence of IP (1, 10 and 100 micrograms/ml). MET I increased PG release in culture medium (10 and 100 micrograms/ml) and PG cluster content (100 micrograms/ml). MET II has no effect on PG production. MET III increased PG in culture medium (100 microgram/ml) but did not influence PG cluster content while MET V (100 micrograms/ml) increased both PG release in culture medium and PG cluster content. COL II release in culture medium and COL II cluster content were significantly (P < 0.025) increased in presence of IP (10 and 100 micrograms/ml), MET III (1, 10 and 100 micrograms/ml) or MET V (100 micrograms/ml). MET I and II did not significantly affect COL II production.

A polypeptide toxin in the sea anemone Actinia equina homologous with other sea anemone sodium channel toxins: Isolation and amino acid sequence

The sea anemone ( Actinia equina) was newly established to contain a polypeptide toxin (named Ae I) having lethal activity to crabs, besides the well-known cytolytic toxins (equinatoxins) of proteinic nature. Ae I, with a minimum lethal dose against crabs of 25 μg/kg, was easily isolated by gel filtration on Sephadex G-50 and reverse-phase HPLC on Nucleosil 300-7C18. Its amino acid composition is characterized by the abundance of Gly, the absence of Ala and the presence of Met. The complete amino acid sequence of Ae I was determined. Ae I has high sequence homology with type 1 sea anemone neurotoxins. Interestingly, the polypeptide chain of Ae I comprises 54 amino acid residues, being 5–8 residues longer than the known type 1 toxins having 46–49 residues.

Effect of some amino acids on the rectal irritation caused by sodium caprate in conscious rats.

The effects of some amino acids such as L-glutamine (Gln), L-arginine (Arg) and L-methionine (Met) on rectal irritation caused by sodium caprate were studied in rats. Rectal irritation was assessed by the balloon method in fasting conscious rats. This method is based on measuring rectal contractions due to possible irritation caused by the presence of drugs and/or adjuvants in the rectum. Strong contractions were observed after rectal administration of an aqueous solution of 100 mM sodium caprate. However, the presence of Gln, Arg or Met (100 mM) in sodium caprate (100 mM) solution resulted in a significant decrease in the intensity of the rectal contraction caused by sodium caprate. The rectal absorption-promoting effect of sodium caprate on 6-carboxyfluorescein (6-CF) was examined following administration with amino acids in rats. The absorption of 6-CF was not influenced by the concurrent administration of amino acids. In addition, the rectal tissue interaction of sodium caprate, with or without Gln, was examined. The concentration of sodium caprate in rectal tissue was reduced by the presence of Gln.

Assessment of homology with the helical mimicry algorithm

Homologies based on structural motifs characterize conserved structures and mechanisms of maintaining function. An algorithm was developed to quantitate homology among segments of two proteins based upon structural characteristics of an amphipathic α-helix. This helical mimicry algorithm scored homology among sequences of two proteins in terms of: (i) presence of Leu, Ile, Val, Phe, or Met in a longitudinal, hydrophobic strip-of-helix at positions n, n + 4, n + 7, n + 11, etc. in the primary sequence, (ii) identity or chemical similarity of amino acids at intervening positions and (iii) exchanges of amino acids from positions n to n − 1, n + 3, n + 4, n + 1, n − 3, n − 4 around n (on the surface of a putative helix). While such exchanges of amino acids on the surfaces of homologous helices may conserve function, they did not maintain specific interactions of those residues with apposing groups.

Amino Acid Limitation and Flow to the Duodenum at Four Stages of Lactation. 2. Extent of Lysine Limitation1,2

Four multiparous Holstein cows with ruminal and duodenal cannulas were assigned to 4 × 4 Latin squares at peak (wk 4), early (wk 14 to 16), mid (wk 21 to 23), and late (wk 29 to 31) lactation to determine, in the presence of supplemental Met, the extent of Lys limitation and its required contribution to total essential AA in duodenal digesta. Treatments were duodenal infusions of 1) water alone or water with 2) 10 g/d of DL-Met plus 10 g/d of L-Lys, 3) 10 g/d of Met plus 20 g/d of Lys, and 4) 10 g/d of Met plus 30 g/d of Lys; quantities were reduced by 20% in late lactation. Rations were corn-based (corn and grass-legume silages, corn meal, wheat middlings, soybean meal, and distillers dried grains with solubles) and most limiting in Lys and Met. Intakes of ruminally degraded and undegraded intake protein (percentage of NRC requirements) were (peak) 115, 97; (early) 112, 83; (mid) 113, 87; and (late) 127, 96. Contribution of Lys to passage of total essential AA to the duodenum without infusions were 13.2, 12.4, 13.8, and 14.8% at the four respective stages of lactation. Extent of Lys limitation determined from responses in content and yield of milk protein approximated 25, 20, and 10 g/d during peak, early, and midlactation. Using measurements of AA flow to the duodenum and assumed intestinal availabilities of 100% for infusate Lys and 80% for microbial and ruminally undegraded intake protein AA, calculated required contributions of Lys to total essential AA passing to the duodenum were 15.2, 13.9, and 14.5% for peak, early, and midlactation, respectively.

Immunohistochemical demonstration of enkephalin-containing nerve fibers in guinea pig and rat lungs.

Met-enkephalin (Met-Enk) and Leu-enkephalin (Leu-Enk), the opioid peptides originally isolated from the brain, are believed to act as inhibitory neuromodulators at various synaptic sites. In this immunohistochemical study, we have investigated the localization and distribution of Met- and Leu-Enk immunoreactivities in airways and pulmonary vessels of guinea pigs and rats. Immunoreactivities to both peptides were found in nerve fibers and nerve terminals distributed mainly to the trachea and major bronchi, and were especially prevalent in the smooth muscle layer, in the lamina propria, and around tracheal and bronchial glands, but not in the epithelium. Few immunoreactive nerve fibers were detected in smaller bronchi, bronchioles, and alveoli. Enkephalin-immunoreactive nerve fibers were also localized in the walls of pulmonary and bronchial vessels. Within airway microganglia, immunoreactivity was observed in a few nerve terminals, but not in ganglion cell bodies. Met- and Leu-Enk immunoreactive nerve fibers showed similar distribution patterns, though minor differences were noted between the two species: Enk-immunoreactive nerve fibers in the smooth muscle layer were more abundant in guinea pigs than in rats, whereas those in mucous glands were richer in rats than in guinea pigs. These results document the presence of Met- and Leu-Enk immunoreactivity in nerve fibers supplying guinea pig and rat airways and pulmonary vessels, and provide a morphologic basis for the view that enkephalins are likely neurotransmitters or neuromodulators in the lung.

Mapping of enkephalin-related peptides in the nervous system of the blowfly, Calliphora vomitoria, and their co-localization with cholecystokinin (CCK)- and pancreatic polypeptide (PP)-like peptides.

The distribution of enkephalin-like immunoreactive material has been studied in the CNS of C. vomitoria. The presence of both Met- and Leu-enkephalin-related peptides is suggested by differential immunostaining with a variety of antisera. Comparisons made between certain of the enkephalin-immunoreactive perikarya, nerve fibres and terminals with cells in corresponding positions as evidenced in previously published neuroanatomical studies of the dipteran brain have suggested specific enkephalinergic pathways. As examples, one Met-enkephalin-immunoreactive neuron appears to link the lobula with the dorsal protocerebrum, and a group of Leu-enkephalin cells in the pars intercerebralis appear to have arborisations in both the central body (fan-shaped body) and the tritocerebral neuropil around the oesophageal foramen. Neuronal pathways of this type indicate that the enkephalin-like peptides of the fly brain are functioning as neurotransmitters and/or neuromodulators. In the thoracic ganglia, symmetrically arranged cells, immunoreactive to both Met- and Leu-enkephalin antisera, are positioned ventrally in pairs on either side of the mid-line in a sagittal plane. Very little immunoreactive material is observed in the neuropil, however, and the source of the accumulation of Leu-enkephalin-immunoreactivity in the dorsal neural sheath is not certain. It is suggested that this material, in contrast to that present in areas of the brain, acts as a neurohormone and that it may have a physiological role following its release into the haemolymph. The enkephalin-like immunoreactive material of certain neurons identified within the brain and thoracic ganglion shows a complex pattern of co-existence with pancreatic polypeptide- and gastrin/cholecystokinin-like peptides.

Identification of nonprotein ligands to the metal ions bound to glutamine synthetase.

Electron paramagnetic resonance (EPR) was used to study the environment of Mn2+ bound to the tight (n1) metal ion binding site of glutamine synthetase in the presence of analogues of the tetrahedral adduct, L-methionine (S)-sulfoximine [Met(O)(NH)-S] and L-methionine (R)-sulfoximine [Met(O)(NH)-R]. The Mn2+ EPR spectrum in the presence of Met(O)(NH)-S is identical with the previously published spectrum obtained from a mixture of isomers [Met(O)(NH)-RS] [Villafranca, J. J., Ash, D. E., & Wedler, F. C. (1976) Biochemistry 15, 544] and is characteristic of a highly octahedral metal ion environment with a small zero field splitting. The presence of Met(O)(NH)-R produces an EPR spectrum that appears characteristic of a more distorted metal ion environment, with a larger zero field splitting. These data demonstrate that the two isomers interact differently with the enzyme-bound Mn2+. Broadening of the Mn2+ EPR spectrum in the presence of Met(O)(NH) is observed in 17O-enriched water due to superhyperfine coupling of water to the metal ion. Deconvolution of the spectrum demonstrates the presence of at least a single water molecule in the inner coordination sphere of the metal ion. Superhyperfine coupling due to the 14N nucleus of the imine nitrogen of the sulfoximine moiety of Met(O)(NH)-S but not of Met(O)(NH)-R has been detected by electron spin-echo envelope modulation spectroscopy. Two intense peaks are evident in the presence of Met(O)(NH)-S with frequencies at 1.7 and 3.3 MHz. These peaks are absent when [15N]imine-labeled Met(O)(NH) is used, indicating the presence of the sulfoximine nitrogen of Met(O)(NH)-S in the inner coordination sphere of the metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth support and toxicity of homocysteine and its effects on methionine metabolism in non-transformed and chemically transformed C3H/10T1/2 cells.

The effects of homocysteine (Hcy) on one non-transformed (Cl 8) and two malignant clones (Cl 16 and Cl T422) of the C3H/10T1/2 mouse embryo fibroblasts, were examined with regard to toxicity, ability to support growth and effects on methionine (Met) metabolism and glutathione level. Homocysteine in its reduced form (Hcy-SH) was toxic to all cell lines, and the LD90 was estimated to be 1.0 X 10(-4) M for Cl 8 and Cl 16 cells measured by plating efficiency, 0.8 X 10(-4) M for Cl 8 and 0.3 X 10(-4) M for Cl 16 when measured by total cell growth. At toxic concentrations, Hcy-SH showed a drastic effect on cell morphology both in the presence and absence of Met. The same effect was demonstrated with L-cysteine. No toxic effect was seen with homocystine (Hcy-SS-Hcy) or homocysteine thiolactone (Hcy-tl) at similar concentrations. Hcy-tl supported growth of both the non-transformed and malignant cells in Met-deficient medium but with decreasing efficiency in the order Cl 8, Cl 16 and Cl T422. The growth rate constant compared to that of Met-supplemented medium was 0.62 for Cl 8, 0.44 for Cl 16 and 0.38 for Cl T422 cells. The intracellular level of S-adenosylhomocysteine (AdoHcy) increased in all three cell lines in Hcy-tl-supplemented medium. The S-adenosylmethionine (AdoMet) content increased in Cl 8 cells, was constant in Cl 16 cells and decreased in Cl T422 cells under the same conditions. This resulted in a constant ratio of AdoMet/AdoHcy in the non-transformed cells (Cl 8) whereas this ratio decreased by 40% in Cl 16 and by 72% in Cl T422 cells when Hcy-tl replaced Met in the medium. The ability of Hcy-tl to support growth thus seemed to correlate well with alteration in Met metabolism in this cell culture system. The intracellular level of glutathione (GSH) was measured during exponential growth, but showed small variations between non-transformed cells and Cl 16 cells. However, Cl T422 cells showed a distinct lower level of GSH in Met-supplemented medium, and this increased 3- to 4-fold when Met was replaced with Hcy-tl.