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PubMed1997

Characterization and glutamyl preferring carboxypeptidase function of prostate specific membrane antigen: a novel folate hydrolase.

We have cloned the gene encoding the prostate-specific membrane (PSM) antigen, which is recognized by the 7E11C-5 antibody. The antigen is strongly expressed in prostate cancer, and the antibody has been approved for use as an imaging agent for detection of prostatic cancer metastasis. The gene was unique and encoded a type II membrane protein. The only clue to its potential function was found in the cDNA coding sequences from 1250 to 1700, which had a modest but significant homology with transferrin-receptor, demonstrating a 54% homology of nucleic acid sequence. In comparing the mRNA obtained from normal prostate with that obtained from cancerous or lymph node carcinoma of the prostate (LNCaP) cells, normal cells produced a shorter alternative spliced species that encoded a cytosolic form of the protein, and not a membrane protein. It appeared that, as the prostatic cells became cancerous, there was a nearly 100-fold difference in expression of the ratio of the messages encoding the 2 forms, with the cytosolic form (PSM') predominant in normal cells and the membrane form (PSM) predominant in cancer cells. The other tissue in which the membrane antigen form of PSM is highly expressed is the membrane brush border of the small intestine of the proximal, but not distal, small intestine. This is the location of a unique membrane form of a folate hydrolase. This membrane folate hydrolase and its location are necessary in human nutrition because humans require folate, and the folate in foods is poly-gamma-glutamated. Polyglutamated folates cannot be taken into the cells by folate-transporter systems. The ability to take up folate from foods requires the membrane folate hydrolase to sequentially remove the gamma-linked glutamates, freeing folate that can then be transported. PSM antigen has a similar folate hydrolase activity. Others have reported finding an enzyme in the rat brain that functions as an alpha-neurocarboxypeptidase and acts on the abundant brain peptide N-acetylaspartylglutamate to generate glutamate and N-acetylaspartate. The 3'-end of the rat brain enzyme had 84% sequence homology with PSM antigen. Because this enzyme liberates glutamate in the brain, the enzyme is considered to have regulatory activity related to glutamate receptors. Current investigations are underway to determine whether glutamate receptors are present in prostate. Thus, PSM antigen is a unique folate hydrolase-carboxypeptidase that can release glutamate with either gamma-or alpha-linkage. Its enzymatic activity raises a number of questions for consideration. In the normal prostate where the protein is intracellular, is PSM' antigen keeping folate in nonglutamated forms? If so, folate should be able to readily diffuse out of prostate cells, making the prostate gland an organ at risk for localized folate deficiency and carcinogenesis. In prostate tumor cells, with the enzyme outside of the cell, can PSM antigen be used for the activation of cytotoxic prodrugs?

Amino Acid Sequence↗
PubMed1994

A novel class of monoglutamated antifolates exhibits tight-binding inhibition of human glycinamide ribonucleotide formyltransferase and potent activity against solid tumors.

Tight-binding inhibition of recombinant human monofunctional glycinamide ribonucleotide formyltransferase by Lometrexol (6R-5,10-dideazatetrahydrofolate) requires polyglutamation. LY254155 and LY222306 differ from 5,10-dideazatetrahydrofolate in the replacement of the 1',4'- phenylene moiety by a 2',5'-thiophene and a 2',5'-furan, respectively. Compared to Lometrexol, the thiophene and furan analogues had 25- and 75-fold greater inhibitory potencies against human monofunctional glycinamides ribonucleotide formyltransferase (Ki = 2.1 and 0.77 nM, respectively). The binding affinities of the thiophene and furan analogues for membrane folate-binding protein from human KB cells were 6- and 350-fold weaker than Lometrexol, respectively. Both the thiophene analogue and 5,10-dideazatetrahydrofolate inhibited the in vivo growth of murine 6C3HED lymphosarcoma, murine C3H mammary carcinoma, and human xenograft HXGC3, HC1, and VRC5 colon carcinomas by 95-100%. The thiophene analogue was efficacious against human xenograft PANC-1, a pancreatic carcinoma which was completely resistant to 5,10- dideazatetrahydrofolate. These novel antifolates represent the first monoglutamated tight-binding inhibitors of glycinamide ribonucleotide formyltransferase. By eliminating the need for polyglutamation, this class of antifolates may have clinical activity in the treatment of solid tumors expressing low levels of folylpolyglutamate synthetase or tumors resistant to antifolate therapy due to increased gamma-glutamyl hydrolase activity.

Acyltransferases↗
PubMed1997

Antifolates in clinical development.

Many novel antifolate compounds with unique pharmacologic properties are currently in clinical development. These newer antifolates differ from methotrexate, the most widely used and studied drug in this class, in terms of their lipid solubility and cellular transport affinity, their level of polyglutamation, and their specificity for inhibiting folate-dependent enzymes, such as dihydrofolate reductase, thymidylate synthase, or glycinamide ribonucleotide formyltransferase. The current status (ie, mechanism of action, clinical response rates, and toxicity) of some of the newer antifolate compounds presently in clinical testing, including edatrexate, piritrexim, raltritrexed, LY 231514, AG337, AG331, 1843U89, ZD 9331, and lometrexol, is reviewed.

Aminopterin↗
PubMed1984

New folate analogs of the 10-deaza-aminopterin series. Basis for structural design and biochemical and pharmacologic properties.

Structural modification of the N10 position of 4-amino folates affects mediated membrane transport in mammalian cells but has little or no effect on target enzyme (dihydrofolate reductase) inhibition. Some of these modifications have been associated with differential effects on transport in various cell types in a manner which favored greater accumulation and persistence of drug in responsive tumor cells than in normal proliferative tissue. With the aid of identifying new structures with greater potential for differential mediated accumulation, we have studied three new 10-alkyl analogs of 10-deaza-aminopterin. Two of these analogs showed therapeutic efficacy substantially greater than 10-deaza-aminopterin, an analog with antitumor properties superior to methotrexate. These analogs, the 10-methyl, 10-ethyl, and 10,10-dimethyl derivatives, were equivalent to the parent compound, 10-deaza-aminopterin, and aminopterin, and slightly more potent than methotrexate, as inhibitors of L1210 cell dihydrofolate reductase. The three new analogs, 10-deaza-aminopterin, and aminopterin exhibited similar transport properties in L1210, Ehrlich, and S180 cells. Efflux and influx Vmax were similar to those of methotrexate, but influx Km was 4- to 14-fold lower than for methotrexate. That is, substitution at N10, but not at C10, reduced influx potential in these tumor cells. These differences in transport properties among this group of analogs which determine net accumulation were reflected in the individual values for growth-inhibitory potency. In contrast to that seen in tumor cells, alkylation at both N10 and C10 reduced influx potential (increased Km) in isolated intestinal epithelial cells from mouse small intestine. Influx was in the order aminopterin greater than 10-deaza-aminopterin with further reduction in each series showing a magnitude in proportion to the size of the 10 substituent. Otherwise, influx Vmax and efflux were similar for the group. Accumulation of polyglutamates in small intestine was greater following aminopterin administration than following administration of other analogs (10-ethyl, 10-deaza-aminopterin less than methotrexate less than 10-deaza-aminopterin). Polyglutamate accumulation for all the analogs was greater in tumor cells, but accumulation of each varied between the two tumors (L1210 and S180) examined. Differences among the analogs were not as great in L1210 as in S180 cells, and their metabolism was not in the same relative order.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminopterin↗
PubMed1997

Folates and one-carbon metabolism in plants and fungi.

Folate-dependent pathways of one-carbon metabolism are essential for the synthesis of purines, formylmethionyl-tRNA, thymidylate, serine and methionine. These syntheses use a cellular source of one-carbon substituted, tetrahydrofolate polyglutamate derivatives which are the preferred substrates of most folate-dependent enzymes. In the last decade, there have been major advances in the folate biochemistry of animal, bacterial, fungal and plant systems. These have included the refinement of methods for folate isolation and characterization, basic work on key enzymes of folate biosynthesis and the detailed characterization of proteins that catalyze the generation and utilization of one-carbon substituted folates.

Folic Acid↗
PubMed1996

Resistance to tomudex (ZD1694): multifactorial in human breast and colon carcinoma cell lines.

ZD1694 (Tomudex; TDX) is a quinazoline antifolate that, when polyglutamated, is a potent inhibitor of thymidylate synthase (TS), the enzyme that converts dUMP to dTMP. Continuous exposure of MCF-7 breast and NCI H630 colon cells to TDX, with stepwise increases in TDX up to 2.0 microM, resulted in stably resistant cell lines (MCFTDX and H630TDX) that were highly resistant to TDX. Initial studies revealed 34-fold increase in TS protein levels in MCFTDX and a 52-fold increase in TS levels in H630TDX cell lines. Despite continued exposure of these cells to 2.0 microM TDX, TS protein and TS mRNA expression decreased to parental levels in H630TDX cells, whereas in MCFTDX cells TS mRNA expression and TS protein levels remained elevated. Southern blot analysis revealed a 20-fold TS gene amplification in the MCFTDX cell line. TDX uptake was 2-fold higher in resistant MCFTDX cells than in parental MCF-7 cells, whereas in H630TDX cells TDX uptake was 50-fold less than that observed in parental H630 cells. In contrast, no change in the transport of either leucovorin or methotrexate into H630TDX cells was noted when compared with the H630 parental cells. In H630TDX cells, folylpolyglutamate synthetase (FPGS) activity was 48-fold less compared to parent H630 cells; however, FPGS mRNA expression was similar in both lines. H630TDX cells were also highly resistant to ZD9331, a novel quinazoline TS inhibitor that does not require polyglutamation, suggesting that defective transport by the reduced folate carrier was also an important mechanism of resistance in these cells. In MCFTDX and H630TDX resistant cells, several mechanisms of resistance are apparent: one increased TS expression; the others evolved over time from increased TS expression to decreased FPGS levels and decreased TDX transport.

Blotting, Northern↗
PubMed2003

Design, synthesis and biological evaluation of 10-CF3CO-DDACTHF analogues and derivatives as inhibitors of GAR Tfase and the de novo purine biosynthetic pathway.

The synthesis and evaluation of analogues and key derivatives of 10-CF3CO-DDACTHF as inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. Polyglutamate analogues of 1 were evaluated as inhibitors of Escherichia coli and recombinant human (rh) GAR Tfase, and AICAR Tfase. Although the pentaglutamate 6 was found to be the most active inhibitor of the series tested against rhGAR Tfase (Ki=0.004 microM), little distinction between the mono-pentaglutamate derivatives was observed (Ki=0.02-0.004 microM), suggesting that the principal role of the required polyglutamation of 1 is intracellular retention. In contrast, 1 and its defined polyglutamates 3-6 were much less inactive when tested against rhAICAR Tfase (Ki=65-0.120 microM) and very selective (> or =100-fold) for rh versus E. coli GAR Tfase. Additional key analogues of 1 were examined (7 and 8) and found to be much less active (1000-fold) highlighting the exceptional characteristics of 1.

Drug Design↗
PubMed1987

Human liver folylpolyglutamate synthetase: biochemical characterization and interactions with folates and folate antagonists.

Folylpolyglutamate synthetase (FPGS) was isolated from human liver cytosol by 0-30% (w/v) ammonium sulfate fractionation and characterized biochemically. Using aminopterin (AMT), L-[3H]glutamate and MgATP as cosubstrates, maximal gamma-L-glutamylation activity was observed in the presence of the activators KCl and NaHCO3. ATP and 2-mercaptoethanol were each required for enzyme activity and stability. In the absence of ATP, human liver FPGS rapidly inactivated at 37 degrees C (t1/2 approximately 8 min), whereas FPGS isolated from rabbit liver was significantly more stable (t1/2 = 68 min). Both folates and antifolates were effectively polyglutamylated by the isolated human liver enzyme. Km parameters determined for AMT (Km = 4.3 microM) were similar to those determined for several reduced folates (tetrahydrofolic acid, dihydrofolic acid, and folinic acid; Km = 3-7 microM), while significantly higher Km values were observed for methotrexate (MTX) and 5-methyltetrahydrofolic acid (Km = 50-60 microM) and for folic acid (Km = 100 microM). All of the substrates examined exhibited Vmax values ranging from 30 to 90% of the AMT value (Vmax = 935 pmol product/mg/h). The order of reactivity for these substrates differed from that determined in parallel studies for FPGS isolated from rat and rabbit liver. In the case of AMT and several reduced folates, inhibition of human liver FPGS was observed at substrate concentrations at or above 50-250 microM. FPGS isolated from six individual human livers exhibited highly similar biochemical and kinetic properties, suggesting the presence of the same or at least highly similar enzyme species in each individual, with a five-fold interindividual range in specific activities observed. Comparison of MTX with its higher polyglutamates (MTX-Glu2 to MTX-Glu6) as FPGS substrates indicated a significant decrease in Vmax values with increasing glutamate chain length which was partially compensated for by a corresponding decrease in Km. Consistent with these observations, the isolated enzyme was unable to synthesize polyglutamates higher than MTX-Glu3 when MTX was supplied as substrate, raising the question as to how MTX polyglutamates containing up to five or six gamma-L-glutamate residues are formed in vivo.

Animals↗
PubMed2002

Influence of processing on total, monoglutamate and polyglutamate folate contents of leeks, cauliflower, and green beans.

Bioavailability of dietary folate might be impaired by the polyglutamate chain to which approximately 70% of dietary folates are bound. This chain must be removed enzymatically in the intestine before folate is absorbed as a monoglutamate. To increase formation of monoglutamate folate in vegetables, the vegetables were subjected to various processing treatments. Treatments included freezing (-18 degrees C, 16 h) and thawing (4 degrees C, 24 h) and hydrostatic high-pressure treatment (200 MPa, 5 min). Both freezing/thawing and high-pressure treatment increased the proportion of folate in the monoglutamate form in leeks, cauliflower, and green beans 2-3-fold. However, loss of total folate after these treatments was >55%. It is concluded that conversion of folate polyglutamate to the monoglutamate form in vegetables is possible by certain processing treatments. Potentially this could lead to vegetables with higher folate bioavailability. However, to prevent folate loss into processing water, processing in a closed system should be applied.

Biological Availability↗
PubMed1985

The binding of folyl- and antifolylpolyglutamates to hemoglobin.

A binding method that detects only the strongest binding site for a ligand on a protein has been used to show that folates and folate analogs, conjugated with poly-gamma-glutamates, are bound to hemoglobin. When the concentration of hemoglobin is much larger than that of the polyglutamate, as is the case in the red cell, the fraction bound is a direct function of the hemoglobin concentration and is independent of the total polyglutamate concentration. Binding to deoxyhemoglobin tetramers is competitive with 2,3-diphosphoglycerate. In oxyhemoglobin the folyl and methotrexate polyglutamates are bound preferentially by free alpha beta dimers, but removal of the pteridine moiety leads to tetramer binding even in oxyhemoglobin. Changes in the length of the polyglutamate side chain and alterations of the pteridine structure such as reduction and/or methylation have a much larger effect on the constant for binding to deoxyhemoglobin tetramers than on that for oxyhemoglobin dimers. The implications of these results for the storage of pteroylpolyglutamates in the erythrocyte and their release from the red cell under the influence of the degree of oxygenation and variations in the 2,3-diphosphoglycerate level are discussed.

Folic Acid↗
PubMed2002

Dietary monoglutamate and polyglutamate folate are associated with plasma folate concentrations in Dutch men and women aged 20-65 years.

Dietary folate consists of monoglutamate and polyglutamate folate species. In the small intestine, folate polyglutamate is deconjugated to the monoglutamate form before absorption takes place. This enzymatic deconjugation might limit the bioavailability of polyglutamate folate. Until now, no data have been available on dietary intake of both folate forms and their associations with folate status. Therefore, we estimated the intake of monoglutamate and polyglutamate folate in the Dutch population and studied whether the association with plasma folate is different for these two folate forms. Dietary intake of monoglutamate and polyglutamate folate from nonfortified foods was estimated for 2435 subjects (1275 men; 1160 women) aged 20-65 y. The intake of monoglutamate folate was about one third of total folate intake, derived mainly from bread (approximately 20%) and meat (approximately 18%), whereas two thirds consisted of polyglutamates, derived mainly from vegetables (approximately 25%). The predictive power of the regression model with total folate intake as the independent variable adjusted for age, smoking and alcohol intake, did not increase when including the ratio of monoglutamate to polyglutamate folate intake. In addition, linear regression models showed that both monoglutamate and polyglutamate folate intake were associated positively with plasma folate levels. However, in men, the monoglutamate folate form appeared to be a threefold stronger determinant of plasma folate levels than polyglutamate folate, whereas in women, both folate forms were equally strong determinants. This might be explained by different food intake patterns of men and women, including alcohol intake. At present, it does not seem necessary to distinguish between food folate forms in advising an increase in folate intake from nonfortified foods.

Adult↗
PubMed1997

The effects of combined antifolates on inhibition of growth of murine leukemia cells cultured in vitro.

The synergistic effect of trimetrexate (TMTX) and sulphonamide derivatives of quinazoline on the cultured 5178Y murine leukemia cells was examined. On exposure to the slightly inhibitory concentrations of TMTX (0.1 nM) in combination with 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroyl-sulphoglyc ine (DMPDDSF) (0.02 microM) a synergistic inhibitory effect of the antifolates on cell growth was observed. These two drugs in the same combination caused also synergistic inhibition of de novo synthesis of thymidylate in intact cells as measured by tritium release from [5-(3)H]deoxyuridylate. This was accompanied by a marked reduction in intracellular concentration of 5,10-methylenetetrahydro-pteroyl-polyglutamate (5,10CH2H4PteGlu(n)) (0.2 microM) and dihydropteroyl-polyglutamate (0.12 microM). In these conditions de novo biosynthesis of purine was decreased by 50%. These observations show that growth inhibition by combined antifolates is mediated by intracellular depletion of the substrate of thymidylate synthase -- 5,10CH2H4PteGlu(n). The results obtained strongly suggest that under certain conditions inhibition of thymidylate synthesis by DMPDDSF is intensified by prior application of TMTX -- an inhibitor of dihydrofolate reductase.

Animals↗
PubMed1997

Cellular and molecular mechanisms of resistance to antifolate drugs: new analogues and approaches to overcome the resistance.

A number of antifolate drugs, which inhibit the key enzymes in the 'thymidylate cycle', dihydrofolate reductase (DHFR) and thymidylate synthase (TS), have been developed as part of the search for analogues with superior antitumor efficacy to a 'classical' antifolate, methotrexate (MTX), and those which are active against the MTX-resistant tumor cells. Recent development of newer classes of antifolate drugs is based on the extensive understanding of the relationship between chemical structures and biological properties and of analogue interactions with target enzymes, transport proteins and folate metabolizing enzyme, folylpolyglutamate synthetase (FPGS). Tumor cells may develop resistance to an antifolate drug by virtue of, (1) amplified activity in its target enzyme, (2) impaired function of drug transport protein, e.g. reduced folate carrier (RFC), (3) induction of mutated target enzyme with low affinity for antifolate(s), and (4) defective polyglutamation of drug(s) in the cells. Recent studies have elucidated in part the molecular events involved in the resistance to antifolates. These include amplification and/or mutation of the gene encoding a target enzyme, reduced or altered gene expression of the RFC, and mutated expression of the FPGS gene. To overcome or circumvent the resistance mechanisms, new antifolates with diverse structures and different biological properties have been designed and developed for clinical use. Trimetrexate (TMQ), a lipophilic DHFR inhibitor which is not a substrate for RFC and FPGS, could overcome the MTX-resistance through impaired RFC and diminished polyglutamation, and partially through amplified DHFR. Selective inhibitors of TS with a folate structure such as raltitrexed could circumvent the resistance by virtue of DHFR overproduction, and this class of compounds which have higher substrate activities for FPGS than MTX may be of value for the treatment of myeloid leukemias in addition to lymphocytic malignancies resistant to conventional chemotherapy. Several strategies to overcome antifolate resistance by using gene therapy are currently under investigation.

Antimetabolites, Antineoplastic↗
PubMed1980

Distribution of pteroylglutamates in rat liver during regeneration after partial hepatectomy.

1. Liver pteroylpolyglutamate distribution was studied during regeneration after partial hepatectomy in rats maintained under controlled feeding conditions. 2. Pteroylhexaglutamate, pteroylpentaglutamate and pteroyltetraglutamate concentrations decrease from 12 to 72 h after operation, then increase and reach normal values at 180 h. Pteroyltriglutamate concentration, already high at 12 h, remains so in the subsequent periods. Pteroyldiglutamate concentration was unchanged. Monoglutamate concentrations at first decrease, and at 180 h exceed normal values. 3. The decrease in polyglutamate derivatives with a high number of glutamate residues, at present considered to be the coenzyme forms of folate, could be related not to a decreased synthesis, but to a greater requirement for these compounds during the early periods of regeneration, when biosynthetic processes are markedly increased. It is indeed probable that the increased availability of the preferred substrate of pteroylpolyglutamate synthetase, i.e. tetrahydrofolate, enhances conversion of folate into coenzyme forms.

Animals↗
PubMed1983

Folylpoly-gamma-glutamate synthetases: properties and regulation.

The substrate specificities of folylpolyglutamate synthetases from Corynebacterium species, Lactobacillus casei, Streptococcus faecalis, and Chinese hamster ovary (CHO) cells are described. The purified Corynebacterium enzyme catalyzes the addition of glutamate moieties to reduced pteroylmonoglutamates, and to 5,10-methylenetetrahydropteroyldi- and triglutamates. The purified protein also possesses a separate dihydrofolate synthetase activity. Binding of pteroate substrates to dihydrofolate synthetase impairs folate binding to folylpolyglutamate synthetase. The in vivo distribution of folylpolyglutamates in bacteria and mammalian cells, which differs from source to source, appears to be a reflection of the ability of folylpolyglutamates to act as substrates for folylpolyglutamate synthetases from different sources. Bacterial synthetases use a variety of pteroylmonoglutamates as their preferred monoglutamate substrate, but use 5,10-methylenetetrahydropteroyl polyglutamates as their preferred, and sometimes only, polyglutamate substrates. Mono- and polyglutamyl forms of tetrahydrofolate are the preferred substrates of the CHO cell synthetase. Products of one carbon metabolism do not affect the in vivo distribution of folylpolyglutamates in CHO cells. Although adenine changes the distribution of folylpolyglutamates in bacteria, this effect is mediated by changing the substrate availability for the synthetase reaction, and not by regulating the levels of the synthetase per se.

Animals↗
PubMed1989

Reconstitution of Escherichia coli DNA photolyase with various folate derivatives.

DNA photolyase from Escherichia coli contains both flavin and pterin. However, the isolated enzyme is depleted with respect to the pterin chromophore (0.5 mol of pterin/mol of flavin). The extinction coefficient of the pterin chromophore at 360 nm is underestimated by a method used in earlier studies which assumes stoichiometric amounts of pterin and flavin. The extinction coefficient of the pterin chromophore, determined on the basis of its (p-aminobenzoyl)polyglutamate content (epsilon 360 = 25.7 x 10(3) M-1 cm-1), is in good agreement with that expected for a 5,10-methenyltetrahydrofolate derivative. Also consistent with this structure, the pterin chromophore could be reversibly hydrolyzed to yield a 10-formyltetrahydrofolate derivative or reduced to yield a 5-methyltetrahydrofolate derivative. The isolated enzyme could be reconstituted with various folate derivatives to yield enzyme that contained equimolar amounts of pterin and flavin. Similar results were obtained in reconstitution studies with the natural pterin chromophore, with 5,10-methenyltetrahydrofolate, and with 10-formyltetrahydrofolate. The results show that the polyglutamate moiety, previously identified in the natural chromophore, is not critical for binding. Reconstitution with the natural pterin chromophore did not affect catalytic activity. The latter is consistent with our previous studies which show that, although the pterin chromophore acts as a sensitizer in native enzyme, it is not essential for dimer repair which can occur at the same rate under saturating light with flavin (1,5-dihydro-FAD) as the only chromophore.

Deoxyribodipyrimidine Photo-Lyase↗
PubMed2004

Raman spectroscopic characterization of secondary structure in natively unfolded proteins: alpha-synuclein.

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.

Amides↗
PubMed1989

Comparison of the conversion of 5-formyltetrahydrofolate and 5-methyltetrahydrofolate to 5,10-methylenetetrahydrofolates and tetrahydrofolates in human colon tumors.

Four hr infusions i.v. of [6RS]5-formyltetrahydrofolate ([6RS]5-CHO-H4PteGlu; 500 mg/m2) and [6RS]5-methyltetrahydrofolate ([6RS]5-CH3-H4PteGlu; 500 mg/m2) were compared for their relative effects on expansion of pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun) in two human colon adenocarcinoma xenografts in mice. Expansion of these pools by 253-661% of control and increase in predominance of di-, tri-, and tetra-glutamate species were observed during [6RS]5-CHO-H4PteGlu infusion. In contrast, only modest pool size expansion (148-164% of control) and limited modulation of polyglutamate species were detected in four tumor lines during infusion with [6RS]5-CH3-H4PteGlu. The data suggest that [6RS]5-CH3-H4PteGlu is less effective than [6RS]5-CHO-H4PteGlu as a precursor for pools of CH2-H4PteGlun and H4PteGlun in colon tumors.

Adenocarcinoma↗