Polyglutamate Chain Research Articles

The distinct initiation sites and processing activities of TTLL4 and TTLL7 in glutamylation of brain tubulin

Mammalian brain tubulins undergo a reversible posttranslational modification-polyglutamylation-which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins, both with preference for the β-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for α-isoform than β-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. Tandem mass spectrometry analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl termini of α1- and β2-tubulins and a recombinant tubulin. Particularly, in the recombinant α1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules premodified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by TTLL4-initiated sites. Finally, we showed that kinesin behaves differentially on microtubules modified by two enzymes. This study underpins the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and sheds light on their distinct rolein vivo.

Combinatorial and antagonistic effects of tubulin glutamylation and glycylation on katanin microtubule severing.

Microtubules have spatiotemporally complex posttranslational modification patterns. How cells interpret this tubulin modification code is largely unknown. We show that C.elegans katanin, a microtubule severing AAAATPase mutated in microcephaly and critical for cell division, axonal elongation, and cilia biogenesis, responds precisely, differentially, and combinatorially to three chemically distinct tubulin modifications-glycylation,glutamylation, and tyrosination-but is insensitive to acetylation. Glutamylation and glycylation are antagonistic rheostats with glycylation protecting microtubules from severing. Katanin exhibits graded and divergent responses to glutamylation on the α- and β-tubulin tails, and these act combinatorially. The katanin hexamer central pore constrains the polyglutamate chain patterns on β-tails recognized productively. Elements distal to the katanin AAA core sense α-tubulin tyrosination, and detyrosination downregulates severing. The multivalent microtubule recognition that enables katanin to read multiple tubulin modification inputs explains invivo observations and illustrates how effectors can integrate tubulin code signals to produce diverse functional outcomes.

Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes.

Glutamylation, introduced by tubulin tyrosine ligase-like (TTLL) enzymes, is the most abundant modification of brain tubulin. Essential effector proteins read the tubulin glutamylation pattern, and its misregulation causes neurodegeneration. TTLL glutamylases post-translationally add glutamates to internal glutamates in tubulin carboxy-terminal tails (branch initiation, through an isopeptide bond), and additional glutamates can extend these (elongation). TTLLs are thought to specialize in initiation or elongation, but the mechanistic basis for regioselectivity is unknown. We present cocrystal structures of murine TTLL6 bound to tetrahedral intermediate analogs that delineate key active-site residues that make this enzyme an elongase. We show that TTLL4 is exclusively an initiase and, through combined structural and phylogenetic analyses, engineer TTLL6 into a branch-initiating enzyme. TTLL glycylases add glycines post-translationally to internal glutamates, and we find that the same active-site residues discriminate between initiase and elongase glycylases. These active-site specializations of TTLL glutamylases and glycylases ultimately yield the chemical complexity of cellular microtubules.

Laboratory assessment of folate (vitamin B9) status

Folate (vitamin B9) plays a crucial role in fundamental cellular processes, including nucleic acid biosynthesis, methyl group biogenesis and amino acid metabolism. The detection and correction of folate deficiency prevents...

Genetically determined folate deficiency is associated with abnormal hepatic folate profiles in the spontaneously hypertensive rat.

Increased levels of plasma cysteine are associated with obesity and metabolic disturbances. Our recent genetic analyses in spontaneously hypertensive rats (SHR) revealed a mutated Folr1 (folate receptor 1) as the quantitative trait gene associated with diminished renal Folr1 expression, lower plasma folate levels, hypercysteinemia, hyperhomocysteinemia and metabolic disturbances. To further analyse the effects of the Folr1 gene expression on folate metabolism, we used mass spectrometry to quantify folate profiles in the plasma and liver of an SHR-1 congenic strain, with wild type Folr1 allele on the SHR genetic background, and compared them with the SHR strain. In the plasma, concentration of 5-methyltetrahydrofolate (5mTHF) was significantly higher in SHR-1 congenic rats compared to SHR (60+/-6 vs. 42+/-2 nmol/l, P<0.01) and 5mTHF monoglutamate was the predominant form in both strains (>99 % of total folate). In the liver, SHR-1 congenic rats showed a significantly increased level of 5mTHF and decreased concentrations of dihydrofolate (DHF), tetrahydrofolate (THF) and formyl-THF when compared to the SHR strain. We also analysed the extent of folate glutamylation in the liver. Compared with the SHR strain, congenic wild-type Folr1 rats had significantly higher levels of 5mTHF monoglutamate. On the other hand, 5mTHF penta- and hexaglutamates were significantly higher in SHR when compared to SHR-1 rats. This inverse relationship of rat hepatic folate polyglutamate chain length and folate sufficiency was also true for other folate species. These results strongly indicate that the whole body homeostasis of folates is substantially impaired in SHR rats compared to the SHR-1 congenic strain and might be contributing to the associated metabolic disturbances observed in our previous studies.

Origins of the difference between food folate analysis results obtained by LC–MS/MS and microbiological assays

To investigate the often reported disagreement in food folate quantitation between the microbiological assay and high-performance liquid chromatography methods, different foods were analyzed both by a microbiological assay and by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. For the LC-MS/MS analysis we emphasize the need for complete deconjugation of polyglutamic folate forms. Moreover, our results revealed no need for an additional enzyme treatment except in the deconjugation step. To check the efficiency of deconjugation without additional sample preparations, the amount of diglutamates was quantified and samples were screened for additional polyglutamates. A thorough investigation of a substance with a polyglutamate chain deconjugated like the folates revealed that it was an oxidation product of 5-methyltetrahydrofolate, a pyrazino-s-triazine called MeFox in previous reports. The latter is not microbiologically active and, therefore, does not contribute to the amount of total folates. But we found it is commonly present in foods, especially in those low in ascorbic acid. The microbiological assay showed different responses to the single vitamers. Therefore, it was necessary to perform calibration with the folate that had the highest portion in the folate distribution. The investigations showed that both methods can provide similar results when they both include a deconjugation step. This is particularly important for LC-MS/MS but probably also for the microbiological assay. Additionally, consideration of the folate distribution was found to be crucial for the accurate calibration of the microbiological assay.

Folylpoly-γ-glutamate synthetase: A key determinant of folate homeostasis and antifolate resistance in cancer

Mammalians are devoid of autonomous biosynthesis of folates and hence must obtain them from the diet. Reduced folate cofactors are B9-vitamins which play a key role as donors of one-carbon units in the biosynthesis of purine nucleotides, thymidylate and amino acids as well as in a multitude of methylation reactions including DNA, RNA, histone and non-histone proteins, phospholipids, as well as intermediate metabolites. The products of these S-adenosylmethionine (SAM)-dependent methylations are involved in the regulation of key biological processes including transcription, translation and intracellular signaling. Folate-dependent one-carbon metabolism occurs in several subcellular compartments including the cytoplasm, mitochondria, and nucleus. Since folates are essential for DNA replication, intracellular folate cofactors play a central role in cancer biology and inflammatory autoimmune disorders. In this respect, various folate-dependent enzymes catalyzing nucleotide biosynthesis have been targeted by specific folate antagonists known as antifolates. Currently, antifolates are used in drug treatment of multiple human cancers, non-malignant chronic inflammatory disorders as well as bacterial and parasitic infections. An obligatory key component of intracellular folate retention and intracellular homeostasis is (anti)folate polyglutamylation, mediated by the unique enzyme folylpoly-γ-glutamate synthetase (FPGS), which resides in both the cytoplasm and mitochondria. Consistently, knockout of the FPGS gene in mice results in embryonic lethality. FPGS catalyzes the addition of a long polyglutamate chain to folates and antifolates, hence rendering them polyanions which are efficiently retained in the cell and are now bound with enhanced affinity by various folate-dependent enzymes. The current review highlights the crucial role that FPGS plays in maintenance of folate homeostasis under physiological conditions and delineates the plethora of the molecular mechanisms underlying loss of FPGS function and consequent antifolate resistance in cancer.

Folate depletion and increased glutamation in juvenile idiopathic arthritis patients treated with methotrexate.

Folates exist as a fluctuating pool of polyglutamated metabolites that may serve as a clinical marker of methotrexate (MTX) activity. This study was undertaken to evaluate circulating folate content and folate polyglutamate distribution in juvenile idiopathic arthritis (JIA) patients and in a cell culture model based on MTX exposure and folate supply. Blood, plasma, and red blood cell (RBC) measurements of MTX and folates were obtained from previously published data sets and an additional analysis of JIA patients receiving MTX (n = 98) and those not receiving MTX (n = 78). Erythroblastoid cells maintained in culture were exposed to MTX and grown under varying levels of folic acid supplementation. Samples were analyzed for cellular folate and MTX content. Circulating folate levels were lower in JIA patients receiving MTX, with reduced levels of blood, plasma, and RBC 5-methyl-tetrahydrofolate (5mTHF) (P < 0.0001). Average polyglutamate chain length (Gluavg ) of RBC 5mTHF was elevated in JIA patients receiving MTX (median ± interquartile range 5.63 ± 0.15 versus 5.54 ± 0.11 in those not receiving MTX; P < 0.001) and correlated with both RBC MTX accumulation (P = 0.02) and reduced plasma 5mTHF levels (P = 0.008). MTX exposure and folate deprivation in erythroblastoid cells resulted in a depletion of bioactive folate species that was associated with a shift to higher Gluavg values for several species, most notably tetrahydrofolate (THF) and 5,10-methylene-tetrahydrofolate (CH2 THF). Increased Gluavg resulted from the depletion of short-chain and the accumulation of long-chain glutamate species. Our findings indicate that folate content and polyglutamate distribution are responsive markers of MTX activity and folate supply in vivo and in vitro, and may provide novel clinical markers of pharmacologic activity of MTX.

Synthetic polyglutamylation of dual-functional MTX ligands for enhanced combined cytotoxicity of poly(I:C) nanoplexes.

The antifolate drug methotrexate (MTX) can serve as a dual-functional ligand in antitumoral drug delivery, inducing both a folate receptor mediated cellular uptake and an intracellular cytotoxic action. Bioactivity of MTX however changes by conjugation; the activity can be affected by the hampered intracellular conversion to more potent poly-γ-glutamyl derivatives. Therefore, in a cancer combination therapy approach for the codelivery of cytotoxic dsRNA polyinosinic-polycytidylic acid poly(I:C), a set of molecularly precise oligo(ethanamino)amides were synthesized comprising poly(ethylene glycol) conjugated MTX ligands. The conjugates differed in the number of additional glutamic acid residues to investigate the effect of different degrees of synthetic "a priori" polyglutamylation. The bioactivity of these compounds concerning dihydrofolate reductase (DHFR) inhibition, cytotoxicity, nucleic acid binding potency, cellular uptake of poly(I:C) polyplexes, and combined antifolate/poly(I:C) toxicity was investigated. Synthetic polyglutamylation had a crucial impact on several stages of efficient poly(I:C) delivery and combined MTX cytotoxicity. DHFR inhibition of the conjugates significantly increased with increasing polyglutamate chain length. The library member with highest glutamylation degree even outperformed free MTX in direct comparison. Studies in KB cells showed the corresponding enhanced cytotoxicity by polyglutamylation. Also poly(I:C) polyplexes of the glutamylated MTX variants exhibited higher cellular uptake in the folate receptor positive cell line. Finally, a synergistic combined cytotoxicity of polyglutamylated MTX ligands and complexed poly(I:C) cargo was observed in transfected KB cells. The present structure-activity relationship study of MTX-based ligands pinpoints the concept of synthetic polyglutamylation as a promising approach for optimizing bioactivity of antifolate conjugates, which might be considered as a useful tool also in context of other drug delivery systems.

Isotope Ratio-Based Profiling of Microbial Folates

Folate metabolism, which is responsible for one-carbon transfer reactions in critical cellular processes including thymidine biosynthesis, is among the most important targets of antibiotic and anticancer drugs. Analysis of intracellular folates is complicated by three different types of folate modification: oxidation/reduction, methylation, and polyglutamylation. Here we present a method for quantifying the full diversity of intracellular folates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method begins with folate extraction using -75 degrees C methanol:water, with ascorbic acid and ammonium acetate added to prevent folate interconversion. The extract is then separated using hydrophilic interaction chromatography with an amino column, ionized by positive mode electrospray, and analyzed on a triple quadrupole instrument using multiple reaction monitoring. The method has been used to profile the folate pools in Escherichia coli and Saccharomyces cerevisiae, with absolute levels of selected folates in E. coli measured by spiking extracts of cells fed uniformly (13)C-glucose with purified, unlabeled folate standards. An isotope-ratio-based approach has been applied to study the effects of trimethoprim, a clinically important antibiotic that blocks bacterial dihydrofolate reductase. In addition to causing the expected increase in oxidized and decrease in reduced folates, trimethoprim triggered a dramatic and previously unrecognized shift towards shorter polyglutamate chain lengths. This finding highlights the potential for analysis of the full spectrum of cellular folates by MS/MS to unveil novel biological phenomena.

Regulation of Folate-mediated One-carbon Metabolism by 10-Formyltetrahydrofolate Dehydrogenase

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes the NADP(+)-dependent conversion of 10-formyltetrahydrofolate to CO(2) and tetrahydrofolate (THF) and is an abundant high affinity folate-binding protein. Although several activities have been ascribed to FDH, its metabolic role in folate-mediated one-carbon metabolism is not well understood. FDH has been proposed to: 1) inhibit purine biosynthesis by depleting 10-formyl-THF pools, 2) maintain cellular folate concentrations by sequestering THF, 3) deplete the supply of folate-activated one-carbon units, and 4) stimulate the generation of THF-activated one-carbon unit synthesis by channeling folate cofactors to other folate-dependent enzymes. The metabolic functions of FDH were investigated in neuroblastoma, which do not contain detectable levels of FDH. Both low and high FDH expression reduced total cellular folate concentrations by 60%, elevated rates of folate catabolism, and depleted cellular 5-methyl-THF and S-adenosylmethionine levels. Low FDH expression increased the formyl-THF/THF ratio nearly 10-fold, whereas THF accounted for nearly 50% of total folate in neuroblastoma with high FDH expression. FDH expression did not affect the enrichment of exogenous formate into methionine, serine, or purines and did not suppress de novo purine nucleotide biosynthesis. We conclude that low FDH expression facilitates the incorporation of one-carbon units into the one-carbon pool, whereas high levels of FDH expression deplete the folate-activated one-carbon pool by catalyzing the conversion of 10-formyl-THF to THF. Furthermore, FDH does not increase cellular folate concentrations by sequestering THF in neuroblastoma nor does it inhibit or regulate de novo purine biosynthesis. FDH expression does deplete cellular 5-methyl-THF and S-adenosylmethionine levels indicating that FDH impairs the folate-dependent homocysteine remethylation cycle.

Cellular folate vitamer distribution during and after correction of vitamin B12 deficiency: a case for the methylfolate trap

Haematological sequellae of vitamin B12 deficiency are attributed to disturbed DNA synthesis, but vitamin B12 itself plays no role in DNA biosynthesis. A proposed explanation for this is the methylfolate trap hypothesis. This hypothesis states that B12 deficiency impairs overall folate metabolism because 5-methyltetrahydrofolate (5MTHF) becomes metabolically trapped. This trap results from the fact that 5MTHF can neither be metabolised via the methionine synthase pathway, nor can it be reconverted to its precursor, methylenetetrahydrofolate. Other manifestations of the methylfolate trap include cellular folate loss because of shorter 5MTHF polyglutamate chains and global hypomethylation. The methylfolate trap has never been demonstrated in humans. We describe a patient with B12 deficiency who was homozygous for the common methylenetetrahydrofolate reductase (MTHFR) C677T mutation. We analysed red blood cell (RBC) folate vitamers and global DNA methylation by liquid chromatography (LC) in combination with tandem mass spectrometry, and 5MTHF polyglutamate length by LC-electrochemical detection. Compared to post-B12 supplementation values, homocysteine was higher (52.9 micromol/l vs. 16.8 micromol/l), RBC folate was lower (268.92 nmol/l vs. 501.2 nmol/l), the 5MTHF fraction of RBC folate was much higher (94.5% vs. 67.4%), polyglutamate chain length was shorter (more tetra- and pentaglutamates), and global DNA methylation was 22% lower. This is the first time that virtually all features of the methylfolate trap hypothesis have been demonstrated in a human with vitamin B12 deficiency.

Crystal Structures of Cobalamin-independent Methionine Synthase Complexed with Zinc, Homocysteine, and Methyltetrahydrofolate

Cobalamin-independent methionine synthase (MetE) catalyzes the synthesis of methionine by a direct transfer of the methyl group of N5-methyltetrahydrofolate (CH3-H2PteGlun) to the sulfur atom of homocysteine (Hcy). We report here the first crystal structure of this metalloenzyme under different forms, free or complexed with the Hcy and folate substrates. The Arabidopsis thaliana MetE (AtMetE) crystals reveal a monomeric structure built by two (betaalpha)8 barrels making a deep groove at their interface. The active site is located at the surface of the C-terminal domain, facing the large interdomain cleft. Inside the active site, His647, Cys649, and Cys733 are involved in zinc coordination, whereas Asp605, Ile437, and Ser439 interact with Hcy. Opposite the zinc/Hcy binding site, a cationic loop (residues 507-529) belonging to the C-terminal domain anchors the first glutamyl residue of CH3-H4PteGlu5. The pterin moiety of CH3-H4PteGlu5 is stacked with Trp567, enabling the N5-methyl group to protrude in the direction of the zinc atom. These data suggest a structural role of the N-terminal domain of AtMetE in the stabilization of loop 507-529 and in the interaction with the poly-glutamate chain of CH3-H4PteGlun. Comparison of AtMetE structures reveals that the addition of Hcy does not lead to a direct coordination of the sulfur atom with zinc but to a reorganization of the zinc binding site with a stronger coordination to Cys649, Cys733, and a water molecule.

Bioavailability of heptaglutamyl relative to monoglutamyl folic acid in healthy adults

The bioavailability of dietary folate has been estimated to be approximately 50% of that of synthetic folic acid. Folate in the diet is linked to a polyglutamate chain that may restrict folate absorption. Our goal was to quantify the bioavailability and bioefficacy of low doses of polyglutamyl folic acid relative to that of monoglutamyl folic acid. In total, 180 men and women aged 50-75 y ingested capsules containing 323 nmol heptaglutamyl folic acid/d or 262 nmol monoglutamyl folic acid/d or placebo in a randomized parallel trial. Serum and erythrocyte folate and plasma homocysteine concentrations were measured after an overnight fast at baseline and after 12 wk of intervention. Mean serum and erythrocyte folate concentrations increased less in the polyglutamyl folic acid group [6.1 (95% CI: 5.3, 7.0) and 155 (122, 188) nmol/L, respectively] than in the monoglutamyl folic acid group [11.8 (10.3, 13.3) and 282 (246, 318) nmol/L, respectively]. Differences remained statistically significant (P < 0.05) after correction for the difference in the amount of folic acid administered. The decrease in plasma homocysteine concentrations did not differ significantly between treatment groups [polyglutamyl: -12.1% (-14.8%, -9.3%); monoglutamyl: -14.1% (-16.3%, -11.9%)]. The relative bioavailability of polyglutamyl folic acid was 64% (52%, 75%) on the basis of serum folate and was 68% (51%, 84%) on the basis of erythrocyte folate concentrations. Bioefficacy, determined by changes in plasma homocysteine concentrations, was 106% (77%, 134%). The polyglutamate chain of folates in the diet reduces their bioavailability.

Methenyltetrahydrofolate Synthetase Regulates Folate Turnover and Accumulation

Cellular folate deficiency impairs one-carbon metabolism, resulting in decreased fidelity of DNA synthesis and inhibition of numerous S-adenosylmethionine-dependent methylation reactions including protein and DNA methylation. Cellular folate concentrations are influenced by folate availability, cellular folate transport efficiency, folate polyglutamylation, and folate turnover specifically through degradation. Folate cofactors are highly susceptible to oxidative degradation in vitro with the exception of 5-formyltetrahydrofolate, which may be a storage form of folate. In this study, we determined the effects of depleting cytoplasmic 5-formyltetrahydrofolate on cellular folate concentrations and folate turnover rates in cell cultures by expressing the human methenyltetrahydrofolate synthetase cDNA in human MCF-7 cells and SH-SY5Y neuroblastoma. Cells with increased methenyltetrahydrofolate synthetase activity exhibited: 1) increased rates of folate turnover, 2) elevated generation of p-aminobenzoylglutamate in culture medium, 3) depressed cellular folate concentrations independent of medium folic acid concentrations, and 4) increased average polyglutamate chain lengths of folate cofactors. These data indicate that folate catabolism and folate polyglutamylation are competitive reactions that influence cellular folate concentrations, and that increased methenyltetrahydrofolate synthetase activity accelerates folate turnover rates, depletes cellular folate concentrations, and may account in part for tissue-specific differences in folate accumulation.

Immobilization of polyglutamate-glucose oxidase onto a cysteamine-modified gold electrode

Polyglutamate–glucose oxidase (GOD) complex was prepared, and the complex was dropped on a cysteamine-modified gold electrode to immobilize the complex; the electrostatic binding between the cysteamine on the electrode surface and the polyglutamate moiety of the complex resulted in the formation of a GOD-attached electrode. The enzyme electrode showed quick response (∼15 s), since the enzyme layer was quite thin (enzyme-complex monolayer structure). When we use un-modified GOD instead of polyglutamate–GOD, the sensitivity of the resulting enzyme electrode was quite low. This clearly shows that the presence of polyglutamate chain of the complex is essential for the immobilization of the modified electrode surface, i.e., the polyglutamate moiety acts as an anchor part for the complex. A linear relationship between the current response on the complex immobilized electrode and the glucose concentration was observed between 5 and 100 μM. The immobilization technique can widely be applied to prepare biosensors and biochips.

Location of the Pteroylpolyglutamate-binding Site on Rabbit Cytosolic Serine Hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) catalyzes the reversible interconversion of serine and glycine with transfer of the serine side chain one-carbon group to tetrahydropteroylglutamate (H(4)PteGlu), and also the conversion of 5,10-methenyl-H(4)PteGlu to 5-formyl-H(4)PteGlu. In the cell, H(4)PteGlu carries a poly-gamma-glutamyl tail of at least 3 glutamyl residues that is required for physiological activity. This study combines solution binding and mutagenesis studies with crystallographic structure determination to identify the extended binding site for tetrahydropteroylpolyglutamate on rabbit cytosolic SHMT. Equilibrium binding and kinetic measurements of H(4)PteGlu(3) and H(4)PteGlu(5) with wild-type and Lys --> Gln or Glu site mutant homotetrameric rabbit cytosolic SHMTs identified lysine residues that contribute to the binding of the polyglutamate tail. The crystal structure of the enzyme in complex with 5-formyl-H(4)PteGlu(3) confirms the solution data and indicates that the conformation of the pteridine ring and its interactions with the enzyme differ slightly from those observed in complexes of the monoglutamate cofactor. The polyglutamate chain, which does not contribute to catalysis, exists in multiple conformations in each of the two occupied binding sites and appears to be bound by the electrostatic field created by the cationic residues, with only limited interactions with specific individual residues.

Effect of varying polyglutamate chain length on the structure and stability of ferricytochrome c

The effect of varying polyglutamate chain length on local and global stability of horse heart ferricytochrome c was studied using scanning calorimetry and spectroscopy methods. Spectral data indicate that polyglutamate chain lengths equal or greater than eight monomer units significantly change the apparent p K a for the alkaline transition of cytochrome c. The change in p K a is comparable to the value when cytochrome c is complexed with cytochrome bc 1. Glutamate and diglutamate do not significantly alter the temperature transition for cleavage of the Met 80-heme iron bond of cytochrome c. At low ionic strength, polyglutamates consisting of eight or more glutamate monomers increase midpoint of the temperature transition from 57.3±0.2 to 66.9±0.2 °C. On the other hand, the denaturation temperature of cytochrome c decreases from 85.2±0.2 to 68.8±0.2 °C in the presence of polyglutamates with number of glutamate monomers n≳8. The rate constant for cyanide binding to the heme iron of cytochrome c of cytochrome c-polyglutamate complex also decreases by ∼42.5% with n≳8. The binding constant for the binding of octaglutamate (m.w.∼1000) to cyt c was found to be 1.15×10 5 M −1 at pH 8.0 and low ionic strength. The results indicate that the polyglutamate ( n≳8) is able to increase the stability of the methionine sulfur–heme iron bond of cytochrome c in spite of structural differences that weaken the overall stability of the cyt c at neutral and slightly alkaline pH.

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.

Dynamic Light Scattering and Small-Angle Neutron Scattering Studies of Ternary Rod/Coil/Solvent Systems

Dynamic light scattering (DLS) and small-angle neutron scattering (SANS) are used to characterize the structure and dynamics of ternary solutions consisting either of rods or “hairy rod” polymers and polystyrene (PS) coils in a solvent composed of chloroform saturated with (<0.5%) formamide (CF). The rods are poly(γ-benzyl-α-l-glutamate) (PBLG), and the hairy rods are two forms of poly(γ-octadecyl-α-l-glutamate)-co-(γ-methyl-α-l-glutamate) with varying amounts of octadecyl substitution. The DLS autocorrelation functions reveal the existence of two relaxation modes that are interpreted in terms of the random phase approximation theory (RPA) as the cooperative and interdiffusive modes. The two relaxation modes are investigated as a function of the total polymer concentration CP and the relative composition x = Crod/CP as well as the percentage of octadecyl groups along the polyglutamate chain. The different diffusion coefficients that characterize the dynamics in these systems are well described by the RPA ...