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PubMed1999

Synthetic peptides identify the minimal substrate requirements of tubulin polyglutamylase in side chain elongation.

The minimal sequence requirement of Crithidia tubulin polyglutamylase is already fulfilled by tubulin-related peptides carrying a free alpha-carboxylate on a glutamic acid residue. Since the product of each glutamylation step fulfills the substrate requirements necessary for the next cycle, very long side chains are generated with brain tubulin as a substrate. Up to 70 mol of glutamic acid was incorporated per alphabeta-heterodimer. We speculate that the strict choice of a particular glutamate residue for the formation of the isopeptide bond initiating a novel side chain is made by a tubulin monoglutamylase which requires the entire tubulin as substrate.

Amino Acid Sequence↗
PubMed2004

Cytotoxic and membrane perturbation effects of a novel amyloid forming model peptide poly(leucine-glutamic acid).

In the present study we have elucidated the toxicity of a novel amyloid forming model peptide, Poly (leucine-glutamic acid). The toxicity of the fibrils prepared from this peptide was analyzed in peripheral blood lymphocytes (PBL). The MTT reduction assay revealed that the viability of PBL decreases significantly upon treatment with Poly(leucine-glutamic acid) (Poly [LE]). Enhanced DCFH-DA fluorescence in treated cells suggests that peptide toxicity is probably mediated by the formation of free radicals. In vivo and in vitro biochemical studies indicated that Poly [LE] inactivates the antioxidant system of cells. Perturbation of Poly [LE] in a membrane lipid environment was assessed by circular dichroism (CD) using phosphotidyl choline-cholesterol bilayers. The CD results revealed that LE enhances its beta sheet content in a bilayer environment. Sequestration of Poly [LE] in lipid rafts demonstrates that it has a binding cleft similar to Abeta in lymphocyte raft domains. Nuclear membrane binding studies showed that Poly [LE] binds to nuclear membranes and may cause genotoxicity.

Adenosine Triphosphatases↗
PubMed1998

Properties of glutamate racemase from Bacillus subtilis IFO 3336 producing poly-gamma-glutamate.

We found glutamate racemase activity in cell extracts of Bacillus subtilis IFO 3336, which abundantly produces poly-gamma-glutamate. The highest activity was obtained in the early stationary phase of growth. The racemase was purified to homogeneity. The enzyme was a monomer with a molecular mass of about 30 kDa and required no cofactor. It almost exclusively catalyzed the racemization of glutamate; other amino acids, including alanine and aspartate but not homocysteinesulfinate, were inactive as either substrates or inhibitors. Although the Vmax value of the enzyme for L-glutamate is 21-fold higher than that for D-glutamate, the Vmax/Km value for L-glutamate is almost equal to that for the D-enantiomer. The racemase gene, glr, was cloned into Escherichia coli cells and sequenced. The racemase was overproduced in the soluble fraction of the E. coli clone cells with the substitution of ATG for TTG, the initial codon of the glr gene. D-Amino acid aminotransferase activity was not detected in Bacillus subtilis IFO 3336 cells. B. subtilis CU741, a leuC7 derivative of B. subtilis 168, showed lower glutamate racemase activity and lower productivity of poly-gamma-glutamate than B. subtilis IFO 3336. These results suggest that the glutamate racemase is mainly concerned in D-glutamate synthesis for poly-gamma-glutamate production in B. subtilis IFO 3336.

Amino Acid Isomerases↗
PubMed1996

Prostate-specific membrane antigen: a novel folate hydrolase in human prostatic carcinoma cells.

A novel monoclonal antibody has been developed that reacts strongly with human prostatic cancer, especially tumors of high grade. This antibody (7E11C-5) is currently in Phase 3 trials as an imaging agent for metastatic disease. We have cloned the gene that encodes the antigen that is recognized by the 7E11C-5 monoclonal antibody and have designated this unique protein prostate-specific membrane (PSM) antigen. PSM antigen is a putative class II transmembranous glycoprotein exhibiting a molecular size of Mr 94,000. Functionally, class II membrane proteins serve as transport or binding proteins or have hydrolytic activity. Preliminary studies have demonstrated binding of pteroylmonoglutamate (folate) to membrane fractions that also cross-reacted with the PSM monoclonal antibody. We observed substantial carboxypeptidase activity as folate hydrolase associated with PSM antigen. The purpose of our study was to demonstrate that human prostatic carcinoma cells expressing PSM antigen exhibit folate hydrolase activity using methotrexate triglutamate (MTXGlu3) and pteroylpentaglutamate (PteGlu5) as substrates. Isolated membrane fractions from four human prostate cancer cell lines (LNCaP, PC-3, TSU-Prl, and Duke-145) were examined for folate hydrolase activity using capillary electrophoresis. After timed incubations at various pH ranges and in the presence and absence of thiol reagents, separation of pteroyl(glutamate)n derivatives was achieved with an electrolyte of sodium borate and SDS, while absorbance was monitored at 300 nm. The results demonstrate clearly that LNCaP cells, which highly express PSM, hydrolyze gamma-glutamyl linkages of MTXGlu3. The membrane-bound enzyme is an exopeptidase, because it progressively liberates glutamates from MTXGlu3 and PteGlu5 with accumulation of MTX and PteGlu1, respectively. The semipurified enzyme has a broad activity from pH 2.5 to 9.5 and exhibits activity maxima at pH 5 and 8. Enzymatic activity is maintained in the presence of reduced glutathione, homocysteine, and p-hydroxymercuribenzoate (0.05-0.5 mm) but was inhibited weakly by DTT (>/=0.2 mm). By contrast to LNCaP cell membranes, membranes isolated from other human prostate adenocarcinoma cells (PC-3, Duke-145, and TSU-Pr1) did not exhibit comparable hydrolase activity, nor did they react with 7E11-C5 monoclonal antibody. After transfection of PC-3 cells with a full-length 2.65-kb PSM cDNA subcloned into a pREP7 eukaryotic expression vector, non-PSM antigen-expressing PC-3 cells developed immunoreactivity to 7E11-C5 monoclonal antibody and demonstrated folate hydrolase activities and optimum pH activity profiles identical to those of LNCaP cells. The membrane-bound enzymes from both LNCaP- and PC-3-transfected cells also have a capacity to hydrolyze an alpha-linked glutamyl moiety from N-acetyl-alpha-aspartylglutamate. We have identified that PSM antigen is a pteroyl poly-gamma-glutamyl carboxypeptidase (folate hydrolase) and is expressed strongly in human prostate cancer. Cancer cells that express this enzyme are resistant to methotrexate therapy. Those developing future therapeutic strategies in the treatment of prostate cancer that utilize folate antagonists need to consider this mechanism of resistance.

Antigens, Surface↗
PubMed1998

Tubulin polyglutamylase: partial purification and enzymatic properties.

In this work, we report on a novel enzyme, tubulin polyglutamylase, which catalyzes the posttranslational formation of polyglutamyl side chains onto alpha- and beta-tubulin. The length of the polyglutamyl side chain regulates the interaction between tubulin and various microtubule-associated proteins. We first developed an in vitro glutamylation assay. Activity measured in brain, a tissue particularly enriched with glutamylated tubulin, decreases during postnatal development. Thus, brains from 3-day-old mice were chosen as the starting material, and the enzyme was purified approximately 1000-fold. Its Mr was estimated to be 360K and its sedimentation coefficient 10 s. The enzyme catalyzes the MgATP-dependent addition of l-glutamate onto tubulin subunits. Microtubules are much better substrates than unpolymerized tubulin, and the reaction is very specific for glutamate, other amino acids or glutamate analogues not being substrates. Moreover, glutamyl units are added sequentially onto tubulin, leading to progressive elongation of the polyglutamyl side chains. Side chains of one to six or seven glutamyl units were obtained with microtubules, whereas much longer side chains (up to 15-20 units) were formed with unpolymerized tubulin. Interestingly, such very long polyglutamyl side chains were recently detected in some situations in vivo.

Adenosine Triphosphate↗
PubMedDate not supplied

Microbial degradation of poly(amino acid)s.

Natural poly(amino acid)s are a group of poly(ionic) molecules (ionomers) with various biological functions and putative technical applications and play, therefore, an important role both in nature and in human life. Because of their biocompatibility and their synthesis from renewable resources, poly(amino acid)s may be employed for many different purposes covering a broad spectrum of medical, pharmaceutical, and personal care applications as well as the domains of agriculture and of environmental applications. Biodegradability is one important advantage of naturally occurring poly(amino acid)s over many synthetic polymers. The intention of this review is to give an overview about the enzyme systems catalyzing the initial steps in poly(amino acid) degradation. The focus is on the naturally occurring poly(amino acid)s cyanophycin, poly(epsilon-L-lysine) and poly(gamma-glutamic acid); but biodegradation of structurally related synthetic polyamides such as poly(aspartic acid) and nylons, which are known from various technical applications, is also included.

Amino Acids↗
PubMed1994

Fibroblast attachment to Arg-Gly-Asp peptide-immobilized poly(gamma-methyl L-glutamate).

The attachment of MRC-5 human fibroblasts was investigated on poly(gamma-methyl L-glutamate) (PMLG), and upon cell adhesion peptides Arg-Gly-Asp-Ser (RGDS)- and Gly-Arg-Gly-Asp-Ser (GRGDS)-immobilized PMLG (RGDS-PMLG and GRGDS-PMLG). The peptides were immobilized by their N-terminal amine to activated PMLG surfaces. Prior to peptide immobilization, the aminolysis of PMLG surfaces was performed with hydrazine hydrate (HA), ethylenediamine (EDA), and hexamethylenediamine (HMDA) and was followed by the activation with hexamethylene diisocyanate. Surface characterization of these films was carried out by means of a Fourier transform IR (FT-IR) spectrometer equipped with an attenuated total reflectance (ATR) attachment. The amount of immobilized RGDS could be controlled by the reaction time of the aminolysis. The effects of HA, EDA, and HMDA as a spacer on the cell attachment were also investigated, and it was suggested that a longer spacer promoted the cell attachment via specific receptor-ligand interaction.

Amino Acid Sequence↗
PubMed1995

Characterization of post-translational modifications of brain tubulin by matrix-assisted laser desorption/ionization mass spectrometry: direct one-step analysis of a limited subtilisin digest.

Matrix-assisted ultraviolet laser desorption/ionization (MALDI) mass spectrometry was used to investigate the molecular masses and heterogeneity patterns caused by post-translational modifications in tubulin from porcine brain. Direct analysis of the limited digest with subtilisn shows that the molecular masses of the majority of the carboxyterminal fragments are below 2 kDa, while the truncated tubulin subunits have lost approximately the same mass. The results confirm the cleavage sites previously postulated for this protease. The mass information on the peptides allows the degree of polyglutamylation to be measured directly and shows that molecules with two glutamyl residues in the side chain are the most abundant species. In addition it identifies the degree of tyrosination of alpha tubulin. This one-step monitoring of a complex digest provides information equivalent to that obtainable from the purified components, while the amount of material required is reduced by three orders of magnitude when compared to previous studies. MALDI spectra partially resolve the alpha and beta subunits of the highly homogeneous tubulin from turkey erythrocytes, which lacks polyglutamylation but does not separate alpha and beta subunits from the heterogeneous brain tubulin. Post-translational modifications of the brain tubulin result in shifting peaks to higher molecular masses, in broadening of the peaks, and in loss of resolution.

Amino Acid Sequence↗
PubMed1984

Taenia crassiceps: temperature, polycations, polyanions, and cysticercal endocytosis.

The effect of various temperatures, poly-L-lysine, and poly-L-glutamic acid on endocytosis of smooth micropinocytotic vesicles (pinosomes) in the tegument of the cysticercus of Taenia crassiceps has been investigated stereologically. The temperature regimes used were 0, 5, 10, 20, 30, and 40 C. Maximum volume, surface density, and number per unit volume were found at 40 C, and minimum surface-to-volume ratio and numbers at 10 C. At 10 C, mean pinosome volume and mean surface area per pinosome were maximal, but volume and surface density did not differ significantly from 40 C. It is proposed that this anomalous finding for 10 C incubations was due to this being a critical temperature at which a slower rate of pinosome formation was compensated for by the formation of larger individual pinosomes. Poly-L-lysine was shown to be a stimulant of pinosome formation, leading to a significant increase in numbers per unit volume. However, volume and surface density, surface-to-volume ratio, mean volume, and mean surface area per pinosome were not significantly different in poly-L-lysine-incubated samples, when compared to controls (fresh from the mouse) or incubations in medium only or samples returned to medium after poly-L-lysine incubation, the only exception being surface to volume ratio and mean volume of pinosomes in the 75-min incubation. These anomalous results were explained by a marked reduction in the form ellipse values, which indicated the production of more elliptical-shaped pinosomes under poly-L-lysine stimulation. Incubation in poly-L-glutamine acid did not have any significant effect at any incubation time.

Animals↗
PubMedDate not supplied

Fine specificities of the murine T cell responses to (Glu Lys Ala) terpolymers.

The T cell proliferative responses to GLA terpolymers among mice of H-2 a, b, d, k, q, r and s haplotypes are heterogeneous. Following immunization with GLA20, GLA40 and GLA60, mouse T cells reacted well with the three terpolymers. The cross-reactions of the above T cells to GLA5 decreased as the alanine content in the terpolymers (immunogens) increased, whereas the trend of the cross-reactions to GA40 did not change. The RIII strain showed no cross-reaction with GLA5. Only mice of H-2 haplotypes q and s failed to show cross-reactions to GA40. Mice of the H-2q haplotype are genetic non-responders to GA40, whereas those of the H-2s haplotype are responders. Immunization of SJL mice with GA40 mixed with GLA40 led to reduction of the specific T cell response to GA40. This reduction of GA40 specific T cell responses in SJL mice by GLA40 occurred during the early stage of the immune response. The kinetic study showed that it was necessary to inject the GLA40 intraperitoneally at least 7 days before GA40 in order for the reduction to take place. The PETLES and lymph node T cells from DBA/1 mice were found to respond to GLA40 differently, and these cells from SJL mice responded differently to GA40.

Alanine↗
PubMed2005

Preparation of dendrimer SAM on Au substrate and adsorption/desorption of poly-L-glutamate on the SAM.

A self-assembled monolayer (SAM) of a dendrimer has been fabricated by covalent bonding of amine-terminated dendrimers to 3-mercaptopropionic acid SAM on an Au substrate, where an amide bond was formed between the amine and the carboxylic acid using amide-coupling reagents. Each reaction step was inspected by transmission surface-enhanced infrared absorption spectroscopy. Reaction was affected by solvents of the coupling reagents and concentrations of the coupling reagents and dendrimers. The pH-dependent adsorption/desorption behavior of sodium poly-L-glutamate (PGA) has been examined on the dendrimer SAM prepared. The adsorbed amount was abundant at acid pH below the pK(a) of carboxylate, while desorption from PGA-adsorbed dendrimer SAM prepared at acid pH proceeded as the pH was raised. The reaction was reversible and reproducible within 30 min under controlled pH, although the carboxylic acid species adsorbed at pH 4.2 was deprotonated to a carboxylate species after more than 30 min of desorption at pH 9.4.

Adsorption↗
PubMed1984

Polyglutamylation, an important element in methotrexate cytotoxicity and selectivity in tumor versus murine granulocytic progenitor cells in vitro.

Methotrexate (MTX) cytotoxicity was assessed by clonogenic assay in agar with granulocytic progenitor cells from mouse bone marrow and in the Ehrlich ascites tumor, the K562 human chronic myelogenous leukemia, and the P388 murine leukemia. After a 2-hr exposure to MTX, the concentrations necessary to produce 50% inhibition of colony formation were 100, 25, 1.2, and 0.25 microM, respectively. This was inversely related to the ability of the tumor cells to accumulate MTX polyglutamyl derivatives and consistent with the observation that no polyglutamyl derivatives were observed in granulocytic progenitor cells after a 2-hr exposure to 5 micron MTX. Continuous exposure to glycine (200 microM)-adenosine (100 microM)-thymidine (10 microM) (GAT), along with MTX, protected cells from MTX cytotoxicity by circumventing the requirement for tetrahydrofolate cofactors. However, while the presence of GAT during a 2-hr exposure to 5 microM MTX is sufficient to protect granulocyte progenitor cells from MTX cytotoxicity, the presence of GAT, even after MTX is removed, is required to protect tumor cells. Indeed, if, after a 2-hr exposure of tumor cells to MTX and GAT, both MTX and GAT are removed before plating in agar, cytotoxicity to tumor cells was expressed. This sustained antitumor effect of MTX correlates with the rapid build-up of polyglutamyl derivatives that are retained in the cell even after extracellular and intracellular monoglutamate is eliminated. This is in contrast to granulocytic progenitor cells which appear to be susceptible to the drug only during the period of exposure to the monoglutamate under these conditions. The data strongly suggest that the marked differences in the accumulation of MTX polyglutamyl derivatives between the tumor cells studied and the murine bone marrow granulocytic progenitor cells are an important element in MTX selectivity.

Animals↗
PubMed2004

Poly(phenylacetylene)s bearing a peptide pendant: helical conformational changes of the polymer backbone stimulated by the pendant conformational change.

Optically active, cis-transoid poly(phenylacetylene) derivatives bearing a poly(gamma-benzyl-L-glutamate) [poly(PBGAm)] or poly(L-glutamic acid) [poly(PGAm)] chain as the pendant were prepared by polymerisation of the corresponding macromonomer with a rhodium catalyst followed by hydrolysis of the pendant ester groups. Their conformational changes in solution, induced by a helix-coil transition of the pendant polypeptides, were investigated using circular dichroism (CD) and absorption spectroscopies. A series of macromonomers with a different peptide chain lengths was synthesised by the polymerisation of the N-carboxyanhydride of gamma-benzyl-L-glutamate with a phenylacetylene bearing an alanine residue as the initiator. The obtained macromonomers (PBGAm) were further polymerised with a rhodium catalyst in N,N-dimethylformamide (DMF) to yield novel poly(phenylacetylene)s [poly(PBGAm)] with a poly(gamma-benzyl-L-glutamate) pendant. The poly(PBGAm) exhibited an induced circular dichroism (ICD) in the UV/Vis region of the polymer backbone in dimethyl sulfoxide (DMSO), probably due to the prevailing one-handed helix formation. The Cotton effect signs of a DMSO solution of the poly(PBGAm) were inverted and accompanied by a visible colour change in the presence of an increasing amount of chloroform or DMF containing lithium chloride. The results suggest that poly(PBGAm) may undergo a conformational change such as a helix-helix transition with a different helical pitch responding to a change in the alpha-helix content of the poly(gamma-benzyl-L-glutamate) pendant. Moreover, a water-soluble poly(PGAm) also showed a similar, but dramatic change in its helical conformation with a visible colour change stimulated by a helix-coil transition of the pendant poly(L-glutamic acid) chains by changing the pH in water.

Acetylene↗