A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins

A selective protonation strategy is described that uses [3-2H] 13C -ketoisovalerate to introduce (1H- methyl)-leucine and (1H- methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of 100 mg/L -ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] -ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-1 methyl)-isoleucine (>90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Epitope Mapping of Human Alpha-Fetoprotein

The epitope structure of human alpha-fetoprotein (AFP) was studied using more than 50 monoclonal antibodies (MAB) to human AFP. These MAB obtained from various world laboratories of the TD-2 AFP Workshops of the International Society for Oncodevelopmental Biology and Medicine (ISOBM-1996-1998-2000) were analyzed by competitive immunoaffinity electrochromatography (IAE) on nitrocellulose membranes (NCM). Five types of interaction of the AFP–MAB complex with the MAB fixed on NCM were found: 1) complete neutralization; 2) partial neutralization; 3) unidirectional neutralization; 4) enhanced binding; 5) lack of interaction. By IAE, 51 MAB were found to recognize 23 different epitopes in the AFP molecule. Based on these findings, an epitope map of AFP was designed which consists of eight epitope clusters and eight individual epitopes. The epitope location is considered with respect to the conformational state of the AFP molecule. Possible causes of the five types of interaction found on neutralization are discussed.     

Isolating substrates for an engineered alpha-lytic protease by phage display

Panning of a substrate phage library with an -lytic protease mutant showed that substrate phage display can be used to isolate sequences with improved protease sensitivity even for proteases of relatively broad specificity. Two panning experiments were performed with an engineered -lytic protease mutant known to have a preference for cleavage after His or Met residues. Both experiments led to the isolation of protease-sensitive phage containing linker sequences in which His and Met residues were enriched compared with the initial library. Despite the relatively hydrophobic substrate binding site of the enzyme, the predominant protease-sensitive sequence isolated from the second library panning had the sequence Asp-Ser-Thr-Met. Kinetic studies showed that this sequence was cleaved up to 4.5-fold faster than rationally designed positive controls. Protease-resistant phage particles were also selected and characterized, with the finding that Gly and Pro appeared frequently at the putative P₄ positions, whereas Asp dominated the putative P₁ position.     

Purification, properties and possible gene assignment of an α1,3-fucosyltransferase expressed in human liver

1,3-Fucosyltransferase solubilized from human liver has been purified 40 000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with 1,3-fucosyltransferase activity and had a specific activity of 5–6 µmol min^–1 mg^–1 and anM _r 44 000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Gal1-4GlcNAc, NeuAc2-3Gal1-4GlcNAc and Fuc1-2Gal1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fuc1-2Gal1-4Glc and the Type 1 compound Gal1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAc2-6Gal1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated 1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with 1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with theFuc-TVI cDNA, suggests a provisional identification ofFuc-TVI as the major 1,3-fucosyltransferase gene expressed in human liver.Died June, 1991     

A low-temperature-responsive translation elongation factor 1α from barley (Hordeum vulgare L.)

A cDNA clone (pBLT63) encoding a protein synthesis elongation factor 1 (EF-1) was isolated from a low-temperature winter barley shoot meristem library by differential screening. The nucleotide sequence of the coding region of the low-temperature-induced barley gene shows very high homology with two EF-1 plant genes from tomato and Arabidopsis. The barley genome contains an EF-1 gene family situated on the short arm of chromosome 2 and the long arm of chromosome 5. The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number Z23130.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

Evaluation of two new coupling agents for incorporation of α,α-dialkylamino acids, such as α-methylalanine, in solid-phase peptide synthesis

Summary The introduction of solid-phase peptide synthesis (SPPS) has greatly facilitated the preparation of peptides containing proteinaceous amino acids. Less common, sterically hindered ,-dialkylamino acids, such as -methylalanine (MeA, aminoisobutyric acid, Aib), have proven a synthetic challenge for incorporation by this approach, especially when present in contiguous sequences. Solution protocols, utilizing highly reactive intermediates such as oxazalones, are generally used during the preparation of peptaibol antibiotics such as alamethicin, emerimicin, etc. which contain such contiguous sequences. Two recently developed coupling strategies (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HATU, and Fmoc-protected amino acid fluorides) allow peptides comprising contiguous sequences of ,-dialkylamino acids to be prepared using SPPS. The present study evaluates the relative merits of these two methods on a set of difficult peptides containing oligo-MeA sequences.     

Purification and partial characterisation of and α-tocopherol-binding protein from rabbit heart cytosol

An -tocopherol-binding protein has been isolated and purified from rabbit heart cytosol. The purified protein had an apparent molecular mass of 14,200, as derived from SDS-PAGE. The content of the protein in rabbit heart was around 11.8 g per g of tissue. The binding of -tocopherol to the purified protein was rapid, reversible, and saturable. Neither nor tocopherol could displace the bound -tocopherol from the protein, suggesting a high specificity for -tocopherol. -Tocopherol-binding protein did not bind oleate. Transfer of -tocopherol from liposomes to mitochodria was stimulated 8-fold in the presence of the binding protein, suggesting that this protein may be involved in the intracellular transport of -tocopherol in the heart.     

Reduction of Glutamate Uptake into Cerebral Cortex of Developing Rats by the Branched-Chain Alpha-Keto Acids Accumulating in Maple Syrup Urine Disease

In the current study we investigated the effect of the branched-chain alpha-keto acids (BCKA) co-ketoisocaproic (KIC), alpha-keto-beta-methylvaleric (KMV), and alpha-ketoisovaleric (KIV) acids, which accumulate in maple syrup urine disease (MSUD), on the in vitro uptake of [3H]glutamate by cerebral cortical slices from rats aged 9, 21, and 60 days of life. We initially observed that glutamate uptake into cerebral cortex of 9- and 21-day-old rats was significantly higher, as compared to that of 60-day-old rats. Furthermore, KIC inhibited this uptake by tissue slices at all ages studied, whereas KMV and KIV produced the same effect only in cortical slices of 21- and 60-day-old rats. Kinetic assays showed that KIC significantly inhibited glutamate uptake in the presence of high glutamate concentrations (50 microM and greater). We also verified that the reduction of glutamate uptake was not due to cellular death, as evidenced by tetrazolium salt and lactate dehydrogenase viability tests of cortical slices in the presence of the BCKA. It is therefore presumed that the reduced glutamate uptake caused by the BCKA accumulating in MSUD may lead to higher extracellular glutamate levels and potentially to excitotoxicity, which may contribute to the neurological dysfunction of the affected individuals.