Synthesis of a new neoglycopeptide for inhibition of lactose permease. Dedicated to Professor Fritz Jähnig,who inspired this work

Summary A new neoglycopeptide was synthesized and tested for its capability to bind to lactose permease ofEscherichia coli and to inhibit the transport of lactose. The free 5′-carboxypentyl-1-thio-β-d-galactopyranoside or the protected 2,3,4,6-tetra-O-acetyl-5′-carboxypentyl-1-thio-β-d-galactopyranoside was linked to the N-terminal α-amino group of the resin bound heptapeptide H-Phe-Phe-Gly-Gly-Gly-Gly-Ala-OH by different activation methods. Upon cleavage from the resin, deacetylation and purification, a neoglycopeptide which showed a significant inhibition of lactose permease was obtained.     

Cyanate is transported by the nitrate permease in Azotobacter chroococcum

Abstract Azotobacter chroococcum cells exhibiting the capacity to take up nitrate actively could transport [¹⁴C]cyanate. This activity was dependent on the nitrogen source present in the culture medium, ammonium acting as a repressor and nitrate as an inducer. The uptake of cyanate required metabolic energy and was absent from A. Chroococcum TR1, a mutant strain lacking the nitrate transport system, but was present at wild-type levels in A. chroococcum E4, a mutant strain deficient in nitrate reductase. These results show that cyanate is transported by the nitrate permease in A. chroococcum and therefore [¹⁴C]cyanate may be useful as a nitrate analogue for studies on nitrate transport.     

Cloning and characterization of an aromatic amino acid and leucine permease of Penicillium chrysogenum

The gene encoding the amino acid permease ArlP (Aromatic and leucine Permease) was isolated from the filamentous fungus Penicillium chrysogenum after PCR using degenerated oligonucleotides based on conserved regions of fungal amino acid permeases. The cDNA clone was used for expression of the permease in Saccharomyces cerevisiae M4054, which is defective in the general amino acid permease Gap1. Upon overexpression, an increase in the uptake of l-tyrosine, l-phenylalanine, l-tryptophan and l-leucine was observed. Further competition experiments indicate that ArlP recognizes neutral and aromatic amino acids with an unbranched β-carbon atom.     

Use of plasmid vectors to show that the uracil and cytosine permeases of the yeast Saccharomyces cerevisiae are electrogenic proton symports

Abstract Strains of Saccharomyces cerevisiae bearing the structural gene for either the uracil permease or the cytosine permease on a multicopy plasmid adsorbed uracil or cytosine at several times the rate exhibited by the wild-type. The basal rate of proton absorption by the yeast increased by at least a factor of 3 in the presence of uracil and a factor of 2 with cytosine. Assays with a voltage-sensitive carbocyanine dye showed that substrate uptake depolarized the plasma membrane. Amplification of the uracil permease activity led to uracil being concentrated about 2000-fold in comparison with 25-fold in the wild-type system. Enhanced cytosine permease activity produced a similar but smaller effect.     

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis

Summary The ptsG gene of Bacillus subtilis encodes Enzyme II^G1c of the phosphoenolpyruvate: glucose phosphotransferase system. The 3 end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes III^Glc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5 end. These results, combined with the sequence of the 3 end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes II^Glc and II^Nag of E. coli but which are present in the II^G1c-like protein encoded by the E. coli malX gene.     

Enzymatic Peptide Synthesis with Fatty Acid-modified Chymotrypsin and Trypsin in Aqueous-organic Media

Equilibrium controlled enzymatic peptide syntheses in aqueous-organic media were done with chymotrypsin and trypsin modified with varied fatty acid. Palmitoyl-modified chymotrypsin and trypsin synthesized the peptide at 1.7 and 2.4 times higher yield than those unmodified. Since an alteration of the substrate specificity was not observed, the chemical modification did not affect the active site of the enzymes.     

Isolation and expression analysis of proline metabolism-related genes in Chrysanthemum lavandulifolium

Proline plays a significant role in plant resistance to abiotic stresses, and its level is determined by a combination of synthesis, catabolism and transport. The primary proteins involved are Δ¹-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PDH) and proline transporter (ProT). To utilise proline metabolism to improve the stress resistance of Chrysanthemum × morifolium, we isolated two P5CS-homologous genes (ClP5CS1 and ClP5CS2), one PDH gene (ClPDH) and four ProT-homologous genes (ClProT1-4) (GenBANK accession numbers: KF743136–KF743142) from Chrysanthemum lavandulifolium, which is closely related to chrysanthemums and exhibits strong resistance to stresses. Expression analysis of these genes in different organs and under various stresses indicated that ClP5CSs showed substantial constitutive expression, while ClPDH was only strongly expressed in the capitulum and was inhibited under most stresses. The expression patterns of four ClProT genes presented characteristics of organ specificity and disparity under stresses. Above all, the expression of ClProT2 was restricted to above-ground organs, especially strong in the capitulum and could be obviously induced by various stress conditions. Promoters of ClPDH and ClProTs contained many cis-acting regulatory elements involved in stress responses and plant growth and development. High levels of free proline were found in flower buds, the capitulum under the non-stress condition and later periods of stress conditions except cold treatment. Interestingly, organ specificity and disparity also exist in the level of free proline under different stress conditions. Our study indicates that ClProTs play significant roles in proline accumulation and stress responses, and that ClProT2 could be used to genetically modify the stress resistance of chrysanthemums. In addition, proline metabolism might be closely related to plant flowering and floral development.