Structural insights into the interplay of protein biogenesis factors with the 70S ribosome

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Expression of leucine genes from an extremely thermophilic bacterium in Escherichia coli

Summary The organisation of the leucine genes in Thermus thermophilus HB8 was analysed by examining the ability of recombinant DNAs to complement Escherichia coli mutations. The arrangement of the genes is different from that in the mesophilic bacteria E. coli and Salmonella typhimurium. The promoter responsible for the expression of the leuB, leuC and leuD genes of Thermus HB8 in E. coli was identified. The sequence of Thermus DNA containing this promoter revealed structural similarities to the promoter and attenuator regions of the E. coli leucine operon.     

Electrophoretic types of leucine aminopeptidase in sheep serum

Leucine aminopeptidase in sheep serum was studied by starch gel electrophoresis. Two phenotypes, A and B, were observed, of which the former was present in 70–90 % of all the sheep examined. These two phenotypes have been shown to be controlled by a single autosomal locus, with two alleles Lay^A and Lap^B . The Lap^A allele is dominant. The frequencies of Lap phenotypes and alleles were determined in eleven Spanish and two foreign breeds. Serum alkaline phospatase and serum leucine aminopeptidase are electrophoretically distinct.     

信号肽对亮氨酸脱氢酶在Bacillus subtilis中分泌表达的影响及酶学性质研究

根据信号肽N端电荷数,选择Sec及Tat两种途径的信号肽构建枯草芽孢杆菌穿梭质粒,首次实现Bacillus cereus源亮氨酸脱氢酶基因在Bacillus subtilis中的分泌表达。Tat途径信号肽Pho D促进蛋白质分泌的效果最好,胞外酶活力达20.25U/ml,为不添加信号肽的2.2倍,信号肽N端较多的电荷数,可能有利于多聚体蛋白的分泌。对表达产物进行纯化和酶学性质测定。结果表明,纯酶比酶活为13U/mg;L-Leucine为底物时酶的K_m为6.17mmol/L,V_(max)为14.49μmol/(L·min);底物特异性研究发现,酶与天然底物L-Leucine的亲和性最好,对一些脂肪族氨基酸也有活性,对芳香族氨基酸L-Phenylalanine无活性;酶的最适pH为10.5~12.0,pH稳定范围为5.0~11.0;最适反应温度为55℃;圆二色谱变温扫描酶二级结构变化,α螺旋含量随温度升高逐渐降低;差示扫描微量热技术(DSC)测定酶的解折叠温度(Tm值)为64.13℃,表明该酶具有较好耐热性。     

Separation and partial characterization of two alkaline peptidases from cotyledons of resting kidney beans, Phaseolus vulagaris

Extracts prepared from cotyledons of resting kidney beans ( Phaseolus vulgaris L., cv. Processor) rapidly hydrolyzed two dipeptides, Leu-Tyr and Ala-Gly, with pH optima at 9.2 and 8.5, respectively. On ion exchange chromatography on DEAE-Sephacel the two activities eluted as separate peaks, showing that they were due to two different peptidases. The extracts also hydrolyzed Leu-β-naphthylamide optimally at pH 6.4; this activity eluted as a third peak berween the other peaks. The activity peak acting on Leu-Tyr and Ala-Gly rapidly hydrolized two tripeptides, showing that it was an aminopeptidase, whereas the Ala-Gly hydrolyzing peak acted only on dipeptides. The activities against Leu-Tyr and Ala-Gly were also separated by gel chromatography on Sephacryl S-300 with elution positions corresponding to M, values of about 360 000 and 105 000. The aminopeptidase was inhibited by bestatin, and the dipeptidase was inhibited by p-hydroxymercuribenzoate. Both enzymes were inhibited by o-phenanthroline. In most of their properties the two kidneys bean enzymes resembled the alkaline aminopeptidase and the dipeptidase earlier characterized from barley grains.     

Cyclic insulin-regulated aminopeptidase (IRAP)/AT4 receptor ligands.

The angiotensin IV receptor (AT4 receptor) is the insulin-regulated aminopeptidase enzyme (IRAP, EC 3.4.11.3). This membrane-spanning enzyme belongs to the M1 family of zinc-dependent metallo-peptidases. It has been proposed that AT4 receptor ligands exert their physiological effects by binding to the active site of IRAP and thereby inhibiting the catalytic activity of the enzyme. The biological activity of a large series of linear angiotensin IV analogs was previously disclosed. Herein, the synthesis and biological evaluation of a series of angiotensin IV analogs, encompassing macrocyclic ring systems of different sizes, are presented. It is demonstrated that disulfide cyclizations of angiotensin IV can deliver ligands with high IRAP/AT4 receptor affinity. One ligand, with an 11-membered ring system (4), inhibited human IRAP and aminopeptidase N (AP-N) activity with similar potency as angiotensin IV but was considerably more stable than angiotensin IV toward enzymatic degradation. The compound provides a promising starting point for further optimization toward more drug-like derivatives. The cyclic constrained analogs allowed us to propose a tentative bioactive conformation of angiotensin IV and it seems that the peptide adopts an inverse gamma-turn at the C-terminal.     

Pituitaries of rats neonatally treated with oestrogen

Summary Pituitaries of male and female rats neonatally treated with oestradiol dipropionate were examined with light and electron microscopy, disc electrophoresis and in vitro C¹⁴-leucine incorporation.Single dose of oestradiol administered shortly after birth to male rats has an prolonged stimulative effect on LTH synthesis and release. This is expressed in the cell hypertrophy, proliferation of ER, increase of corresponding band density and higher rate of ¹⁴C-leucine incorporation. In females this effect was less pronounced.STH cell in the treated male pituitaries were degranulated, corresponding hormone band density significantly lowered and incorporation of ¹⁴C-leucine was reduced. This could be considered as an inhibitory effect of oestradiol on STH synthesis and release. Such effect was more expressed two than four months after treatment and is more pronounced in male than in female rats.The technical assistence of Slobodanka Selimovi is gratefully acknowledged.     

Increased lymphocytic aminopeptidase N/CD13 promoter activity after cell‐cell contact

Aminopeptidase N (APN)/CD13 is a transmembrane ectoenzyme expressed on a wide variety of cells. With respect to haematopoietic cells, APN/CD13 has been considered specific for the myeloid lineage, because granulocytes and monocytes/macrophages, but not lymphocytes of peripheral blood, show a surface expression of CD13 antigen. However, we could recently show that cell‐cell contact of lymphocytes with endothelial cells, monocytes, and fibroblast‐like synoviocytes (SFCs) results in an increase of steady‐state APN/CD13 mRNA and a rapid expression of cell‐surface protein on the lymphocytes. In this study using the Dual‐Luciferase reporter assay, we demonstrate that interaction of the T‐cell line Jurkat with SFCs results in a higher activity of the APN/CD13 myeloid promoter in T cells. An enhancer located between the myeloid and epithelial APN/CD13 promoter increases the response of the promoter to the cell‐cell contact‐induced expression of APN/CD13 in lymphocytes. Adhesion of lymphocytes to extracellular matrix did not result in increased promoter activity. The lymphocytic promoter response induced by direct cell‐cell contact with SFCs is not affected by mutations of a proximal promoter element (nucleotides −48 to −35), which has a possible functional role in the basal APN/CD13 gene expression in lymphocytes. Upregulated peptidase‐promoter activity via cell‐cell contact shown in this study for the first time is discussed as a general mechanism in peptidase induction. J. Cell. Biochem. 80:115–123, 2000. © 2000 Wiley‐Liss, Inc.     

N-hydroxy-amide analogues of MHC-class I peptide ligands with nanomolar binding affinities

A novel class of major histocompatibility complex class I (MHC-I) ligands containing an N-hydroxy-amide bond was designed on the basis of the natural epitope SIINFEKL, and synthesized on solid phase. The capacity of these compounds to bind to the MHC-I molecule H-2K^b and to induce T cell responses was analysed in comparison with the corresponding glycine containing variant of SIINFEKL. Binding to the MHC molecule was diminished by the N-hydroxy group at positions 2 and 3 of the oligomer and improved in the case of positions 4, 5, 6 and 7. No change was seen for position 1. The efficacy of T cell stimulation was strongly reduced by the modification of all positions except for position 1. A complete loss of activity was found for the N-hydroxy variant in positions 4 and 6. N-Hydroxy amide-containing peptides displayed an enhanced stability to enzymatic degradation. This new class of MHC ligand can become instrumental as immunomodulatory reagent in various disease situations. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Novel reversible methionine aminopeptidase-2 (MetAP-2) inhibitors based on purine and related bicyclic templates

The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC₅₀: 1 μM) led to the most potent compound 27 (IC₅₀: 0.038 μM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.