Molecular cloning,expression and characterization of three distinctive genes encoding methionine aminopeptidases in cyanobacterium<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. strain PCC6803

Methionine aminopeptidase, known to be encoded by single genes in prokaryotes, is a cobalt-dependent enzyme that catalyzes the removal of N-terminal methionine residues from nascent polypeptides. Three ORFs encoding putative methionine aminopeptidases from the genome of cyanobacterium Synechocystis sp. strain PCC6803, designated as slr0786 (map-1), slr0918 (map-2) and sll0555 (map-3) were cloned and expressed in Escherichia coli. The purified recombinant proteins encoded by map-1 and map-3 had much higher methionine aminopeptidase activity than the recombinant protein encoded by map-2. Comparative analysis revealed that the three recombinant enzymes differed in their substrate specificity, divalent ion requirement, pH, and temperature optima. The broad activities of the iso-enzymes are discussed in light of the structural similarities with other peptidase families and their levels of specificity in the cell. Potential application of cyanobacterial MetAPs in the production of recombinant proteins used in medicine is proposed. This is the first report of a prokaryote harboring multiple methionine aminopeptidases.Abbreviations map Gene encoding methionine aminopeptidase - MetAP Methionine aminopeptidase - eMetAP-Ia Escherichia coli methionine aminopeptidase type Ia - yMetAP-Ib Yeast methionine aminopeptidase type Ib - yMetAP-IIa Yeast methionine aminopeptidase type IIa - hMetAP-IIb Human methionine aminopeptidase type IIb - pfMetAP–IIa Pyrococcus furiosis methionine aminopeptidase type Ia - bst MetAP-Ia Bacillus stearothermophilus methionine aminopeptidase type Ia - c1MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-1 - c2MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-2 - c3MetAP-Ib Cyanobacterial methionine aminopeptidase type Ib, ncoded by map-3     

Analysis of CLDN14 gene in deaf Moroccan patients with non-syndromic hearing loss

Mutations in the CLDN14 gene, encoding the tight junction claudin 14 protein has been reported to date in an autosomal recessive form of isolated hearing loss DFNB29. In order to identify the contribution of CLDN14 to inherited deafness in Moroccan population, we performed a genetic analysis of this gene in 80 Moroccan familial cases. Our results show the presence of 7 mutations: 6 being conservative and one leading to a missense mutation (C11T) which was found at heterozygous and homozygous states, with a general frequency of 6.87%. The pathogenicity of the resulting T4M substitution is under discussion.     

Circadian Rhythm in Gamma Glutamyltranspeptidase and Leucine Aminopeptidase Urinary Activity in Rats

Urinary gamma glutamyltranspeptidase (GGT) and leucine aminopeptidase (LAP), renal tubular brush border enzymes, have been shown to be sensitive indicators of renal tubular functions. This study documents circadian rhythms in the urinary activity of GGT and LAP, statistically validated and quantified by the cosinor method, in 15 male Wistar rats standardized to a LD 12:12 illumination schedule (light from 0800 hr to 2000 hr) and fed ad libitum. The acrophase of the circadian rhythms in urinary GGT and LAP activity occurred at the end of the rest span of the animals: between 1730 and 1915 for GGT (depending on the mode of expression of the activity) and between 1700 and 1910 for LAP. Of striking resemblance in their timing, both these rhythms were also of large amplitude (about 50% of the mesor for urinary GGT activity and about 45% for LAP one). The circadian acrophases of urinary GGT and LAP activity led in timing the circadian rhythms in urine volume and creatinine excretion by about 13hr. Such findings consistent with the circadian variations found by other investigators in GGT in kidney homogenates or in LAP in human urine thus reflect a periodicity in renal tubular function. The reasons for these circadian variations, still unknown at this time, are discussed. The influence recently demonstrated of the hormonal context on protein and enzyme synthesis at the tubule, and its phase relations to urinary enzyme excretion emphasize how much the circadian rhythm in urinary GGT and LAP activity is well included in the murine time structure. Therefore it should be of interest to consider the circadian rhythm in urinary GGT and LAP release as a marker rhythm of predictive value as to the side effects of nephrotoxic drugs.     

Dipeptidyl Aminopeptidase IV from Pseudomonas sp. WO24

Dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24 was purified as two molecular forms of 84 and 82-kDa by SDS–PAGE. Peptide mapping and N-terminal sequence analyses indicated that both proteins might be derived from the same protein, and that the 82-kDa molecule might be a truncated form from the 84-kDa molecule at least at the N-terminus. The DAP IV gene of Pseudomonas sp. WO24 was cloned and expressed in E. coli. The enzyme expressed in E. coli JM109 harboring a hybrid plasmid, pYO-6A, with about a 3-kbp fragment containing the DAP IV gene, was purified with an activity recovery of 24%. The recombinant enzyme also had the same two molecular forms, though the ratio of the two forms (about 1:1) was different from that of the native ones (about 1:4). The native and recombinant enzyme preparations had similar specific activities, suggesting that the 84 and 82-kDa molecules are in an active form and have almost the same specific activity. The molecular mass, the subunit number, the substrate specificity, and the effects of various inhibitors of the native enzyme indicated that this enzyme was a typical DAP IV and had properties similar to those of Flavobacterium meningosepticum rather than others.     

Heterodimeric Aminopeptidase A from Bacillus licheniformis NS115

An aminopeptidase A (EC 3.4.11.7) was purified to homogeneity from Bacillus licheniformis NS115 and its enzymatic properties were characterized. The enzyme had an apparent molecular mass of 64 kDa, consisting of heterodimeric 42 kDa and 22 kDa subunits, and is a new enzyme from N-terminal analysis of heavy and light subunits. The light suhunit had no catalytic activity against the substrate and apparent K_m values of heavy and whole enzyme were 0.26 and 0.087 mM of γ-glutamyl-p-nitroanilide, respectively.     

Application of Axial-haloketone Rule to Lactones

Antifungal activities were examined and compared for some 40 kinds of aliphatic and aromatic aldehydes, alcohols, phenolic compounds, ether compounds and hydrocarbons from essential oils and for some related compounds, using seven fungi.     

一种新型亮氨酸5-羟化酶NmLEH的异源表达、纯化及酶学性质分析 *

羟基化氨基酸是一种新型氨基酸衍生物,可广泛用作化工材料的前体物及医药合成的中间体。将来源于Nostoc minutum的新型L-亮氨酸5-羟化酶 (NmLEH) 通过重组质粒在大肠杆菌中异源表达。结果表明,在BL21(DE3) 宿主细胞中,诱导温度为25℃,IPTG诱导浓度为0.5mmol/L,诱导10h时,蛋白质表达量最高 (0.45mg/ml);通过Ni-亲和层析和凝胶过滤层析两步分离纯化获得了高度纯化的重组NmLEH蛋白;对NmLEH的酶学性质进行了表征,该酶的最适反应温度为25℃,最适pH 为7.5,在pH 7.0~9.0较为稳定,最适底物为亮氨酸和甲硫氨酸;同源序列分析表明NmLEH属于亚铁和α-酮戊二酸依赖性双加氧酶家族[Fe(II)/αKG-Dos],并预测了该酶的保守催化活性位点(H150、D152、H236);通过同源建模得到了该蛋白质的模拟结构,分析了该蛋白质催化活性中心的形成机制。     

A leucine zipper-based peptide hybrid delivers functional Nanog protein inside the cell nucleus

We synthesized a pair of compounds containing leucine zipper peptides to deliver protein cargo into cells. One is a cell-penetrating peptide (CPP) with Lz(E), a leucine zipper peptide containing negatively charged amino acids, and the other is a Nanog protein with Lz(K), a leucine zipper peptide containing positively charged amino acids. When cells were treated with these equimolar mixtures, Nanog-Lz(K) hybridized with Lz(E)-CPP was successfully delivered into the cells. Furthermore, Nanog-Lz(K) exerted its proper function after nuclear transport.     

Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization,cDNA cloning,and expressed sequence tag analysis

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.     

Mass spectrometric differentiation of leucine and isoleucine in proteins derived from bacteria or cell culture

The differentiation of leucine and isoleucine is a well known difficulty in mass spectrometric peptide sequencing. A technique has been developed which allows these two amino acids to be distinguished by growing a bacterial or cell culture in a medium containing γ,δ-dl-dideuteroleucine. The isotopically labelled residue is incorporated into the cell's proteins, and the resulting mass spectra of leucine containing peptides exhibit sequence ions 2 amu higher than the corresponding isoleucine peptides.