Biological chemistry Hoppe-Seyler最新文献

The cysteine proteases cathepsin B and cathepsin L are very likely involved in invasive processes of normal and malignant cells, they become relevant for a number of diseases and are possibly prognostic markers for the outcome of human lung cancer. Therefore, we have determined activities of these related enzymes in cells and in cell extracts of human lung carcinoma cell lines of different cathepsin composition by flow cytometry and by spectrophotometry, respectively. To this end we applied the synthetic dipeptidyl substrates benzoxycarbonyl-arginyl-arginine- and benzoxycarbonyl-phenyl-arginine- coupled to 4-methoxy-beta-naphthylamide, aminomethyl-coumarine or rhodamine R110. The apparent enzymatic activities were differentially defined by protease inhibitors, particularly E-64 and CA-074. Independent of the dipeptidyl-composition more than 99 per cent of the apparent activity was due to cathepsin B when 4-methoxy-beta-naphthylamide or aminomethylcoumarine were the leaving groups. The 4-methoxy-beta-naphthylamide precipitate used for detection of cell associated activities revealed a wide spectrum of excitation to fluorescence thwarting the application of other possible fluorescent tags. Therefore, its application is restricted to uniparametric fluorescence investigations. Both dipeptidylgroups coupled to rhodamine R110 were promiscuous: only 25 to 30% of the apparent activity were due to cathepsin B; the predominant activity came from cathepsin L, irrespective whether intracellular or activities of cellular extracts were analyzed. However, rhodamine R110-coupled substrates open the way for multiparametric fluorescent analysis of cathepsins B and L containing cells if appropriate inhibitors for specification of the enzymatic activities are additionally applied. In very contrast to 4-methoxy-beta-naphthylamide, which causes irreparable damage to the cells, the rhodamine substrates permit studies with living cells and live cell sorting.

Fragments from various proteolytically degraded precursor proteins can form beta-amyloid fibrils. We studied, by electron microscopy and quantitative Congo red binding, the ability of three synthetic peptides, corresponding to residues 359-374 (C-36), 370-374 (C-5) and 375-394 (C-20) from the C-terminal part of alpha 1-antitrypsin (AAT) to form beta-amyloid fibrils in vitro. The peptides C-36 and C-5 had a pronounced tendency to form fibrils. C-20 lacked this property, suggesting that residues 359-375 and/or 370-374 are most critical for fibril formation. Native AAT added to peptide 125I-C-36 could bind and form complexes with the peptide, resulting in inhibition of amyloid fibril formation. Moreover, native AAT added to preformed fibrils induced disaggregation of fibrillar structures. The structural rearrangements of AAT that occurred during this 'autointeraction' included polymerization of the serpin, and an increase of its thermal stability. Also, following interaction, an increase (20-40%) of AAT's antielastase activity was noted. The demonstration of an in vitro beta-amyloid fibril formation from the AAT derived C-terminal peptides C-36 and C-5 and its regulation by the intact AAT molecule may have important in vivo implications.

Two hemoglobin components are recognized in erythrocytes of the adult turkey (Meleagris gallopavo). We determined the amino acid sequences of turkey alpha A-, alpha D- and beta-globin from intact globin chains and chemical cleavage fragments. The sequences are highly similar to the hemoglobin of the Phasianidae, chicken, Japanese quail and pheasant. Turkey and pheasant beta-globin are identical. The amino acid sequence of turkey alpha A-globin differs by only one residue from chicken alpha A-globin. Phylogeny trees from alpha A-, alpha D- and beta-globin were constructed by the neighbor-joining method. Although the trees generated from alpha A- and beta-globin were similar, that from turkey alpha D-globin differed.

In addition to cystine aminopeptidase (oxytocinase) alanine aminopeptidase is present at high levels in the serum of pregnant women. In this study we compared the enzyme with membrane-bound aminopeptidase N purified from human placenta. Comparison of catalytic and immunological properties and N-terminal sequence analyses revealed that the enzymes were differentially processed derivatives of the same protein, and that the N-terminal 68 residues of aminopeptidase N were deleted in the alanine aminopeptidase. The deleted sequence contains a small cytoplasmic region, a hydrophobic transmembrane domain and a junctional domain. These results suggest that the enzyme may be released into the maternal circulation as a result of lacking these three domains.

Outer dense fibers (ODF) or accessory fibers are filamentous structures of the sperm tail of many eumetozoan organisms endowed with internal fecundation. The bovine and porcine cDNA of an outer dense fiber protein was cloned, sequenced and compared to the previously characterized human and rat cDNA sequences. The coding sequences and the 5' and 3' untranslated regions of the ODF cDNAs are highly conserved. A comparison of the bovine, porcine, human and rat ODF protein sequences revealed that the protein displays a high degree of similarity, ranging from 87% to 98%. The ODF protein is rich in cysteine and contains the C.X.P. repeat at the C-terminal which is different in number among mammalian species. All the 27 cysteine residues in the ODF sequence except those in the C.X.P. repeat are conserved in the four species. We report here also the organization of the bovine ODF gene which is similar to that of human and rat. The transcription start site in the bovine ODF gene is localized 98 bp upstream of the translation start site. Alignment of the 5' flanking region of bovine ODF with the rat gene reveals that the first 130 nucleotides upstream of the transcription start site exhibit an overall sequence similarity of 83%. This conserved region contains a TATA-like box (TTTAAA) and binding sites for AFT/CREB and EGR-1 transcription factors.

Tissue kallikrein is well known to liberate the vasoactive peptide kallidin from L-kininogen. Recently it was reported to activate matrix degrading metalloproteinases in vitro and to be present in gastric carcinoma cells. By immunohistochemistry we localized tissue kallikrein in the cytoplasm of ductal breast cancer cells. In addition, two-dimensional Western blotting was used to further characterize its biochemical properties. By this method immunoreactive tissue kallikrein was found to have a molecular mass of 25 kDa and an isoelectric point close to pH 6. Furthermore its presence in human milk could be demonstrated.

Matrix metalloproteinases (MMPs) represent a family of structurally and functionally related enzymes responsible for the proteolytic degradation of extracellular matrix (ECM) components such as basement membrane or interstitial stroma. MMPs are important participants of normal tissue remodeling. Due to their potential hazardous effects MMPs are highly regulated at different levels. At the transcriptional level, MMP expression is precisely controlled by various cytokines acting through positive or negative regulatory elements of its genes. Moreover, MMP activity is post-transcriptionally regulated by proteolytic activation of the latent proenzymes and by interaction with specific tissue inhibitors of metalloproteinases (TIMPs). Expression and secretion of both MMP activating enzymes and TIMPs are also influenced by cytokines. Dysregulation of MMP production and activation may cause altered extracellular proteolysis that is associated with a number of diseases such as rheumatoid arthritis and tumor metastasis. Thus, the molecular analysis of the regulatory mechanisms of gene expression and activity of MMPs and their inhibitors is essential for understanding the complex scenario of tissue remodeling and ECM degradation under both normal and pathological conditions.

Cathepsin G was isolated from granules of rabbit bloodstream leukocytes and purified to apparent homogeneity by a multi-step procedure consisting of ammonium sulphate precipitation, affinity chromatography on elastin-Sepharose, and finally by ion-exchange chromatography on a CM-52 column. The molecular weight of the enzyme, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), was 27,000. The first 24 N-terminal amino-acids were determined and showed 96%, 92% and 79% identity respectively to those of human, dog and rat cathepsin G. Despite the difference in the total amino-acid composition of cathepsin G between rabbit and other mammalian species, close similarities have been found in their substrate specificity and inhibition profile. The kcat/Km values of rabbit cathepsin G with Suc-Ala2-Pro-Phe-NA and Suc-Ala2-Pro-Leu-NA are quite similar to those reported for human cathepsin G under the same conditions. The inhibition profile of the isolated enzyme indicates that cathepsin G from rabbits, like that from other mammalians species belongs to the group of serine proteinases. Finally, like human cathepsin G, catalytically active rabbit enzyme is able to induce platelet aggregation.

From human embrional lung fibroblasts mRNA was obtained and converted to cDNA. The procathepsin L coding region was amplified by PCR, inserted into pALTER and, after checking the nucleotide sequence, transferred into pET81F1+. Procathepsin L was expressed by induction of recombinant E. coli strain BL21[DE3](pLysS) with IPTG and was found to be deposited into inclusion bodies. These were isolated and solubilized in guanidinium hydrochloride. The soluble proteins were sulphonated and procathepsin L was obtained after gel filtration. Purified proenzyme was refolded by dialysis and autoactivated into a form of the expected size and enzymatic activity against a fluorogenic substrate.