Biological chemistry Hoppe-Seyler最新文献

The hemoglobin of the lobe-lipped bat (Chalinolobus morio, Vespertilionidae) is composed of 45% HbI and 55% HbII. Both components show identical alpha-chains but differ at the following three positions of their beta-chains: beta I/beta II 21: Glu/Asp, 70: Ser/Ala, and 135: Gln/Leu. High performance liquid chromatography revealed pure alpha-chains and a mixture of only partly separated beta-chains. Based on this material, the primary structures of all three globin chains could be achieved by automatic Edman degradation of the whole chains and peptides obtained by trypsin hydrolysis. Compared to human hemoglobin, Chalinolobus shows 17 replacements in the alpha-chains and 24/22 in the beta-chains. A sequence comparison of the globin chains from the three vespertilionid bats Chalinolobus morio and Myotis velifer (Vespertilioninae) as well as Antrozous pallidus (Nyctophilinae) supports a close relationship of the former only for the beta-chains. Molecular modeling showed that the replacements involved in three alpha 1/beta 1 and one alpha 1/beta 2 subunit interface contacts do not cause any interruption. All phosphate binding sites and amino acid residues responsible for the Bohr effect are unchanged. Thus normal physiological properties should be expected for Chalinolobus morio hemoglobin.

Deferoxamine (DFO)-induced iron deprivation caused an increase in p53 expression in ML-1 and Raji cells. In ML-1 cells, with express wild type p53, p53 protein levels were transiently increased 6 h after addition of 10(-4)M DFO. In Raji cells, which carry a mutant p53 allele, p53 increased 6 h after addition of 10(-4)M DFO and remained elevated for 24 h. Growth inhibition was observed in both cell types 6 h after addition of 10(-4)M DFO. In both cells, p53 mRNA levels did not increase following incubation with DFO, suggesting that increased p53 expression is the result of a post-transcriptional mechanism. Although increases in wild type p53 protein in ML-1 cells resulted in increases in a p53 target gene, p21cipl/wafl/sdil, this effect was not observed in Raji cells which express a mutant p53 protein.

Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a functional cysteine protease of the papain family which is expressed after infection in Spodoptera fruglperda Sf9 cells. The protease displays an inhibition profile typical for cysteine proteases and is highly active against synthetic peptide substrates. The pH optimum of the bell-shaped pH-activity curve is between 5.0 and 5.5. The best substrate tested is Z-Arg-Arg-MCA which is specific for cathepsin B. The specificity constant (Kcat/Km) of AcNPV protease for this substrate is approximately two times higher than for human cathepsin B. In contrast to human cathepsins, AcNPV protease does not exhibit a discriminating specificity towards neutral hydrophobic residues in the P2 position. These substrates are hydrolysed at a ten-fold lower rate than the P2 arginine containing substrate. The pH activity profile against the Z-Arg-Arg-MCA substrate reveals a pK of 5.35 which can be assigned to a glutamate residue in the S2 subsite pocket. Like in cathepsin B, this residue facilitates the binding of positively charged P2 residues in the primary binding pocket. In this respect, the AcNPV protease resembles cathepsin B more than cathepsins L and S.

Callus cultures of Taxus baccata L. cv. stricta were induced from hypocotyl and leaf explants on Woody Plant medium (hormone-free or additionated with phytohormones). Under continuous dark condition, adventitious roots were regenerated from hypocotyl- and leaf-derived callus cultures. Antibodies raised in rabbits against 10-succinyl-10-deacetylbaccatin III were used for the detection and the semi-quantitative determination of 10-deacetylbaccatin III in Taxus cultures. The presence of 10-deacetylbaccatin III in callus extracts was confirmed by TLC, HPLC using a photodiode array detector and mass spectrometry (CI-MS). The highest equivalent content of the taxoid derivatives (7.83 mg/100 g dry wt.) was detected in an extract from leaf-derived callus.

The phytopathogenic fungus Ustilago maydis exists in a yeast-like haploid form and as a filamentous dikaryon. Only the dikaryon can infect corn plants. We have isolated a gene, egl1, that is not expressed in haploid cells but strongly induced in the filament. Molecular and biochemical analyses revealed that egl1 encodes a cellulase. By immunogold labelling, secreted protein could be detected at the hyphal tip. Mutants deleted for egl1 are viable and are affected neither in filament formation nor in pathogenic development under the conditions tested.

We have characterized the gene of SVSP109, specifying the bovine secretory protein SVSP109, which is synthesized in a tissue- as well as species-specific manner. Approximately 1.3 kb upstream of the SVSP109 gene, the 3'-end of another gene designated HG5 was identified. The HG5 gene fragment comprises exon (n-1) and exon (n), separated by an intron. Exon (n) contains the complete 3'-untranslated region, whereas exon (n-1) encodes the C-terminal part of a hitherto unknown protein with high homology to SVSP109. The sequenced region of 6956 bp of a bovine genomic clone comprised the complete SVSP109 gene, which is made up of five exons and four introns. Primer extension analysis and RTPCR of poly(A+)RNA from seminal vesicle revealed that the first exon 1 extends to a position 34 bp downstream of the TATA sequence. Employing a CAT assay, a definitive but weak promoter activity was detected in pCATeSVSP15 (base pairs -639 to +574) and pCATeSVSP10 (base pairs -639 to +198); pCATeSVSP6 (base pairs -262 to +65) displayed promoter activity similar to the positive control. We conclude from these results that the TATA sequence located at position -34 is part of the functional promoter of the SVSP109 gene.

Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin which degrades various extracellular matrix components. uPA is focused to the cell surface via binding to a specific receptor (uPAR, also termed CD87). uPAR-bound uPA mediates pericellular proteolysis in a variety of biological processes, e.g. cell migration, tissue remodeling and tumor invasion. We have developed a competitive microtiter plate-based chromogenic assay which allows the analysis of uPA/uPAR interaction. The plates are coated with recombinant uPAR expressed in Chinese hamster ovary (CHO) cells. Proteolytically active uPA (HMW-uPA) is added to the microtiter plate-attached uPAR. The amount of receptor-bound uPA is then determined indirectly via addition of plasminogen, which is activated to plasmin, followed by cleavage of a plasmin-specific chromogenic substrate. Substances interfering with binding of HMW-uPA to uPAR diminish the generation of plasmin, as indicated by a reduction of cleaved chromogenic substrate. This assay was used to analyze the inhibitory capacity of a variety of proteins and peptides, respectively, on the uPA/uPAR interaction: i) uPAR and uPAR-variants expressed in CHO cells, yeast or E. coli, ii) the aminoterminal fragment (ATF) of human uPA or yeast recombinant pro-uPA, iii) synthetic peptides derived from the sequence of the uPAR-binding region of uPA, and iv) antibodies directed against uPAR. This assay may be helpful in identifying uPA and uPAR analogues or antagonists which efficiently block uPA/uPAR interaction.

During growth with formate as the sole energy source the autotrophic bacterium Alcaligenes eutrophus synthesizes a cytoplasmic formate dehydrogenase. The enzyme is a molybdo-iron-sulfur-flavo protein and the major NADH-producing system under these growth conditions, although it was estimated to constitute only 0.65% of the soluble cell protein. An electron microscopic analysis of the purified enzyme revealed that the particle is made up of four nonidentical submasses, corroborating previous structural data. The NH2-terminal amino acid sequences of the enzyme subunits exhibited significant similarities to those of only one other heteromeric formate dehydrogenase, the enzyme from the methane-utilizing bacterium Methylosinus trichosporium. Metal analyses yielded 21.5 g-atom iron, 2.18 g-atom nickel, 0.76 g-atom molybdenum, and 0.59 g-atom zinc per mol of enzyme. Initial electron paramagnetic resonance spectroscopic studies showed at least three distinct signals which appeared upon reduction of the enzyme with NADH or formate. The corresponding spin systems could be attributed to iron-sulfur centers of the enzyme. Comparative immunostaining and activity-staining experiments using cell extracts from various bacteria established immunological similarities between the soluble formate dehydrogenase of A. eutrophus and the soluble enzymes from all tested facultative autotrophs as well as from M. trichosporium.

The current status of calpain research is summarized on the basis of the most recent results. The main points are as follows. (i) Calpain constitutes a large family. (ii) Ca2+ ions cause the dissociation of calpain into subunits and the resulting free 80 kDa subunit is the active form of the enzyme. This dissociation corresponds to the activation of calpain. (iii) Some powerful clues have been obtained that will be helpful for analyzing the physiological function.