Showing papers in "Biological chemistry Hoppe-Seyler in 1996"

Mechanisms of intracellular protein transport

Abstract: Recent advances have uncovered the general protein apparatus used by all eukaryotes for intracellular transport, including secretion and endocytosis, and for triggered exocytosis of hormones and neurotransmitters. Membranes are shaped into vesicles by cytoplasmic coats which then dissociate upon GTP hydrolysis. Both vesicles and their acceptor membranes carry targeting proteins which interact specifically to initiate docking. A general apparatus then assembles at the docking site and fuses the vesicle with its target.

Cystatins in health and disease

Abstract: Proteolytic enzymes have many physiological functions in plants, bacteria, viruses, protozoa and mammals. They play a role in processes such as food digestion, complement activation or blood coagulation. The action of proteolytic enzymes is biologically controlled by proteinase inhibitors and increasing attention is being paid to the physiological significance of these natural inhibitors in pathological processes. The reason for this growing interest is that uncontrolled proteolysis can lead to irreversible damage e.g. in chronic inflammation or tumor metastasis. This review focusses on the possible role of the cystatins, natural and specific inhibitors of the cysteine proteinases, in pathological processes.

Synthetic peptides corresponding to alpha-lactalbumin and beta-lactoglobulin sequences with angiotensin-I-converting enzyme inhibitory activity.

Abstract: Novel angiotensin-I-converting enzyme (ACE) inhibitory activities were detected in synthetic peptides corresponding to sequences of beta-lactoglobulin and alpha-lactalbumin and which are known to possess opioid activity. Using hippuryl-histidyl-leucine as substrate, the tetrapeptides beta-lactorphin (Tyr-Leu-Leu-Phe), alpha-lactorphin (Tyr-Gly-Leu-Phe) and beta-lactotensin (His-Ile-Arg-Leu) were shown to have IC50 values of 171.8, 733.3 and 1153.2 microM, respectively. Related dipeptides also inhibited ACE, with Tyr-Leu being the most potent, having an IC50 value of 122.1 microM.

Inactivation of the Very Strong HCMV Immediate Early Promoter by DNA CpG Methylation In Vitro

Abstract: The influence of DNA methylation in vitro on the activity of the very strong human cytomegalovirus (HCMV) major immediate early (IE) modulator/enhancer/promoter region was investigated by transient transfection experiments of premonocytic HL-60 cells. While sequence-specific methylation of the major IE enhancer and/or modulator with the cytosine methyl-transferases FnuDII, HhaI and HaeIII had no significant effect, the promoter activity was completely repressed by methylation of the cytosine in 5'-CpG sites with the Spiroplasma methyltransferase SssI. Addition of TNF-alpha or PMA which are strong stimulators of HCMV major IE enhancer/promoter activity in premonocytic HL-60 cells had no effect on repression. Inactivation of the IE enhancer/promoter via methylation by M.SssI could be partially alleviated by co-transfection with an excess of untranscribable highly methylated DNA. These results indicate that a methyl-CpG binding factor is involved as mediator in the inhibitory effect of HCMV enhancer/promoter methylation. Taken together, the HCMV major IE enhancer/ promoter has been shown to be susceptible to transcriptional inactivation by methylation of the cytosines in CpG dinucleotides, a process that is proposed to play a modulatory role in viral latency.

Pathogenic mechanisms induced by microbial proteases in microbial infections.

Abstract: Most bacterial and fungal proteases excreted into infected hosts exhibit a wide range of pathogenic potentials ranging from pain, edema or even shock to translocation of bacteria from the site of infection into systemic circulation, thus resulting in septicemia. The basic mechanism or principle common to all these phenomena is explained by kinin generation, either directly from high- and/or low-molecular weight kininogens or indirectly via activation of the bradykinin generating cascade: i.e. Hageman factor-->activated Hageman factor-->prekallikrein-->kallikrein-->high-molecular weight kininogen-->bradykinin. Some bacterial proteases are also involved in activation of other host protease zymogens such as plasminogen, procollagenase (matrix metallo proteases) and proenzymes of the clotting system. Furthermore, most bacterial proteases are not only resistant to plasma protease inhibitors of the hosts, most of which belong to a group of serine protease inhibitors called serpins (serine protease inhibitors), but they also quickly inactivate serpins. Some bacterial proteases may also activate bacterial toxins thus rendering toxigenic pathogenesis. They are also capable of degrading immunoglobulins and components of the complement system and facilitate propagation of micro organisms. All in all, microbial proteases are very critical in enhancing pathogenesis of severe diseases. It is also noteworthy that bacterial cell wall components themselves, i.e. endotoxin (or lipopolysaccharide) of gram negative bacteria and teichoic/lipoteichoic acid of gram positive bacteria, are also able to activate the bradykinin generating cascade-involving activation of Hageman factor as mentioned above.

The biostructural pathology of the serpins: critical function of sheet opening mechanism.

Abstract: The serpins illustrate the way in which the study of a protein family as a whole can clarify the functions of its individual members. Although the individual serpins have become remarkably diversified by evolution they all share a common structural pathology. We have previously shown how plotting of the dysfunctional natural mutations of the serpins on a template structure defines the domains controlling the mobility of the reactive centre loop of the molecule. Here we compare these natural mutations with reciprocal mutations in recombinants that restore the inhibitory stability of a labile member of the family, plasminogen activator inhibitor-1 (PAI-1). The combined results emphasise the critical part played by residues involved in the sliding movement that opens the A-sheet to allow reactive loop insertion. It is concluded that changes in these residues provide the prime explanation for the ready conversion of PAI-1 to the inactive latent state. The consistency of the overall results gives confidence in predicting the likely consequences of mutations in individual serpins. In particular the two common polymorphic mutations present in human angiotensinogen are likely to affect molecular stability and hence may be contributory factors to the observed association with vascular disease.

GSH transporters: molecular characterization and role in GSH homeostasis.

Abstract: Considerable progress has been made in the last few years in the molecular identification and characterization of hepatic GSH transporter-associated polypeptides. We are now poised to determine their precise mechanisms of action and regulation at the transcriptional and post-translational level. It is also anticipated that molecular characterization of the mitochondrial GSH transporter and sodium GSH co-transporters will be accomplished in the near future. With this information, a more complete understanding of GSH/cysteine homeostasis can be achieved which can be applied to furthering the prevention and treatment of the diseases of oxidative stress, such as aging, HIV, cataract, atherosclerosis, cancer and alcoholic liver disease.

Sialic acids structure-analysis-metabolism-occurrence-recognition.

Abstract: Sialic acids are commonly positioned at non-reducing termini of complex carbohydrates. Steady refinements of analytical techniques have enabled detailed mapping of the complexity of sialic acids, unravelling a number of possibilities for substitutions. These developments have aided the description of the required enzymatic activities. In view of the physiological significance of this intriguing extent of variability of one sugar unit, the assumption that distinct types of sialic acids can serve as ligands in recognitive interactions is gaining support. It is reinforced by the discovery of several classes of mammalian lectins that bind sialo-glycoconjugates. Notably, an often encountered modification of sialic acids, namely O-acetylation, can be considered as a modulatory signal in recognition, either serving as contact point or masking a ligand structure. The increased knowledge of the physiological roles of sialic acids, for example in selectin-mediated leukocyte recruitment to sites of inflammation or in influenza virus propagation, even points to clinical applications. This perspective has led the field from the inherently descriptive beginning to technically sophisticated attempts for deliberate drug design.

Prognostic Value of Cathepsins B, H, L, D and Their Endogenous Inhibitors. Stefins A and B in Head and Neck Carcinoma

Abstract: To estimate the prognostic value of cathepsins B, H, L, D and stefins A and B in head and neck carcinoma, their concentrations in cytosols of primary tumours and adjacent normal tissue were measured (cathepsins B, D stefins A, B in 45, cathepsin L in 24 and cathepsin H in 21 patients). Median concentrations of cathepsins B, L, and D were significantly higher in tumour than in the adjacent normal tissue (B and D: p < 0.0001; L: p = 0.004); cathepsin H concentration was higher in normal tissue (p = 0.001). Concentrations of either stefin did not differ significantly between normal and tumour tissue. Concentrations of cathepsins B, H, L, and D were higher in laryngeal than in non-laryngeal normal and tumour tissues. The difference was statistically significant for cathepsin B in tumour tissue (p = 0.045), and marginally significant in normal tissue (p = 0.07). Early tumours had lower concentrations of stefins A and B than locally advanced tumours (stefin A: p = 0.04; stefin B: p = 0.07). Disease-free and disease-specific survival rates were better in patients with concentrations of cathepsin L in tumour tissue below or equal to the cut-off values (p = 0.035; p = 0.05), whereas for cathepsin B the difference was established only for disease-free survival (p = 0.07). The opposite was true for stefin A (p = 0.0002; p = 0.002) and stefin B (p = 0.009; p = 0.003), and in disease-free survival also for cathepsin H (p = 0.055). The concentration of cathepsin D did not correlate with survival. Our data indicate that cathepsins B, H, L and stefins A and B might have prognostic value in head and neck carcinoma.

DNA-binding specificity of PAR and C/EBP leucine zipper proteins: a single amino acid substitution in the C/EBP DNA-binding domain confers PAR-like specificity to C/EBP.

Abstract: PAR and C/EBP family proteins are liver-enriched basic leucine zipper (bZip) transcription factors that bind similar sites on the promoters of albumin and cholesterol 7 alpha hydroxylase genes. However, C/EBP proteins have a more relaxed binding specificity than PAR proteins, in that they recognize many sites within promoter or randomly selected rat genomic DNA sequences that are ignored by PAR proteins. Thus, DNAse I protection experiments suggest that C/EBP recognizes a binding site with an affinity similar to the one of the cholesterol 7 alpha hydroxylase gene promoter every 200 to 300 bp. The frequency of PAR protein binding sites with comparable affinities is about 20-fold lower in the rat genome. By using a PCR-based amplification assay we selected high affinity DNA-binding sites for C/EBP beta and the PAR protein DBP from a pool of oligonucleotides. Both proteins indeed recognize similar sequences with the optimal core binding sequences 5'RTTAY.GTAAY3'. However, as expected, DBP, is considerably less tolerant to deviations from the consensus site. Here we have characterized a single amino acid substitution mutant of C/EBP beta that increases its target site specificity. This protein, C/EBP beta V > A, contains a valine to alanine substitution at position 13 of the basic domain (residue 216 of C/EBP beta). C/EBP beta V > A selectively binds only the subset of C/EBP sites that are also DBP sites, both as oligonucleotides and within the natural contexts of the albumin and cholesterol hydroxylase promoters.

Glycyl-tRNA synthetase.

Abstract: Glycyl-tRNA synthetase, a class II aminoacyl-tRNA synthetase, catalyzes the synthesis of glycyl-tRNA, which is required to insert glycine into proteins. In a side reaction the enzyme also synthesizes dinuceloside polyphosphates, which probably participate in regulation of cell functions. Glycine is the smallest amino acid occurring in natural proteins, probably established as a protein component very early in evolution. Besides the amino and the carboxyl groups there is no functional group in the molecule. Alanine, the amino acid which is structurally most similar to glycine, possesses an additional methyl group as 'side chain'. Glycyl-tRNA synthetase is one of the few synthetases which exhibit different oligomeric structures in different organisms (alpha 2 beta 2 and alpha 2). The alpha 2 beta 2 enzymes exhibit similarities to PheRS (also an alpha 2 beta 2 enzyme). The alpha 2 forms belong to the subclass IIa enzymes with regard to sequence homologies. In eukaryotes the polypeptide is weakly associated with multienzyme complexes consisting of aminoacyl-tRNA synthetases. In the aminoacylation reaction a 'half-of-the-sites' mechanism as found for GlyRS from Bombyx mori is probably used by all glycyl-tRNA synthetases under in vivo conditions. Essentially, tRNAGly is recognized by GlyRS through standard identity elements in the anticodon region and in the acceptor stem. The last three facts may indicate that GlyRS is an enzyme which still possesses properties of a primordial aminoacyl-tRNA synthetase. Nine genes of glycyl-tRNA synthetases from six organisms have been sequenced. They encode synthetase subunits of chain lengths ranging from 300-700 amino acids. One crystal structure, that of the alpha 2 enzyme from Thermus thermophilus, has also been determined. The two subunits each possess three domains: the active site resembling that of aspartyl and seryl enzymes, a C-terminal anticodon recognition domain, and one domain which almost certainly interacts with the acceptor stem of tRNAGly. Antibodies against glycyl-RNA synthetase occur in the sera of patients suffering from polymyositis and interstitial lung disease.

Minimally modified oligonucleotides : combination of end-capping and pyrimidine-protection

Abstract: The nuclease resistance of an oligonucleotide sequence that was phosphorothioate (PS)-modified in various positions and patterns was examined. We present a new ?minimal' protection strategy for antisense oligonucleotides which is a combination of the end-capping technique and the protection of internal pyrimidine residues which are the major sites of endonuclease degradation. This strategy reduces the number of modifications needed to make a nuclease resistant oligonucleotide and therefore should minimize the non-sequence-specific effects that are frequently observed with uniformly modified oligonucleotides.

Repeat-induced gene silencing: common mechanisms in plants and fungi

Abstract: One of the most surprising observations made in plant science in recent years is the inactivation of transgenes triggered by interactions between DNA repeats. In plants, we can differentiate between transcriptional silencing, most likely reflecting a regulation at the DNA level, and post-transcriptional silencing that affects steady state RNA levels. In the filamentous fungi Ascobolus immersus and Neurospora crassa, we find two premeiotic silencing processes that are also based on the interaction of repeated sequences. A common feature of transcriptional silencing in plants and premeiotic gene inactivation in filamentous fungi is that the repeated sequences undergo cytosine methylation. DNA methylation, which is either the cause or the consequence of gene silencing, can be associated with changes in chromatin structure. These structural changes are reminiscent of homology-based silencing mechanisms in Drosophila, an organism that lacks DNA methylation. Repeat-induced silencing may therefore reflect the activity of an endogenous mechanism, present in some species, which screens for homology and has significant implications for the organization and evolution of the genome.

Human Immunodeficiency Virus type 1 reverse transcriptase.

Abstract: The Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus and a causative agent of the Acquired Immuno Deficiency Syndrome (AIDS). Retroviruses are distinct from other viruses in their ability to encode an enzyme called reverse transcriptase (RT). The RT is the enzyme mainly involved in replication. It performs RNA- as well as DNA-dependent DNA synthesis in order to convert the single-stranded viral RNA genome into double-stranded DNA. The double-stranded DNA is stably integrated into the host cell genome and is used as a template for the production of a new viral generation. The HIV-1 RT is partially encoded by the POL open reading frame of the HIV-1 genome and consists of two subunits of 66 kDa (p66) and 51 kDa (p51). The p66 polypeptide encodes the reverse transcriptase and the RNase H domain. Half of the p66 molecules are further processed to generate the p51 protein with an identical N-terminus, but lacking the C-terminus which encodes the RNase H domain. In vivo both polypeptides are found in equimolar amounts thus forming a heterodimer. This dimerization is critical for the enzymatic activity. In this review we summarize (i) the replication cycle of HIV-1, (ii) the enzymatic properties of HIV-1 RT and (iii) the structure-function relationship of the HIV-1 RT in view of the known three dimensional structure.

Selective inhibition of cyclic AMP-dependent protein kinase by isoquinoline derivatives.

Abstract: A large series of isoquinoline derivatives was synthesised including derivatives of isoquinoline, isoquinolino[3,4-c]furazan, 1,2-dihydro-1-oxoisoquinoline, 6-oxopyrimido[1,2-d]isoquinoline, benzo[c][1,8]-naphthyridine, pyrazino[2,3-c]isoquinoline and benzimidazo[2,1-a]isoquinoline as well as further structurally related isoquinoline derivatives and pyrido-2,3-furazans. Representatives of all of these classes of isoquinolines are potent and selective inhibitors of the cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK) from rat liver. The most effective cAK inhibitors are a series of 1,3-di-substituted and 1,3,4-tri-substituted isoquinolines (IC50 values 30-50 nM) (compounds A1, A2, A3, A4 and A5) and 2-ethylcarboxy-3-amino-5,6-dihydro-6-oxobenzo[c] [1,8]naphthyridine (E1) (IC50 0.08 microM). Compounds A1-A5 inhibit cAK in a fashion that is competitive with respect to ATP as substrate. The isoquinoline inhibitors A1-A5 are ineffective or very poor inhibitors of wheat embryo Ca(2+)-dependent protein kinase (CDPK) and rat brain Ca(2+)-dependent protein kinase C (PKC), chicken gizzard myosin light chain kinase (MLCK) and potato tuber cyclic nucleotide-binding phosphatase (Pase). E1 is a moderately effective inhibitor of CDPK and PKC (IC50 values 30 and 61 microM, respectively). The bisisoquinoline-1(2H)-one compound B7 inhibits cAK, CDPK, PKC and MLCK (IC50 values 8, 95, 24 and 7 microM, respectively) as does J1 [2-(p-bromophenyl)pyrrolo-[2,3-c]isoquinoline-5(4H)-one] (IC50 values 2, 50, 44 and 7 microM, respectively). The very potent isoquinoline-derived cAK inhibitors found here involve substitution of the N-containing isoquinoline ring system and these inhibitors show high specificity for cAK.

Dramatic changes in the ratio of homologous recombination to nonhomologous DNA-end joining in oocytes and early embryos of Xenopus laevis.

Abstract: We have developed a versatile plasmid vector (pReco-sigma) for recombination studies. When linearized and introduced into the cells of interest, pReco-sigma allows the simultaneous determination of the relative frequencies of homologous recombination versus nonhomologous DNA-end joining (also termed end-to-end joining), the latter an example of illegitimate recombination processes. As a system we made use of stage VI oocytes and fertilized eggs of the African clawed frog Xenopus laevis, which were previously described to support homologous recombination and DNA-end joining, respectively. Extending these earlier findings, we show that oocytes yield > 80% of the homologously recombined product, whereas in eggs a highly efficient DNA-end joining activity predominates (> 95%). Both reactions, homologous recombination and DNA-end joining, are shown to occur quickly, with the majority of the respective products being formed within the first 20 minutes of incubation under optimal conditions. In fertilized eggs, up to 50% of all injected linear DNA molecules are recircularized by DNA-end joining. With high amounts of injected DNA per fertilized egg, DNA-end joining is reduced, presumably due to competition for essential factors, and homologous recombination becomes readily detectable. As there is a sequence of rapid cleavage divisions after fertilization of the egg, the fast and highly efficient DNA-end joining, even though it is error-prone at the junction site, seems to be best suited to cope with DNA double-strand breaks that might occur in the genome during early embryogenesis. On the other hand, the long-lived oocytes seem to repair DNA double-strand breaks via homologous recombination. This latter property may be exploited both in Xenopus and in other organisms to achieve homologous integration of exogenous DNA into germ cells for gene targeting.

Differential sensitivity of zinc finger transcription factors MTF-1, Sp1 and Krox-20 to CpG methylation of their binding sites.

Abstract: Cytosine methylation at CpG sites is often negatively correlated with mammalian gene activity. Many transcription factors whose DNA binding site contains one or more CpG dinucleotides are no longer able to efficiently bind DNA when the site is methylated. A notable exception is the zinc finger factor Sp1 which binds DNA and activates transcription even when its binding site is methylated. Here we show that two other zinc finger factors, MTF-1 and Krox-20, can also bind to CpG methylated sites. MTF-1 regulates metallothionein gene transcription by binding to a number of metal responsive elements (MREs), and Krox-20 regulates Hox genes during hindbrain segmentation. However, a refined analysis of MTF-1/MRE binding shows that methylation is not tolerated at every binding site: the highest affinity site in the mouse metallothionein I gene, MREd, is unaffected by methylation, while two other MRE sites with CpGs at different positions are rendered partially or completely nonfunctional by methylation. Both methylation sensitive and insensitive factors/binding sites are likely to determine the developmental expression pattern of a gene.

Anisoosmotic regulation of hepatic gene expression.

Abstract: The effect of anisoosmolarity on the abundance of various mRNA species was examined in perfused rat liver and H4IIE rat hepatoma cells. Hyperosmotic exposure (385 mosmol/l) of isolated rat livers increased mRNA levels for tyrosine aminotransferase (TAT) by 246% and those for phosphoenolpyruvate carboxykinase (PEPCK) by 186%, whereas hypoosmotic exposure (225 mosmol/l) decreased their levels to 43% and 42%, respectively. mRNA levels for fructose-1,6-bisphosphatase (FBP), argininosuccinate lyase (ASL), argininosuccinate synthetase (ASS), glutamine synthetase (GS), glutaminase (GA) and glucokinase (GK) were largely unaffected. In H4IIE cells the modulation of TAT and PEPCK mRNA levels by anisoosmotic exposure was similar to that found in perfused rat liver. ASL and glutaminase mRNA levels were influenced in an opposite manner. The effects of anisoosmolarity on PEPCK mRNA levels in H4IIE cells were largely abolished in the presence of the protein kinase inhibitors H-7, H-89 and HA-1004. Other protein kinase inhibitors such as Go-6850, KN-62, Rp-8-CPT-cAMPS, rapamycin, wortmannin, genistein or herbimycin did not prevent the osmosensitivity of PEPCK mRNA levels. Also pertussis and cholera toxin, vanadate and colchicine did not affect the osmosensitivity of PEPCK mRNA levels. The data suggest that anisoosmotic exposure acts on the levels of some but not all mRNA species and that this action may involve changes in protein phosphorylation. They further indicate that the recently identified osmosensitive signal transduction pathway which involves a G-protein and tyrosine kinase dependent activation of mitogen-activated protein kinases is apparently not involved in the osmoregulation of PEPCK mRNA levels.

Role of endocrine cell microvesicles in intercellular chemical transduction.

Abstract: Microvesicles (MVs) in endocrine cells are morphologically similar to neuronal synaptic vesicles. MVs were shown to contain proteins involved in neurotransmitter storage such as vacuolar H(+)-ATPase and neurotransmitter transporters, and ones in vesicular trafficking such as synaptobrevins and N-ethylmaleimide-sensitive fusion protein. Isolated MVs accumulate cell-specific neurotransmitters in an energy-dependent manner. Upon stimulation, the MVs may fuse with the plasma membrane and secrete the internal neurotransmitters. Thus, endocrine cells possess an MV-mediated secretion system as an intercellular signal transducing system.

Structure of recombinant human parathyroid hormone in solution using multidimensional NMR spectroscopy.

Abstract: The solution structure of human parathyroid hormone, in the form of recombinant prolyl-hPTH(1-84), has been investigated by multidimensional NMR spectroscopy under conditions (aqueous trifluoroethanol) which favour the structured-state of the protein. Spin systems were identified from 3D 1H DQF (double-quantum filtered)-COSY and TOCSY spectra and sequence-specific assignments were from 2D 1H phase-sensitive NOESY spectra. Signal overlap was resolved in a 3D-NOESY-TOCSY spectrum and assignments were confirmed with 2D NOESY-15N-HMQC (heteronuclear multiple-quantum coherence) spectra taken of a sample universally labeled with 15N. A satisfactory set of final structures was calculated from the quantitative NOE data using restrained molecular dynamics and energy minimization calculations. The N-terminus is dominated by three, well defined helices between Ser-3 to Asn-10, Ser-17 to Lys-27 and Asp-30 to Leu-37, while the most significant structural features in the C-terminus are a short, less-well defined helix between Asn-57 to Ser-62 and a series of loose turns. These two terminal units are joined by an unstructured mid-region. The molecule shows a tendency towards tertiary structure, defined by a number of long-range NOEs. A detailed RMS deviation analysis allowed the final refined structures to be classified into a limited ensemble of stable conformations that reflect the inherent flexibility of the hormone in solution.

Reductive activation markedly increases the stability of cathepsins B and L to extracellular ionic conditions.

Abstract: Cathepsins B and L are thought to function extracellularly in pathological conditions. pH-Activity profiles of cathepsin B, measured in phosphate and acetate-Mes-Tris buffers of constant ionic strength, indicated that cathepsin B is sensitive to specific buffer ions, as previously reported for cathepsin L. In assessing the activity of these enzymes in vitro the influence of the buffer must therefore be taken into account. In Hank's balanced salt solution, a buffer modeling the extracellular fluid, the half-life of activated human liver cathepsin B at 37 degrees C is 245 +/- 11.3 s, at pH 7.2, and 857 +/- 50.1 s, at pH 6.8 (the peritumor pH), indicating that cathepsin B is markedly stable under these conditions. The stability was increased by the additional presence of proteins. Without immediate activation, however, the stabilities of both cathepsins B and L were markedly decreased, a large proportion of their activity being lost before it could be measured. Enzymes injected into the extracellular space in the unactivated state would therefore survive for only a very short time in their native conformation. It is proposed that the active site thiolate-imidazolium ion pair contributes substantially to the stability of cathepsins B and L to extracellular ionic conditions.

Human Colon Carcinoma Cells Synthesize and Secrete α1-Proteinase Inhibitor

Abstract: Our previous results have shown that tumor cell-secreted procathepsin B can be activated by neutrophil elastase in vitro. In the present study, we addressed two questions: 1. Can neutrophil elastase be detected in human colon carcinomas, and 2. Does the co-culture of human colon carcinoma cells with neutrophils generate a cathepsin B-dependent pericellular proteolysis as assessed with radiolabeled laminin? We show that neutrophil elastase is present in colon carcinoma tissue and that its level is in good agreement with the degree of tissue infiltration by neutrophils. In co-culture experiments, elastase is released by neutrophils in a cell number dependent way, but no activation of tumor cell-secreted procathepsin B could be observed. In addition, the degradation of radiolabeled laminin by neutrophil proteinases was markedly decreased in the presence of tumor cells. These findings prompted us to search for a tumor cell-secreted elastase inhibitor. We show by enzyme activity measurements, gelatin-zymography, immunoblotting and RT-PCR that colon carcinoma cells synthesize and secrete alpha 1-proteinase inhibitor, a functional inhibitor of neutrophil elastase. The importance of this finding in the context of pericellular activation of tumor cell-secreted procathepsin B by neutrophil elastase is discussed.

Isolation and characterization of sialate lyase from pig kidney.

Abstract: Sialate lyase (sialate aldolase; systematic name N-acetylneuraminate pyruvate-lyase, EC 4.1.3.3) was isolated as soluble enzyme from pig kidney and purified 630-fold using a heating step, gel filtration, and chromatography on immobilized neuraminic acid beta-methyl glycoside in 14% yield to apparent homogeneity as tested by SDS-gel electrophoresis. The molecular mass is 58 kDa and the pH-optimum is at pH 7.2. Kinetic parameters were determined with N-acetyl-neuraminic acid as substrate: Km 3.7 mM and Vmax 37.1 mU. The lyase cleaves only free sialic acids with relative rates of 100% for N-acetylneuraminic acid, 55% for N-glycolylneuraminic acid and 32% for N-acetyl-9-O-acetylneuraminic acid, whereas N-acetyl-4-O-acetylneuraminic acid or 2-deoxy-2,3-didehydro-N-acetylneuraminic acid are not substrates. Enzyme activity was inhibited with p-chloromercuribenzoate, o-phenanthroline, cyanide, 5-diazonium-1-H-tetrazole, 5,5'-dithiobis(2-nitrobenzoic acid), diethylpyro-carbonate, and Rose Bengal in the presence of light and O2. Reduction with sodium borohydride in the presence of N-acetylneuraminic acid or pyruvate resulted in irreversible inhibition of enzyme activity. The inhibition experiments suggest the involvement of histidine, lysine and SH-residues in enzyme catalysis. Thus, this mammalian lyase most probably belongs to the Class I aldolases, and has properties similar to the same enzyme from Clostridium perfringens and is active with the alpha-form of N-acetylneuraminic acid.

Substrate and inhibitor specificity of glutamine cyclotransferase (QC).

Abstract: This paper reports a systematic study of the substrate and inhibitor specificity of papaya latex glutamine cyclotransferase (QC). The results showed that the second amino acid residue in N-terminal glutaminyl peptides significantly accelerated papaya latex QC-catalyzed reactions while the third residue provided no further rate enhancement. Substrate binding was shown to be the main specificity-determining step. Fifteen proline derivatives and dipeptides containing an N-terminal proline were tested and found to inhibit papaya latex QC. This supports our previous molecular modeling study of the QC catalytic pathway which suggested a structure of the reaction intermediates similar to that of L-proline.

Characterization of cystatin C from bovine parotid glands: cysteine proteinase inhibition and antiviral properties.

Abstract: Cystatin C, a low Mr cysteine proteinase inhibitor was isolated from bovine parotid glands by a procedure which includes alkaline treatment of the homogenate, affinity chromatography, gel filtration and ion exchange chromatography. The purified inhibitor has a pl of 8.0 and Mr of 14500. The identity with bovine cystatin C from colostrum was confirmed by N-terminal sequence of the inhibitor and amino acid composition. Cystatin C rapidly (kass = 5.5 x 10(7) M-1s-1) and tightly inhibits papain (Ki = 0.02 nM), whereas its interaction with bovine cathepsin B is substantially weaker (Ki = 4.4 nM). Bovine cystatin C also shows a weak antiviral effect on poliovirus infected human Hela cells.

Heterologous expression and characterisation of mouse brain fatty acid binding protein.

Abstract: A novel brain-type member of the fatty acid binding protein family (B-FABP) was heterologously expressed in Escherichia coli, either as inclusion bodies at 37 degrees C or in soluble form at 22 degrees C. Both B-FABP renatured from inclusion bodies and the solubly expressed protein could be purified to homogeneity by anion exchange chromatography and gel filtration in a functional conformation as they bound oleic acid with high affinity. None of the five cysteines of B-FABP was involved in disulphide bond formation. Isoelectric focusing revealed heterogeneity of the renatured protein but not of the solubly expressed protein. By Western blotting using affinity purified rabbit antibodies raised against the recombinant B-FABP it was demonstrated that in adult mice, B-FABP is predominantly expressed in the olfactory bulb.

The production and characterization of artificial heterodimers of the restriction endonuclease EcoRV.

Abstract: A novel approach to studying the inter- and intrasubunit communication required for the activity of homodimeric proteins is described. It was developed for the restriction endonuclease EcoRV, but should also be useful for other homodimeric enzymes. Two ecorV genes encoding different EcoRV mutants are coexpressed in the same Escherichia coli cell leading to homo- and heterodimeric variants of the enzyme. The two ecorV genes carry either a 5' extension coding for the glutathione-S-transferase or a His6-tag. The EcoRV heterodimer produced in vivo is separated from the two EcoRV homodimers and purified to homogeneity by affinity chromatography. Purified EcoRV heterodimers are stable and are not subject to reassortment of the subunits. To investigate the interdependence of the two catalytic centers, EcoRV heterodimers consisting of one subunit with wild type sequence and one subunit with amino acid substitutions in the PD...(D/E)XK motif, characteristic for the active sites of many restriction endonucleases, were produced. While the homodimeric EcoRV active site mutants are catalytically inactive, the heterodimeric EcoRV variants with one active and one inactive catalytic center display a twofold reduced activity toward oligodeoxynucleotide substrates compared to the wild type, and preferentially nick supercoiled plasmid DNA. From these results we conclude that in the wild type enzyme both catalytic centers function independently of each other.

Purification and characterisation of the pyruvate decarboxylase from a haploid strain of saccharomyces cerevisiae

Abstract: A novel purification procedure was developed for pyruvate decarboxylase (PDC, E.C. 1.1.1.4) from the haploid yeast strain YSH 4.127-1A expressing only one (PDC1) of the three structural genes for PDC. The purified enzyme is homotetrameric with a molecular mass of about 240,000 whereas PDC from brewer's yeast is a dimer of dimers composed of subunits of different size (alpha 2 beta 2) with the same molecular mass as the tetramer. Despite these structural variations there are no significant differences in the kinetic behaviour of the two enzyme species. PDC purified from the haploid yeast mutants shows a sigmoid dependence of the reaction rate from the substrate concentration due to the substrate activation. In the presence of the substrate surrogate pyruvamide the shape of the v/S plot is transformed into a hyperbolic one. As expected, polyclonal antibodies react with both the enzyme from haploid yeast strain mutants and that from brewer's yeast.

Evaluation of HBV promoters for use in hepatic gene therapy.

Abstract: Strategies for in vivo hepatic gene therapy will require regulatory elements which allow for long-term expression of therapeutic genes and restriction of expression to hepatocytes. This study investigates the suitability of promoters derived from hepatitis B virus (HBV) for liver-specific gene expression in vectors for hepatic gene therapy. We provide three hepatocyte-specific promoters, the HBV core promoter, the HBV core promoter linked directly to the HBV enhancer I, and a hybrid promoter containing the HBV enhancer II and a basic CMV promoter, which are hepatocyte-specific and allow for increasing levels of reporter gene expression. Moreover, in long-term expression studies using our promoter constructs in the context of an EBV based expression system we found that expression from these promoters remained nearly unchanged over a period of at least two months in hepatocyte-derived cell lines.

Purification of a new ribosome-inactivating protein from the seeds of Cinnamomum porrectum and characterization of the RNA N-glycosidase activity of the toxic protein.

Abstract: Porrectin, a new type II ribosome-inactivating protein (RIP), was purified from the seeds of the camphor tree (Cinnamomum porrectum) by affinity chromatography on acid-treated Sepharose 4B. Porrectin is a glycoprotein (M(r)64,500, sugar content 2.5%) consisting of an A-chain (M(r)30,500) and a B-chain (M(r)33,500) linked by the disulfide bond. The terminal sugar of glycan in porrectin B-chain is determined to be mannose. By non-denaturing polyacrylamide gel electrophoresis, porrectin displayed three isoforms that have different pl values with the same molecular weight. Porrectin is a potent inhibitor of eukaryotic protein synthesis in the rabbit reticulocyte lysate system. The molecular mechanism of action of porrectin on rat liver ribosomes is demonstrated to be specific for RNA N-glycosidase. The cleavage site is the adenosine at position 4324 (rat liver 28S rRNA) embedded in the highly conserved ricin/alpha-sarcin ('R/S') domain.