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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
31

Kallikrein-related peptidase 14 is the second KLK protease targeted by the serpin vaspin

Ulbricht, David, Tindall, Catherine A., Oertwig, Kathrin, Hanke, Stefanie, Sträter, Norbert, Heiker, John T. 27 January 2020 (has links)
Kallikrein-related peptidases KLK5, KLK7 and KLK14 are important proteases in skin desquamation and aberrant KLK activity is associated with inflammatory skin diseases such as Netherton syndrome but also with various serious forms of cancer. Previously, we have identified KLK7 as the first protease target of vaspin (Serpin A12). Here, we report KLK14 as a second KLK protease to be inhibited by vaspin. In conclusion, vaspin represents a multispecific serpin targeting the kallikrein proteases KLK7 and KLK14, with distinct exosites regulating recognition of these target proteases and opposing effects of heparin binding on the inhibition reaction.
32

Efficient extracellular recombinant production and purification of a Bacillus cyclodextrin glucanotransferase in Escherichia coli

Sonnendecker, Christian, Wei, Ren, Kurze, Elisabeth, Wang, Jinpeng, Oeser, Thorsten, Zimmermann, Wolfgang 13 April 2018 (has links)
Background: Cyclodextrin glucanotransferases (CGTases) catalyze the synthesis of cyclodextrins, cyclic oligosaccharides composed of glucose monomers that find applications in the pharmaceutical, food, and cosmetic industries. An economic application of these industrially important enzymes requires their efficient production and recovery. In this study, the effect of Sec-type signal peptides on the recombinant expression of a CGTase derived from Bacillus sp. G825-6 was investigated in Escherichia coli BL21(DE3) using a codon-adapted gene. In addition, a novel purification method for the CGTase using starch adsorption was developed. Results: Expression vectors encoding N-terminal PelB, DacD, and the native Bacillus sp. G825-6 CGTase signal peptides (SP) were constructed for the recombinant CGTase. With the DacD SP derived from E. coli, a 3.9- and 3.1-fold increase in total enzyme activity was obtained compared to using the PelB and the native CGTase SP, respectively. DacD enabled a 7.3-fold increase of activity in the extracellular fraction after induction for 24 h compared to the native CGTase SP. After induction for 48 h, 75% of the total activity was detected in the extracellular fraction. By a batch wise adsorption to starch, the extracellular produced CGTase could be purified to homogeneity with a yield of 46.5% and a specific activity of 1637 U/mg. Conclusions: The signal peptide DacD promoted the high-level heterologous extracellular expression of a recombinant CGTase from Bacillus sp. G825-6 with a pET20b(+) vector in E. coli BL21(DE3). A protocol based on starch adsorption enabled a fast and efficient purification of the recombinant enzyme.
33

Basic Residues of β-Sheet A Contribute to Heparin Binding and Activation of Vaspin (Serpin A12)

Ulbricht, David, Oertwig, Kathrin, Arnsburg, Kristin, Saalbach, Anja, Pippel, Jan, Sträter, Norbert, Heiker, John T. 06 March 2019 (has links)
Many members of the serine protease inhibitor (serpin) family are activated by glycosaminoglycans (GAGs). Visceral adipose tissue-derived serpin (vaspin), serpin A12 of the serpin family, and its target protease kallikrein 7 (KLK7) are heparin-binding proteins, and inhibition of KLK7 by vaspin is accelerated by heparin. However, the nature of GAG binding to vaspin is not known. Here, we measured vaspin binding of various glycosaminoglycans and low molecular weight heparins by microscale thermophoresis and analyzed acceleration of protease inhibition by these molecules. In addition, basic residues contributing to heparin binding and heparin activation were identified by a selective labeling approach. Together, these data show that vaspin binds heparin with high affinity (KD = 21 ± 2 nm) and that binding takes place at a basic patch on top of β-sheet A and is different from other heparin-binding serpins. Mutation of basic residues decreased heparin binding and activation of vaspin. Similarly, reactive center loop insertion into sheet A decreased heparin binding because it disturbs the basic cluster. Finally, using vaspin-overexpressing keratinocyte cells, we show that a significant part of secreted vaspin is bound in the extracellular matrix on the cell surface. Together, basic residues of central β-sheet A contribute to heparin binding and activation of vaspin. Thus, binding to GAGs in the extracellular matrix can direct and regulate vaspin interaction with target proteases or other proteins and may play an important role in the various beneficial functions of vaspin in different tissues.
34

Bedeutung nicht-kodierender RNAs im Immunsystem

Hösler, Nadine 19 June 2015 (has links)
Immer mehr Berichte deuten darauf hin, dass nicht-kodierende RNAs an der Regulation des Immunsystems beteiligt sind. In dieser Arbeit wurden nicht-kodierende RNAs identifiziert, die durch zwei unterschiedliche immunologische Prozesse in zwei verschiedenen Zelltypen reguliert wurden. Zum einen wurde das Transkriptom von Multiplen Myelom-Zellen in Abhängigkeit von der Interleukin 6-Stimulation untersucht. Dabei wurden einige sehr lange, IL 6-regulierte macroRNAs identifiziert, die STAIRs (STAT3-induced RNAs). Bei den STAIRs handelt es sich wahrscheinlich um funktionelle, kontinuierliche, nicht-kodierende macroRNAs, die im Zellkern angereichert sind. Einige STAIRs dienen eventuell zusätzlich oder ausschließlich als Primärtranskript für gespleißte, lange ncRNAs (lncRNAs), die weitere Funktionen in der Zelle ausüben können. Die STAIRs weisen eine große Bandbreite an Gewebsspezifität auf und bei den Untersuchungen in dieser Arbeit zeigten sich Hinweise, dass sie sich für verschiedene Krebserkrankungen als Biomarker eignen könnten. Die zweite Transkriptomanalyse wurde bei der Aktivierung naiver T Zellen durchgeführt. Dabei offenbarte sich, dass die Zellen bei diesem Prozess einen dramatischen Wechsel ihres Transkriptionsprogrammes vollziehen und eine Vielzahl nicht Protein-kodierender Gene reguliert werden. Es wurde die Regulation von ncRNAs, die bisher noch nicht im Zusammenhang mit T Zellen beschrieben wurden, beobachtet und erneut unbekannte, differentiell exprimierte Bereiche identifiziert. Im Anschluss wurde STAIR18, eine nicht-kodierende RNA, die durch die beiden untersuchten Signalwege reguliert wird, eingehender untersucht. Es zeigte sich, dass STAIR18 im menschlichen Genom dupliziert ist und beide Loci die gespleißte, lange ncRNA152 in diversen Varianten transkribieren. ncRNA152 ist hauptsächlich im Zytoplasma lokalisiert und befindet sich dort anscheinend in perinukleären Aggregaten. Die verschiedenen ncRNA152-Isoformen scheinen unter-schiedliche Funktionen auszuführen. Einerseits ist eine Wirkung als competing endogenous RNA wahrscheinlich. Eine weitere Aufgabe der ncRNA152 scheint darin zu bestehen, das STAT3-Primärtranskript zu stabilisieren oder dessen Prozessierung zu fördern.:1 Einleitung 1 1.1 Nicht-kodierende RNAs 1 1.1.1 Funktionen langer nicht-kodierender RNAs 2 1.1.2 Lange nicht-kodierende RNAs in Krebserkrankungen 4 1.2 Die Signaltransduktion von IL-6 und STAT3 5 1.2.1 Die IL-6/STAT3-Signalkaskade 5 1.2.2 Der Transkriptionsfaktor STAT3 7 1.2.3 Interleukin 6 und STAT3 in Krebserkrankungen 9 1.2.4 STAT3-regulierete nicht-kodierende RNAs 12 1.3 T-Zellen 13 1.3.1 T Zellaktivierung 14 1.3.2 Lange nicht-kodierende RNAs in T Zellen 16 1.4 Zielstellung 18 2 Material und Methoden 20 2.1 Bioinformatische Methoden 20 2.1.1 Evaluierung von Tiling Array-Daten 20 2.1.2 Evaluierung von Hochdurchsatz-Sequenzierungen 21 2.1.3 Auswertung von Mikroarray-Daten 21 2.1.4 DNA-Sequenzanalysen 22 2.1.5 Design von Oligonukleotiden 23 2.1.6 Statistische Auswertung 24 2.2 Molekularbiologische Methoden 25 2.2.1 Zellfraktionierung 25 2.2.2 Isolation von RNA 25 2.2.3 Reverse Transkription 26 2.2.4 Quantitative real-time PCR 27 2.2.5 Klonierung 29 2.3 Zellbiologische Methoden 36 2.3.1 Präparation primärer T-Helferzellen 36 2.3.2 Zellkultur und Stimulation eukaryontischer Zellen 38 2.3.3 Durchflusszytometrie 40 2.3.4 Transiente Transfektion eukaryontischer Zellen 42 2.3.5 Reportergenanalysen 44 2.3.6 Fluoreszenz in situ Hybridisierung 45 2.3.7 Gewebe- und Patientenproben 48 3 Ergebnisse 50 3.1 Identifizierung und Charakterisierung neuer STAT3-regulierter ncRNA-Gene 50 3.1.1 Genomweite Untersuchung STAT3-regulierter ncRNAs 50 3.1.2 Validierung der IL-6-Tiling Array-Daten 65 3.1.3 Intrazelluläre Lokalisation der STAIRs 67 3.1.4 Expressionsprofile der STAIRs 68 3.2 Identifizierung regulierter ncRNA-Gene während der T Zellaktivierung 72 3.2.1 Etablierung der in vitro Aktivierung primärer T Zellen 72 3.2.2 Genomweite Untersuchung regulierter RNAs während der T-Zellaktivierung 74 3.2.3 Validierung der T-Zellaktivierungs-Genomdaten 83 3.3 Untersuchung der nicht-kodierenden RNA STAIR18 / ncRNA152 83 3.3.1 STAIR18 ist im humanen Genom dupliziert 84 3.3.2 Von beiden STAIR18-Loci werden gespleißte Transkripte generiert 86 3.3.3 Regulation der ncRNA152 90 3.3.4 Untersuchung des STAIR18/ncRNA152-Promotors 96 3.3.5 Intrazelluläre Lokalisation von STAIR18 und ncRNA152 101 3.3.6 Überexpression der ncRNA152 in XG-1-Zellen 106 3.3.7 Knockdown der ncRNA152 in XG-1-Zellen 107 3.3.8 Identifizierung putativer Zielgene der ncRNA152 109 4 Diskussion 111 4.1 IL 6/STAT3 regulierte macroRNAs 111 4.1.1 Charakterisierung der STAIRs 114 4.1.2 STAIRS als potentielle Biomarker 121 4.2 Regulation von lncRNAs während der T Zellaktivierung 122 4.3 Untersuchung von STAIR18/ncRNA152 128 4.3.1 Regulation von STAIR18 und der ncRNA152 129 4.3.2 Lokalisation von STAIR18 und der ncRNA152 130 4.3.3 Manipulation der ncRNA152-Expression 131 4.3.4 Bedeutung der ncRNA152 133 5 Zusammenfassung 135 6 Summary 138 7 Literaturverzeichnis 141 8 Anhang 153 Danksagung 168 Publikationen 16969 Selbstständigkeitserklärung 170
35

Ligand-dependent tRNA processing by a rationally designed RNase P riboswitch

Ender, Anna, Etzel, Maja, Hammer, Stefan, Findeiß, Sven, Stadler, Peter, Mörl, Mario 16 February 2022 (has links)
We describe a synthetic riboswitch element that implements a regulatory principle which directly addresses an essential tRNA maturation step. Constructed using a rational in silico design approach, this riboswitch regulates RNase P-catalyzed tRNA 5'-processing by either sequestering or exposing the single-stranded 5'-leader region of the tRNA precursor in response to a ligand. A single base pair in the 5'-leader defines the regulatory potential of the riboswitch both in vitro and in vivo. Our data provide proof for prior postulates on the importance of the structure of the leader region for tRNA maturation. We demonstrate that computational predictions of ligand-dependent structural rearrangements can address individual maturation steps of stable non-coding RNAs, thus making them amenable as promising target for regulatory devices that can be used as functional building blocks in synthetic biology.
36

N-Terminal Ile-Orn- and Trp-Orn-Motif repeats enhance membrane interaction and increase the antimicrobial activity of Apidaecins against Pseudomonas aeruginosa

Bluhm, Martina E. C., Schneider, Viktoria A. F., Schäfer, Ingo, Piantavigna, Stefania, Goldbach, Tina, Knappe, Daniel, Seibel, Peter, Martin, Lisandra L., Veldhuizen, Edwin J. A., Hoffmann, Ralf January 2016 (has links)
The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.
37

Functional relevance of naturally occurring mutations in adhesion G protein-coupled receptor ADGRD1 (GPR133)

Fischer, Liane, Wilde, Caroline, Schöneberg, Torsten, Liebscher, Ines January 2016 (has links)
Background: A large number of human inherited and acquired diseases and phenotypes are caused by mutations in G protein-coupled receptors (GPCR). Genome-wide association studies (GWAS) have shown that variations in the ADGRD1 (GPR133) locus are linked with differences in metabolism, human height and heart frequency. ADGRD1 is a Gs protein-coupled receptor belonging to the class of adhesion GPCRs. Results: Analysis of more than 1000 sequenced human genomes revealed approximately 9000 single nucleotide polymorphisms (SNPs) in the human ADGRD1 as listed in public data bases. Approximately 2.4 % of these SNPs are located in exons resulting in 129 non-synonymous SNPs (nsSNPs) at 119 positions of ADGRD1. However, the functional relevance of those variants is unknown. In-depth characterization of these amino acid changes revealed several nsSNPs (A448D, Q600stop, C632fs [frame shift], A761E, N795K) causing full or partial loss of receptor function, while one nsSNP (F383S) significantly increased basal activity of ADGRD1. Conclusion: Our results show that a broad spectrum of functionally relevant ADGRD1 variants is present in the human population which may cause clinically relevant phenotypes, while being compatible with life when heterozygous.
38

Applicability of a computational design approach for synthetic riboswitches

Domin, Gesine, Findeiß, Sven, Wachsmuth, Manja, Will, Sebastian, Stadler, Peter F., Mörl, Mario January 2016 (has links)
Riboswitches have gained attention as tools for synthetic biology, since they enable researchers to reprogram cells to sense and respond to exogenous molecules. In vitro evolutionary approaches produced numerous RNA aptamers that bind such small ligands, but their conversion into functional riboswitches remains difficult. We previously developed a computational approach for the design of synthetic theophylline riboswitches based on secondary structure prediction. These riboswitches have been constructed to regulate ligand dependent transcription termination in Escherichia coli. Here, we test the usability of this design strategy by applying the approach to tetracycline and streptomycin aptamers. The resulting tetracycline riboswitches exhibit robust regulatory properties in vivo. Tandem fusions of these riboswitches with theophylline riboswitches represent logic gates responding to two different input signals. In contrast, the conversion of the streptomycin aptamer into functional riboswitches appears to be difficult. Investigations of the underlying aptamer secondary structure revealed differences between in silico prediction and structure probing. We conclude that only aptamers adopting the minimal free energy (MFE) structure are suitable targets for construction of synthetic riboswitches with design approaches based on equilibrium thermodynamics of RNA structures. Further improvements in the design strategy are required to implement aptamer structures not corresponding to the calculated MFE state.
39

A unique serpin P1′ glutamate and a conserved β-sheet C arginine are key residues for activity, protease recognition and stability of serpinA12 (vaspin)

Ulbricht, David, Pippel, Jan, Schultz, Stephan, Meier, René, Sträter, Norbert, Heiker, John T. 06 March 2019 (has links)
SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1′) of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1′ glutamate (Glu379) with an arginine residue (Arg302) of the β-sheet C. A P1′ glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1′ residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin–protease interaction. Arg302 is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1′ Glu379, which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu379 and Arg302 influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin.
40

Enzymatischer Abbau von Amadori-Produkten durch intestinale Disaccharidasen und intrazelluläre Ketosaminkinasen: Enzymatic degradation of Amadori products by intestinal disaccharidases and intracellular ketosamine kinases

Seidowski, Anne 06 December 2010 (has links)
Amadori-Produkte werden spontan während der ersten Phase der Maillard-Reaktion aus reduzierenden Zuckern und Aminen wie Lysin gebildet. Sie entstehen während der Erhitzung von Lebensmitteln und in vivo. Der enzymatische Abbau solcher spontan gebildeten Produkte ist Thema dieser Arbeit. Ein Teil untersuchte die Rolle von Oligosaccharid-Amadori-Produkten während der Verdauung von Kohlenhydraten im Dünndarm. Aufgrund ihrer strukturellen Ähnlichkeit mit bekannten Glycosidase-Inhibitoren wurde eine hemmende Wirkung der Amadori-Produkte auf die Kohlenhydratverdauung vermutet. Der andere Teil beschäftigte sich mit Fructosamin-3-kinase (FN3K) und dessen verwandtem Enzym Fructosamin-3-kinase-related Protein (FN3K-RP) aus humanen Erythrocyten. Diese Ketosaminkinasen werden als Proteinreparaturenzyme betrachtet, sogar als enzymatische Verteidigung gegen Glykierung in vivo diskutiert. Durch ihre Reaktion entstehen jedoch auch hoch-reaktive 1,2-Dicarbonylverbindungen, die weitere Proteinschäden bewirken können. Noch ist nicht klar, ob die Ketosaminkinasen die pathophysiologischen Folgen der Glykierung verhindern oder fördern. In dieser Arbeit wurde die Substratspezifität von Ketosaminkinasen mit einer Reihe von Amadori-Produkten untersucht. Damit könnten Inhibitoren zur weiteren Enzymcharakterisierung oder sogar für pharmazeutische Anwendungen identifiziert werden. Außerdem wurde die Variabilität der Enzymaktivitäten von Mensch zu Mensch in einer Kohorte von 100 Probanden untersucht. Als Modell für die menschliche Kohlenhydratverdauung im Dünndarm wurden Caco-2-Zellen als Monolayer etabliert. Deren Sucrase-Isomaltase kann die alpha-glycosidische Bindung in Amadori-Produkten von Maltose und Maltotriose mit Lysin und auch in Maltulose hydrolysieren. Trotz der Aminogruppe hemmen diese Amadori-Produkte die Maltosehydrolyse nur schwach als konkurrierende Substrate. Lactulosyllysin konnte nicht durch die Lactase der Caco-2-Zellen hydrolysiert werden. Tagatosyllysin und die Heyns-Produkte Glucosyllysin und Mannosyllysin hemmten die Lactosehydrolyse schwach. Alle beobachteten Hemmeffekte sind wahrscheinlich zu schwach, um während der Verdauung in vivo bedeutsam zu sein. Für FN3K konnte Desoxypiperidinofructose als kompetitiver Inhibitor identifiziert werden (Kic 0,006 mM). FN3K zeigte nur geringe Selektivität gegenüber Amadori-Produkten verschiedener Amine, ausgenommen aromatischer Amine. FN3K-RP war in Erythrocyten wesentlich aktiver als FN3K, auch wenn die Aktivität nicht selektiv inhibiert werden konnte. Beide Enzymaktivitäten unterscheiden sich unter den 100 Probanden, mit einer Spannweite von 3 bis 12 mU/g Hämoglobin für FN3K und 60 bis 135 mU/g Hb für FN3K und FN3K-RP zusammen. Es scheint eine Verbindung zwischen der Ketosaminkinase-Aktivität in Erythrocyten und Nierenerkrankungen, familiär auftretendem Diabetes mellitus, sowie familiär aufgetretenen Herzinfarkten oder Schlaganfällen zu bestehen, wie orientierende Auswertungen zeigten. Deshalb ist eine genauere Untersuchung der physiologischen Bedeutung der Ketosaminkinasen nötig. / Amadori products are formed spontaneously from reducing sugars and amines, e.g. lysine, during the first phase of the Maillard reaction. They occur in heated food and in vivo. The thesis focuses on the enzymatic degradation of such spontaneously formed compounds. One part of this work investigated the faith and impact of oligosaccharide derived Amadori products during small intestinal carbohydrate digestion. Due to their structural similarity with known glycosidase inhibitors, an inhibitory action of Amadori products towards carbohydrate digestions was assumed. The other part dealt with fructosamine-3-kinase (FN3K) and its related protein (FN3K-RP) from human erythrocytes. Such ketosamine kinases are regarded as protein repair enzymes, maybe even an enzymatic defence against glycation in vivo. While deglycating protein bound Amadori products, however, they produce highly reactive 1,2-dicarbonyl compounds, which can lead to further protein damage. It is unclear, whether the ketosamine kinase action prevents or supports the pathophysiological effects of glycation. This work studied the substrate specifity of ketosamine kinases with a variety of Amadori products, which could result in inhibitors for further enzyme characterisation or even pharmaceutical uses. Further, the variability of both enzyme activities in a cohort of 100 subjects was examined. As a model for human small intestinal carbohydrate digestion, a Caco-2 cell monolayer was employed. Their sucrase-isomaltase is able to hydrolyse the alpha-glucosidic linkage in Amadori products of maltose and maltotriose with lysine, as well as in maltulose. Despite their amino group, those amadori products inhibited maltose hydrolysis merely weakly as competing substrates. Lactulosyl lysine on the other hand could not be hydrolysed by Caco-2 lactase. Tagatosyl lysine and the Heyns products glucosyl lysine and mannosyl lysine showed weak inhibition of lactose hydrolysis. All observed inhibitory effects are probably too weak to be of importance during carbohydrate digestion in vivo. Deoxypiperidinofructose was identified as a competitive inhibitor of FN3K (Kic 0,006 mM). FN3K acted rather non-specific towards Amadori products of different amines, except aromatic amines. FN3K-RP showed much higher activity in erythrocytes than FN3K, although its activity could not be inhibited selectively. Both enzyme activities vary among 100 subjects, with a range of 3 to 12 mU/g hemoglobin for FN3K and 60 to 135 mU/g hb for FN3K and FN3K-RP together. Relations of ketosamine kinase activity in erythrocytes with renal diseases, familial diabetes mellitus and familial cardiovascular events seem to exist. Thus, investigating the physiological impact of ketosamine kinases is necessary.

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