Pyridoxal-5′-phosphate-dependent catalytic antibodies

Cofactors—i.e., metal ions and coenzymes—extend the catalytic scope of enzymes and might have been among the first biological catalysts. They may be expected to efficiently extend the catalytic potential of antibodies. Monoclonal antibodies (MAbs) against Nα-phosphopyridoxyl-l-lysine were screened for 1) binding of 5′-phosphopyridoxyl amino acids, 2) binding of the planar Schiff base of pyridoxal-5′-phosphate (PLP) and amino acids, the first intermediate of all PLP-dependent reactions, and 3), catalysis of the PLP-dependent α, β-elimination reaction with β-chloro-D/L-alanine. Antibody 15A9 fulfilled all criteria and was also found to catalyze the cofactor-dependent transamination reaction of hydrophobic D-amino acids and oxo acids (k′ cat=0.42 min−1 with D-alanine at 25°C). No other reactions with either D- or L-amino acids were detected. PLP markedly contributes to catalytic effecacy—it is a 104 times more efficient acceptor of the amino group than pyruvate. The antibody ensures reaction specificity, stereospecificity, and substrate specificity, and further accelerates the transamination reaction (k′ cat(Ab)/k′ cat(PLP)=5×103). The successive screening steps simulate the selection criteria that might have been operative in the evolution of protein-assisted psyridoxal catalysi

The influence of soil gravel content on compaction behaviour and pre-compression stress

AbstractMany arable soils have significant horizon-specific gravel content levels. Just how these influence compaction behaviour, and in particular precompression stress as an important criterion of a soil's susceptibility to compaction, has yet to be sufficiently clarified. This article is intended to contribute towards answering this question.Firstly, three different fine earths, from the “Clay”, “Silt Loam” and “Sandy Loam” soil texture classes were mixed with staggered proportions (0, 10, 20, 30, 40% by volume) of a quartz gravel (the shape of which was subrounded to rounded, average weighted diameter 6mm). Soil core samplers were filled with the mixtures at a typical density for a natural site. In the case of the 30% by volume variant only, in addition to the quartz gravel an angular to subangular limestone gravel with the same size graduation was also used. The tests were supplemented by 20 samples from a natural site; the gravel content of these varied between 0.1 and 23.5% by volume. All of the disturbed and natural samples were adjusted to a water content at a matric potential of −6kPa. Subsequently, an oedometer test was used to apply loads to them in stages (5–550kPa). Precompression stress was calculated using the resulting stress–bulk density functions.While fine earth bulk density remained constant, the staggered addition of quartz gravel led to an increase in the whole soil density after packing, and thus also to a vertical shift in overall stress–bulk density functions. However, the stress–density functions of the fine earth do show that the overall compaction of fine earth decreased as gravel content increased. In the case of low gravel content levels of no more than 10% by volume, the increase in precompression stress (log) in the disturbed samples was, on the whole, very low. In the disturbed samples, however, as gravel content increased precompression stress (log) increased exponentially. Contrary to this, a continuous linear increase in precompression stress (log) could be observed with increasing gravel content in the natural samples. The angular to subangular shape of the gravel only resulted in greater precompression stress (log) in the “Silt Loam”.At gravel-rich sites, gravel content influences soil compaction behaviour and precompression stress very strongly. For this reason, it is essential that it be considered when assessing such sites' risk of compaction damage

Statistical Methods for Identifying Small Differences in the Thermal Interruption Performance of SF6 Alternatives

This contribution will present thermal current interruption measurements performed in pure CO2 with a puffer circuit-breaker test device and establish a statistical method to assess the reignition probability as a function of the prospective current slope. Its efficacy will be demonstrated with measurements of the interruption limit scaling with respect to the pressure buildup inside the test device. A separate contribution will apply these methods to evaluate the influence of fluorinated additives on the switching performance

Bodenstrukturzustand der unteren Ackerkrume auf langjährig konservierend und tiefenreduziert bewirtschafteten Ackerflächen in Mitteldeutschland

Bisher liegen nur wenige Untersuchungen vor, die den Einfluss langjähriger konservierender und tiefenreduzierter Bodenbearbeitung auf den Bodenstrukturzustand der unteren, nicht mehr bearbeiteten, Ackerkrume unter Praxisbedingungen erfasst haben. Daher wurden im Frühjahr 2011 auf 24 langjährig pfluglos bewirtschafteten Flächen in Mitteldeutschland Bodenproben entnommen, um daran den Bodenstrukturzustand (Parameter: Trockenrohdichte, effektive Lagerungsdichte, Spatendiagnose, gesättigte Wasserleitfähigkeit, Luftkapazität) im Bereich unterhalb des Bearbeitungshorizontes zu bestimmen. Es sollte überprüft werden, ob sich in der unteren Ackerkrume Bodenverdichtungen einstellen, welche die Funktionalität der Bodenstruktur einschränken. Die Ergebnisse zeigen, dass die in der Literatur getroffenen Aussagen zur besseren Tragfähigkeit und geringeren Verdichtungsgefährdung konservierend bearbeiteter Flächen auf die realen Bedingungen in der Praxis nur bedingt übertragbar sind. Vielmehr werden die funktionellen Anforderungen an den Bodenstrukturzustand in der beprobten Schicht auf einem Großteil der untersuchten Flächen nicht vollständig erfüllt

Einfluss des Grobbodengehaltes in Böden auf das Verdichtungsverhalten und die mechanische Vorbelastung

Vorliegende Arbeit soll einen Beitrag zu der Frage leisten, inwieweit zunehmende Grobbodengehalte das Verdichtungsver-halten und die mechanische Vorbelastung beeinflussen. Dazu wurden drei unterschiedliche Feinböden (Tu2, Ut3, Sl3) mit gestaffelten Anteilen (0, 10, 20, 30, 40 Vol.-%) eines Quarzkieses versetzt und mit standortüblicher Dichte des Feinbodens in Stechzylinder gefüllt. Ergänzt wurden die Versuche durch Proben eines natürlichen Standortes Grobbodengehalte 0,1-23,5 Vol.-%). Alle Proben wurden auf einen Wassergehalt bei einer Wasserspannung von 6 kPa eingestellt und im Ödometer stufenweise belastet (5-550 kPa). An den resultierenden Druck-Dichtefunktionen wurde die Vorbelastung bestimmt. Die gestaffelten Grobbodenanteile führten sowohl bei den künstlichen wie bei den natürlichen Proben bei konstanter Feinbodendichte zur Zunahme der Trockenrohdichte des Gesamtbodens und damit zur Verschiebung der gesamten Druck-Dichtefunktionen. Die Druck-Dichtefunktionen des Feinbodens zeigen bei den künstlichen Proben jedoch eine mit zunehmendem Grobbodenanteil ab-nehmende Gesamtverdichtung des Feinbodens. Bei geringen Grobbodenanteilen von maximal 10-20 Vol.-% ist die Zunahme der Vorbelastung (log) bei den gestörten Proben insgesamt sehr gering. Mit steigendem Grobbodenanteil nimmt die Vorbelastung (log) bei diesen Proben jedoch exponentiell zu. Abweichend davon ist bei den natürlichen Proben eine kontinuierlich lineare Zunahme der Vorbelastung (log) zu verzeichnen

High affinity binding of hydrophobic and autoantigenic regions of proinsulin to the 70 kDa chaperone DnaK

BACKGROUND: Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. Complexes of peptides with chaperones induce peptide-directed immunity. Here we analyzed the interaction of (pre)proinsulin with the best characterized chaperone of the hsp70 family, bacterial DnaK. RESULTS: Of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to DnaK was found for the signal peptide and one further region in each proinsulin domain (A- and B-chain, C-peptide). Among the latter, peptides covering most of the B-chain region B11-23 exhibited strongest binding, which was in the range of known high-affinity DnaK ligands, dissociation equilibrium constant (K'd) of 2.2 ± 0.4 μM. The B-chain region B11-23 is located at the interface between two insulin molecules and not accessible in insulin oligomers. Indeed, native insulin oligomers showed very low DnaK affinity (K'd 67.8 ± 20.8 μM) whereas a proinsulin molecule modified to prevent oligomerization showed good binding affinity (K'd 11.3 ± 7.8 μM). CONCLUSIONS: Intact insulin only weakly interacts with the hsp70 chaperone DnaK whereas monomeric proinsulin and peptides from 3 distinct proinsulin regions show substantial chaperone binding. Strongest binding was seen for the B-chain peptide B 11-23. Interestingly, peptide B11-23 represents a dominant autoantigen in type 1 diabetes

SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease

Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-beta (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD(+))-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications. The pathomechanism of succinyl-CoA ligase (SCL) deficiency, a hereditary mitochondrial disease, is not fully understood. Here, the authors show that increased succinyl-CoA levels contribute to SCL pathology by causing global protein hyper-succinylation.Peer reviewe