Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody

Stöcklein, Walter; Behrsing, Olaf; Scharte, Gudrun; Micheel, Burkhard; Benkert, Alexander; Schössler, W; Warsinke, Axel; Scheller, Frieder W.

doi:10.1016/s0956-5663(00)00094-4

Cited by 11 publications

(10 citation statements)

References 10 publications

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“…The enzyme assay used in this study was modified from methods previously described (79). Purified SiaB was immobilized onto a Series S CM5 sensor chip by amine coupling.…”

Section: Methodsmentioning

confidence: 99%

Nontypeable Haemophilus influenzae Has Evolved Preferential Use of N- Acetylneuraminic Acid as a Host Adaptation

Day

Atack

et al. 2019

mBio

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Nontypeable Haemophilus inﬂuenzae (NTHi) is a Gram-negative bacterial pathogen that is adapted exclusively to human hosts. NTHi utilizes sialic acid from the host as a carbon source and as a terminal sugar on the outer membrane glycolipid lipooligosaccharide (LOS). Sialic acid expressed on LOS is critical in NTHi biofilm formation and immune evasion. There are two major forms of sialic acids in most mammals, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the latter of which is derived from Neu5Ac. Humans lack the enzyme to convert Neu5Ac to Neu5Gc and do not express Neu5Gc in normal tissues; instead, Neu5Gc is recognized as a foreign antigen. A recent study showed that dietary Neu5Gc can be acquired by NTHi colonizing humans and then presented on LOS, which acts as an antigen for the initial induction of anti-Neu5Gc antibodies. Here we examined Neu5Gc uptake and presentation on NTHi LOS. We show that, although Neu5Gc and Neu5Ac are utilized equally well as sole carbon sources, Neu5Gc is not incorporated efficiently into LOS. When equal amounts of Neu5Gc and Neu5Ac are provided in culture media, there is ∼4-fold more Neu5Ac incorporated into LOS, suggesting a bias in a step of the LOS biosynthetic pathway. CMP-Neu5Ac synthetase (SiaB) was shown to have ∼4,000-fold-higher catalytic efficiency for Neu5Ac than for Neu5Gc. These data suggest that NTHi has adapted preferential utilization of Neu5Ac, thus avoiding presentation of the nonhuman Neu5Gc in the bacterial cell surface. The selective pressure for this adaptation may represent the human antibody response to the Neu5Gc xenoantigen. IMPORTANCE Host-adapted bacterial pathogens such as NTHi cannot survive out of their host environment and have evolved host-specific mechanisms to obtain nutrients and evade the immune response. Relatively few of these host adaptations have been characterized at the molecular level. NTHi utilizes sialic acid as a nutrient and also incorporates this sugar into LOS, which is important in biofilm formation and immune evasion. In the present study, we showed that NTHi has evolved to preferentially utilize the Neu5Ac form of sialic acid. This adaptation is due to the substrate preference of the enzyme CMP-Neu5Ac synthetase, which synthesizes the activated form of Neu5Ac for macromolecule biosynthesis. This adaptation allows NTHi to evade killing by a human antibody response against the nonhuman sialic acid Neu5Gc.

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“…The enzyme assay used in this study was modified from methods previously described (79). Purified SiaB was immobilized onto a Series S CM5 sensor chip by amine coupling.…”

Section: Methodsmentioning

confidence: 99%

Nontypeable Haemophilus influenzae Has Evolved Preferential Use of N- Acetylneuraminic Acid as a Host Adaptation

Day

Atack

et al. 2019

mBio

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“…As an example, alternative methods to characterize binding constants (e.g. binding affinity assays ) as well as insights from structural biology may add to the understanding of enzymatic function. This review is by no means an exhaustive summary, focusing only on the most commonly used data types in network modeling.…”

Section: New Data New Perspectivesmentioning

confidence: 99%

New types of experimental data shape the use of enzyme kinetics for dynamic network modeling

et al. 2013

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Since the publication of Leonor Michaelis and Maude Menten's paper on the reaction kinetics of the enzyme invertase in 1913, molecular biology has evolved tremendously. New measurement techniques allow in vivo characterization of the whole genome, proteome or transcriptome of cells, whereas the classical enzyme essay only allows determination of the two Michaelis–Menten parameters V and Km. Nevertheless, Michaelis–Menten kinetics are still commonly used, not only in the in vitro context of enzyme characterization but also as a rate law for enzymatic reactions in larger biochemical reaction networks. In this review, we give an overview of the historical development of kinetic rate laws originating from Michaelis–Menten kinetics over the past 100 years. Furthermore, we briefly summarize the experimental techniques used for the characterization of enzymes, and discuss web resources that systematically store kinetic parameters and related information. Finally, describe the novel opportunities that arise from using these data in dynamic mathematical modeling. In this framework, traditional in vitro approaches may be combined with modern genome‐scale measurements to foster thorough understanding of the underlying complex mechanisms.

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“…The equilibrium dissociation constant of the antibodies was determined using a BIACORE ® 2000 (Amersham Pharmacia, Uppsala, Sweden) as described previously [11].…”

Section: Methodsmentioning

confidence: 99%

Monoclonal anti‐diuron antibodies prevent inhibition of photosynthesis by diuron

et al. 2002

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Two monoclonal anti-diuron antibodies were generated that bind to diuron with an extremely low equilibrium dissociation constant. The antibodies prevented and restored in vitro and in vivo the diuron-dependent inhibition of photosynthesis. In isolated thylakoids prepared from spinach leaves (Spinacia oleracea L.) the diuron-inhibited Hill reaction was reconstituted immediately after the addition of the monoclonal antibodies. The antibodies also restored the diuron-dependent inhibition of the photosynthetic oxygen evolution of the cell wall-de¢cient mutant cw15 of the green alga Chlamydomonas reinhardtii Dangeard. ß

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Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody

Cited by 11 publications

References 10 publications

Nontypeable Haemophilus influenzae Has Evolved Preferential Use of N- Acetylneuraminic Acid as a Host Adaptation

Nontypeable Haemophilus influenzae Has Evolved Preferential Use of N- Acetylneuraminic Acid as a Host Adaptation

New types of experimental data shape the use of enzyme kinetics for dynamic network modeling

Monoclonal anti‐diuron antibodies prevent inhibition of photosynthesis by diuron

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