real-time-metabolic-detection-living-cells-using-hyperpolarized-13c

Real-Time Metabolic Detection in Living Cells Using Hyperpolarized <sup>13</sup>C NMR

The protocol presents a method to integrate an in-house-built bioreactor system with hyperpolarized 13C nuclear magnetic resonance (NMR) spectroscopy to ...

National Institutes for Quantum Science and Technology

university of tsukuba

Biological properties of protein molecules depend on their interaction with other molecules, and enzymes are no exception. Enzyme activities are controlled by their interaction with other molecules in living cells. Enzyme activation and their catalytic properties in the presence of different types of polymers have been studied in vitro, although these studies are restricted to only a few enzymes. In this study, we show that addition of poly-l-lysine (PLL) can increase the enzymatic activity of multiple oxidoreductases through formation of enzyme assemblies. Oxidoreductases with an overall negative charge, such as l-lactate oxidase, d-lactate dehydrogenase, pyruvate oxidase, and acetaldehyde dehydrogenase, each formed assemblies with the positively charged PLL via electrostatic interactions. The enzyme activities of these oxidoreductases in the enzyme assemblies were several-folds higher than those of the enzyme in their natural dispersed state. In the presence of PLL, the turnover number (k) improved for all enzymes, whereas the decrease in Michaelis constant (K) was enzyme dependent. This type of enzyme function regulation through the formation of assemblies via simple addition of polymers has potential for diverse applications, including various industrial and research purposes.

Activation of oxidoreductases by the formation of enzyme assembly.

We have studied binding properties of three detergents, i.e., sodium dodecyl sulfate (SDS), Sarkosyl and sodium lauroyl glutamate (SLG), to model proteins based on their effects on electrophoretic mobilities of the proteins using agarose native gel electrophoresis and circular dichroism (CD). This simple technology can evaluate the dissociative properties of bound detergents from the proteins and their effects on protein structure. SDS influenced the electrophoretic mobilities of all model proteins more strongly than the other two detergents, implying a stronger inclination for protein binding and subsequent alterations in protein structure or reductions in activity, which are supported by CD analysis. On the contrary, Sarkosyl and SLG showed weaker binding and interfered less with the structure and biological activities, indicating that these detergents may be useful for protein purification and analysis. It appeared that SLG was weaker in protein binding than Sarkosyl, although the effects of these two detergents appeared to depend on the proteins.

Effects of sodium dodecyl sulfate, Sarkosyl and sodium lauroyl glutamate on the structure of proteins monitored by agarose native gel electrophoresis and circular dichroism.

The Ferguson plot is a simple method for determining the molecular weight of native proteins and their complexes. In this study, we tested the validity of the Ferguson plot based on agarose native gel electrophoresis using multimeric chaperone protein, ClpB, derived from a moderate halophile that forms a native hexamer. The Ferguson plot showed a single band with a molecular weight of 1,500 kDa, approximately twice the size of the native hexamer. This result is consistent with the structure of other chaperons that form a double ring assembly comprising two hexameric units, i.e., a dodecamer. Supporting this, dynamic light scattering experiment showed two peaks, which likely correspond to the hexamer and dodecamer structures.

Article	Year
Activation of oxidoreductases by the formation of enzyme assembly. Scientific reports\| PubMed ID: 37658129	2023
Effects of sodium dodecyl sulfate, Sarkosyl and sodium lauroyl glutamate on the structure of proteins monitored by agarose native gel electrophoresis and circular dichroism. Biophysical chemistry\| PubMed ID: 39168056	2024
Ferguson Plot Analysis of Chaperone ClpB from Moderate Halophile. The protein journal\| PubMed ID: 39755991	2025

Tomoto Ura

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