Аңдатпа
Molybdenum enzymes (Mo-enzymes) contain a molybdenum cofactor (MoCo) in the active site. These enzymes are potentially interesting for studying the survival mechanism of fish under hypoxic water conditions. This is because Mo-enzymes can synthesize nitric oxide from nitrates and nitrites, which are present in high concentrations under hypoxic water conditions. However, there is currently no method for assessing the Mo-enzymes content in the fish internal organs. Methods capable of determining Mo-enzymes content in the fish are of major importance. For this purpose, a method for quantitative determination of MoCo from plant tissues was modified. We demonstrated the Mo-enzyme content assessment by isolated MoCo from the fish's internal organs and the Neurospora crassa nit-1 extract containing inactive NADPH nitrate reductase. The Mo enzyme content was calculated using a calibration curve in nM of nitrites as a product of restored NADPH reductase activity after complementation with MoCo. Here we present a robust laboratory method which can be used to assess the content of Mo-enzymes in the internal organs of fish. Mo-enzymes play a crucial role in detoxifying toxic compounds. Therefore, it is important to develop a method to accurately determine the amount of Mo-enzymes present. Notably, the method demonstrated the efficiency and accuracy as detected high content of Mo-enzymes in the liver and intestines (P <0.0001). The obtained data on the distribution of Mo-enzymes in the internal organs of this species correspond to that of other vertebrates. Here, we present a rapid, sensitive, accurate and accessible method. The developed method is simple and easy to use. Importantly, the protocol does not require complex manipulations, and the equipment used is available in most laboratories. The article provides step-by-step instructions for reproducing the method.