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    • br Material and methods br Results Table shows that

    2024-03-20


    Material and methods
    Results Table 1 shows that in addition to Leu/CysAP and DPPIV, genes for AspAP, ArgAP, MetAP, APM and PSA are expressed in adipocytes. The kinetic parameters of these AP are also shown in Table 1, revealing that APM has the highest Vmax and catalytic efficiency (Kcat/Km), whereas AspAP has the highest affinity (bromocriptine mesylate among animal groups are shown in Fig. 1. The noteworthy changes are that AspAP is absent in high and low density microsomal and plasma membrane fractions from obese and control under food deprivation (Fig. 1-II), MetAP in plasma membrane is higher in obese than in food deprived obese (Fig. 1-VII), and PSA in low density microsomal fraction is higher in obese than in control, as well as in obese and control under food deprivation, while it is negligible in plasma membrane from all groups (Fig. 1-VIII).
    Discussion Gene expressions of ArgAP, AspAP, MetAP, APM and PSA and corresponding catalytic activities in different subcellular compartments of adipocytes isolated from a depot of visceral adipose tissue were demonstrated for the first time. The choice of visceral adipose tissue depot was based on data showing that the excessive of visceral fat is the main factor related to obesity and metabolic syndrome (Després and Lemieux, 2006). Additionally, the choice of retroperitoneal depot of visceral adipose tissue was based on data suggesting that the increase of the absolute mass and energy's demand from retroperitoneal (not periepididymal) fat pad should contribute to the later development of type 2 diabetes mellitus in MSG obesity (Alponti and Silveira, 2010, Alponti et al., 2008). The kinetic analysis provided semi-quantitative values of parameters needed to establish the optimal conditions for the assay of these new enzymes in adipocytes. It is well known that each enzyme has it own Vmax, Km and Kcat that reflect the cellular environment, the concentration of substrate typically found in vivo by the enzyme and the chemistry of reaction. However, in a mixture of proteins only a prospective analysis is possible, since the accuracy of values of kinetic parameters is directly related to the enzyme purity (specific activity) (Alponti et al., 2011a, Marschner and Klein, 2015). Vmax is the maximum velocity that the enzyme reaches at very high concentrations of substrate. Vmax value is critical to perform the enzyme assay with saturate concentration of substrate. Km (Michaelis–Menten constant) describes the amount of substrate needed for the enzyme to obtain half of its Vmax. Km is important to determine the rate of the enzymatic catalysis reaction with specific substrate (binding affinity). The first step of catalysis is the binding between substrate and enzyme. Thus, low Km indicates a large binding affinity, as the reaction will approach Vmax more rapidly, such is the case for AspAP, DPPIV and PSA. On the other hand, high Km indicates that the enzyme does not bind as efficiently with the substrate, and Vmax will only be reached if the substrate concentration is high enough to saturate the enzyme, such is the case for ArgAP, CysAP, APM, LeuAP and MetAP. The second step of catalysis is the forming of product. The second step is more precisely the number of times that each enzyme converts substrate to product per unit of time and is indicated by the turnover number or Kcat. Kcat/Km is the specificity constant that show us the catalytic efficiency, which means how fast the enzyme reacts with the substrate once both collide. This measure of efficiency is helpful in determining whether the rate is limited by the formation of product or the amount of substrate in the environment. If the rate of efficiency is high, Kcat is much larger than Km and the enzyme complex converts a greater proportion of the substrate it binds into product, as occurs for APM and PSA. On the other hand, when Kcat is much smaller than Km, the enzyme complex converts a lesser proportion of the substrate it binds into product, as occurs for CysAP, ArgAP and DPPIV.