Our conclusion was confirmed by two experiments The addition

Our conclusion was confirmed by two experiments. The addition of aminopeptidase-specific inhibitors at high concentrations allowed us to exclude the possibility of substrate hydrolysis by other proteases present in the samples, for example, endoproteases. After the application of inhibitors, we were not able to observe any substrate hydrolysis. Moreover, we determined the Km values in the lysate for two substrates: natural (Ala) and unnatural (the well-recognized hPhe). The obtained values were the same as the Km constants measured for isolated APN. Our results indicate that in the cell membranes of kidneys from rat, pig and human, APN has the predominant aminopeptidase activity. Previous studies also showed the activity of other enzymes [8], [13], [14], [15]. However, those studies did not indicate the precise fraction where the enzyme activity occurred.
Author contributions
Acknowledgments This research was supported by the National Science Centre and the State for Scientific Research in Poland (grants N N401 042838 and 2013/09/N/NZ/01859) and the Foundation for Polish Science (grant TEAM/2011-7/5). Project supported by Wroclaw Centre of Biotechnology, programme. The Leading National Research Centre (KNOW) for years 2014–2018.
Introduction Identifying the function of potential new enzymes that global genome sequencing efforts have revealed is one of the great challenges nowadays. Adipocytes are α-Naphthoflavone that depend on multiple enzymatic pathways to carry out their functions. The lack of a complete assembly of the genomic sequence and the presence of many predicted enzymes with unknown or unsure catalytic activities have hampered the complete view of the adipocyte's metabolic pathways (Rutkowski et al., 2015). Cys/Leu aminopeptidase (IRAP, insulin-regulated aminopeptidase, EC 3.4.11.3) in adipocytes is known to traffic between high and low density microsomal fractions toward the plasma membrane under stimulation by insulin (Keller et al., 1995, Keller et al., 2002 and Keller, 2004). The roles of IRAP in targeting AS160 protein to specialized vesicles containing GLUT4 and in regulating the retention of GLUT4 within the adipocytes were proposed based on data showing that the AS160 protein controls the amount of GLUT4 in plasma membrane and interacts with cytoplasmic domain of IRAP (Jordens et al., 2010). IRAP deficiency leads to suppression of plasminogen activator inhibitor type 1 expression in adipocytes and upregulation of uncoupling protein-1-mediated thermogenesis in brown adipose tissue and increased energy expenditure to prevent the development of obesity, and these facts suggest a therapeutic potential of IRAP/angiotensin (ANG) IV receptor blockade in diet-induced obesity (Niwa et al., 2015). IRAP has a preference for substrates containing the N-terminal Cys (Alponti et al., 2015), such as oxytocin. Indeed the obesity has been associated with reduced plasma oxytocin due to increased peptide degradation by liver and adipose tissue rather than changes in hormone synthesis (Gajdosechova et al., 2014). Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) was recently identified in adipocytes from visceral and subcutaneous fat pads, being considered a new adipokine with local and systemic effects respectively in adipose tissue and bloodstream (Sell et al., 2013). Circulating levels of this aminopeptidase in obese patients are higher in insulin-resistant subjects than in insulin-sensitive ones, suggesting that DPPIV is like to be a marker of insulin resistance, independently of obesity (Sell et al., 2013). However, the functional role of DPPIV in adipose tissue remains to be elucidated. Besides these two aminopeptidase (AP) enzymes, there are no reports about other AP enzymes in adipocytes. The relationship of AP enzymes with peptides involved in sensing the nutritional status and energy control balance is still poorly explored. Recently, the perspectives in this field were expanded by the demonstration that plasma activity levels of puromycin-insensitive neutral AlaAP (APM, EC 3.4.112) are correlated with body mass, Lee index and retroperitoneal fat pad mass in food deprived rats (Alponti and Silveira, 2010). On the other hand, activity levels of diprotin A-insensitive DPPIV are correlated with body mass, Lee index and periepididymal adipose tissue mass and decreased 33% in monosodium glutamate (MSG) obese rats that are also food deprived (Alponti and Silveira, 2010). APM mapping in the hypothalamus and hippocampus (Alponti et al., 2011b), as well as of DPPIV/CD26 in the hypothalamus (Alponti et al., 2011c) showed the regulation of these two proteins in obese and food deprived rats. Many peptides involved in the intracellular machinery and/or in the regulation of energy homeostasis are substrates of AP enzymes such as bradykinin, polyglutamine (polyQ) sequences and ANG I, II and III (Martínez-Martos et al., 2011, Menzies et al., 2010, Prieto et al., 2003). The inhibitors of certain AP enzymes seem to be promising drug candidates to treat and prevent obesity-related diseases. Inhibition of DPPIV and APM and/or puromycin-sensitive neutral AlaAP (PSA, EC 3.4.11.14) has been preconized to suppress inflammatory immune responses (Reinhold et al., 2007). Gliptin class DPPIV inhibitors are effective to treat diabetes mellitus. Glucagon-like peptide-1 receptor agonists resistant to DPPIV hydrolysis are also effective to treat diabetes mellitus and have been associated with weight loss and reductions in systolic blood pressure (Russell, 2013). Basic AP (ArgAP, EC 3.4.11.6)/leukotriene A4 hydrolase inhibitor holds promise for improved anti-inflammatory properties (Stsiapanava et al., 2014). Acid AP (AspAP, EC 3.4.11.7) inhibitors that cross the blood–brain barrier have been believed to lead the way of a new class of antihypertensive agents (Marc et al., 2012). Furthermore, it is already known that fumagillin, a MetAP (EC 3.4.11.18) inhibitor, reduces the formation of adipose tissue in mice with diet-induced obesity (Lijnen et al., 2010). Beloranib, another MetAP inhibitor, is currently under phase 2a of clinical evaluation for the treatment of obesity (Joharapurkar et al., 2014).