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  • br Materials and methods br Results and discussion br Conclu

    2019-10-09


    Materials and methods
    Results and discussion
    Conclusions Codon-optimized MGSK-Lys-C was expressed at a high level in an optimized expression system as inclusion bodies. The Lys-C activity reached its maximum with about 9340 nkat/Lculture under standard (chemical) refolding conditions. The refolding of MGSK-Lys-C could be increased remarkably using native pre-N-pro peptide of L. enzymogenes. The concentration of the pre-N-pro peptide as well as the refolding time had a strong influence on the Lys-C activity achieved and could be increased 58-fold under optimal conditions. Our study showed that the choice of the expression system as well as the refolding conditions had a strong influence on the expression rate and enzyme activity achieved. With the results presented, the interesting, but expensive lysyl endopeptidase can be produced in high activity and high amounts. Now, the potential of Lys-C for tailor-made protein hydrolysates with bioactive and/or techno functional properties can be investigated in future time studies.
    Acknowledgements This research project was supported by the Federal Ministry for Economic Affairs and Energy (via AiF) and the FEI (Forschungskreis für Ernährungsindustrie e.V., Bonn): Project AiF 16541 N.
    Introduction
    Material and Methods
    Results
    Discussion While numerous studies have investigated the signaling mechanisms and key Cathepsin S inhibitor involved in the control of the male penile erectile tissue, the pathways mediating the function of vaginal vascular and nonvascular smooth muscle are still poorly understood. In accordance with the situation in the human penile erectile tissue (corpus cavernosum), it has been assumed that an imbalance in the production and degradation of vasoconstrictor and vasodilator peptides may contribute to the impairment of female sexual function, including the arousal response. There is growing evidence that the function of genital vascular and nonvascular smooth muscle is associated with the subjective arousal sensation. For example, reduced responsiveness of genital smooth muscle might be an underlying cause in the pathophysiology of female sexual arousal disorder (FSAD), leading to a reduction in genital blood flow and, subsequently, a lack in vaginal lubrication and genital sensation [1]. Among the endogenous peptides assumed to be involved in the peripheral control of female genital organs, ET‐1 has been shown to contract human vaginal smooth muscle much more effectively than does oxytocin, serotonin, or the alpha‐adrenergic agonist norepinephrine [14]. ET‐1 is formed by proteolysis of preproET‐1 through the intermediate product, Big ET‐1 [16]. Big ET‐1 also produces vasoconstrictor activity; however, the effect is weaker than that brought about by ET‐1 17, 18. The conversion of Big ET‐1 into ET‐1 is catalyzed by the ECE, a membrane‐bound metalloendopeptidase, structurally and functionally related to NEP [16]. Recently, endopeptidase enzymes have attracted growing attention of researchers and clinicians. In our study, Big ET‐1 induced dose‐dependent contractile responses of human vaginal wall smooth musculature. This observation is in agreement with the results from tissue bath experiments using isolated rabbit aorta: Big ET‐1 mediated a contraction response of 61% (of calibrated scale) and 74%, respectively, in the presence or in the absence of the endothelial layer [19]. The contraction response elicited by Big ET‐1 is in support of a role of the endothelin system in the control of human vaginal wall smooth muscle. Preexposure of the vaginal tissue to the endopeptidase inhibitor KC 12615 significantly reduced the contraction induced by Big ET‐1. Similar responses were observed in a study using isolated rat basilar arteries: phosphoramidon, an endopeptidase inhibitor, attenuated the contractions induced by Big ET‐1 of the vascular smooth muscle [20]. In anesthetized rats, the compound attenuated the hypertensive effect produced by the administration of Big ET‐1 [21]. Since ET‐1 has been proposed to contribute to the maintenance of tension of the vaginal wall, the inhibition of the endopeptidase ECE can suppress the transformation of Big ET‐1 to ET‐1, thereby counteracting the contraction brought about by Big ET‐1/ET‐1. This might facilitate the relaxation of vaginal nonvascular (and vascular) smooth muscle and, finally, the arousal response.