02457nas a2200289   4500008004100000022001400041245008200055210006900137260000800206490000900214520156800223653001301791653001601804653001401820653001301834653001001847653000701857653003901864653001601903100002701919700001901946700001501965700002401980700002702004700002502031856011102056       2023                        eng d  a0304-416500aMechanistic insights on anserine hydrolyzing activities of human carnosinases0 aMechanistic insights on anserine hydrolyzing activities of human  cMAR0 v18673 a<p>
	Anserine and carnosine represent histidine-containing dipeptides that exert a pluripotent protective effect on human physiology. Anserine is known to protect against oxidative stress in diabetes and cardiovascular diseases. Human carnosinases (CN1 and CN2) are dipeptidases involved in the homeostasis of carnosine. In poikilothermic vertebrates, the anserinase enzyme is responsible for hydrolyzing anserine. However, there is no specific anserine hydrolyzing enzyme present in humans. In this study, we have systematically investigated the anserine hydrolyzing activity of human CN1 and CN2. A targeted multiple reaction monitoring (MRM) based approach was employed for studying the enzyme kinetics of CN1 and CN2 using carnosine and anserine as substrates. Surprisingly, both CN1 and CN2 can hydrolyze anserine effectively. The observed catalytic turnover rate (Vmax/[E]t) was 21.6 s-1 and 2.8 s-1 for CN1 and CN2, respectively. CN1 is almost eight-fold more efficient in hydrolyzing anserine compared to CN2, which is comparable to the efficiency of the carnosine hydrolyzing activity of CN2. The Michaelis constant (Km) value for CN1 (1.96 mM) is almost three-fold lower compared to CN2 (6.33 mM), representing higher substrate affinity for anserine-CN1 interactions. Molecular docking studies showed that anserine binds at the catalytic site of the carnosinases with an affinity similar to carnosine. Overall, the present study elucidated the inherent promiscuity of human carnosinases in hydrolyzing anserine using a sensitive LCMS/MS approach.</p>
10aAnserine10aCarnosinase10aCarnosine10aDiabetes10aLC-MS10aMS10aMultiple reaction monitoring (MRM)10aPromiscuity1 aPandya, Vaibhav, Kumar1 aShankar, Shiva1 aRajesh, S.1 aRathore, Rajeshwari1 aKumaran, Sangaralingam1 aKulkarni, Mahesh, J.  uhttps://library.ncl.res.in/content/mechanistic-insights-anserine-hydrolyzing-activities-human-carnosinases02515nas a2200277   4500008004100000022001400041245011700055210006900172260000800241300001100249490000800260520162800268653001301896653002201909653001201931653001201943653002201955100001501977700001901992700002002011700001902031700002002050700001902070700002502089856012302114       2025                        eng d  a1046-592800aSimple freeze-thaw based method for efficient purification of recombinant human proinsulin from inclusion bodies0 aSimple freezethaw based method for efficient purification of rec  cMAR  a1066450 v2273 a<p>
	Insulin is a pivotal peptide hormone essential for regulating glucose homeostasis. It has been known for over 100 years, but its production and purification methods are still under improvement. Escherichia coli based bacterial expression system is primarily used for insulin production. The human insulin protein expressed in bacteria usually forms inclusion bodies, complicating the purification process. Traditionally, insulin purification is a timeconsuming process involving urea-based denaturation methods, and various refolding techniques, followed by extensive chromatographic methods. Here, we report an easy and efficient purification of human proinsulin involving freeze-thaw based solubilization method. The extracted proinsulin inclusion bodies are treated with different concentrations of urea, followed by a freeze-thaw based solubilization. The freezing was carried out at various temperatures, mainly -80 degrees C, -20 degrees C, and -196 degrees C to determine the optimum condition for solubilization. Highest solubilization of proinsulin from the inclusion body was achieved with 0.5M urea and -20 degrees C. Further Nickel NTA-based purification was performed, and the purified protein was characterized for disulfide mapping by high-resolution mass spectrometer (HRMS). We also performed glucose uptake assays to validate the functional properties of purified proinsulin. This freeze-thaw based mild solubilization approach is a fast and effective method for getting bioactive proinsulin, which will help further design better purification and processing strategies for insulin production.</p>
10aDiabetes10aDisulfide mapping10aglucose10ainsulin10aMass spectrometry1 aRajesh, S.1 aJathar, Swaraj1 aBanarjee, Reema1 aSharma, Monika1 aPalkar, Shivani1 aShankar, Shiva1 aKulkarni, Mahesh, J.  uhttps://library.ncl.res.in/content/simple-freeze-thaw-based-method-efficient-purification-recombinant-human-proinsulin