की गतिविधि को मापने के लिए एक परख Escherichia कोलाई Lysine Decarboxyase inducible

Summary

inducible lysine decarboxylase की गतिविधि सब्सट्रेट एल lysine और उत्पाद cadaverine 2,4,6-trinitrobenzensulfonic एसिड के साथ प्रतिक्रिया adducts कि टोल्यूनि में अंतर विलेयता फार्म द्वारा नजर रखी है.

Abstract

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 – 9)¹ and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 – 2². To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB³, the arginine decarboxylase AdiA⁴, the lysine decarboxylase LdcI^{5, 6} and the ornithine decarboxylase SpeF⁷. All of these enzymes utilize pyridoxal-5′-phospate as a co-factor⁸ and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate². In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 Å, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5′-diphosphate,3′-diphosphate (ppGpp) ¹⁴. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses⁹. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth¹⁰. We have demonstrated that ppGpp is a specific inhibitor of LdcI ¹⁴. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.¹¹, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the α-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)⁵. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N’-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N′-bistrinitrophenyllysine (TNP-lysine), respectively¹¹. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

Protocol

1) अभिकर्मकों और उपकरण सबसे पहले, निम्नलिखित तीन समाधान तैयार: समाधान के 1 एमएल जो 8 मिमी एल lysine, 100 मिमी 2 सोडियम से बना है – (एन morpholino) ethanesulphonic एसिड (एमईएस) 6.5 पीएच, 0.2 nucleotide मिमी, जहां nucleotide है या तो: ग्वानोसिन diphosphate (जीड?…

Discussion

Lysine decarboxylase परख में, TNBS एल lysine और cadaverine के प्राथमिक amines के साथ प्रतिक्रिया व्यक्त की है TNP-lysine और TNP-cadaverine adducts (चित्रा 1) के रूप में. TNP-lysine पर carboxylic एसिड समूह की उपस्थिति के कारण, इस adduct पानी में घुलनशील रहता है, जबकि TNP-cadaverine, carboxylic ए…

Materials

Material Name	Type	Company	Catalogue Number	Comment
2,4,6-trinitrobenzensulfonic acid		Sigma Aldrich	P2297
0.1 mM pyridoxal 5′-phosphate (PLP)		Sigma Aldrich	P9255
Cadaverine		Sigma Aldrich	D22606
96 well polystyrene plates		Sarstedt
96-well quartz plate		Hellma
VWR Digital Heatblock		VWR
ThermoStat Plus		Eppendorf
2.0 mL 96-well polypropylene plates		Axygen	P-DW-20-C
Handy Step Repeat Pipettor		Brand
12.5 mL Repeat Pipettor Tips		Brand (Plastibrand)	702378
SpectraMax 340PC Plate Reader		SpectraMax