Abstract
Mycobacterial genomes encode a plethora of genes that are involved in the synthesis, utilization and degradation of cAMP. The genome of M. tuberculosis H37Rv, for example, encodes 16 adenylyl cyclases and 10 genes harbouring the cyclic nucleotide-binding (CNB) domain (Shenoy and Visweswariah, 2006). Cyclic AMP is efficiently secreted by mycobacteria, and cytosolic as well as extracellular levels of cAMP can reach hundreds of micromolar. We have recently reported that an abundantly expressed universal stress protein (USP; Rv1636 in M. tuberculosis H37Rv and MSMEG_3811 in M. smegmatis, respectively) binds cAMP (Banerjee et al., 2015). Given the number of cAMP-binding proteins present in mycobacteria, it is expected that a significant fraction of intracellular cAMP may be bound to protein. The methods typically employed to measure cAMP are radioimmunoassay (RIA) and ELISA. However, these procedures include prior acidification of samples that would dissociate cAMP ‘bound’ to protein, and therefore represent the ‘total’ cAMP present in the sample. In this protocol, we describe a method to separate the fraction of cAMP ‘bound’ to protein from what is ‘free’ or not associated with protein. This is performed by subjecting the cytosolic fraction or the culture supernatant to filtration through a membrane with a 3 kDa cut-off. Only ‘free’ cAMP is able to pass through the membrane. Therefore, cAMP concentrations in the filtrate represent the ‘free’ cAMP in the sample. Cyclic AMP levels in the original cytosolic fraction or the culture supernatant represent the ‘total’ cAMP concentration. Subtracting the ‘free’ from the ‘total’ provides the amount of cAMP bound to protein.
Keywords: CAMP, CAMP-binding protein, Mycobacterium, Universal stress protein
Materials and Reagents
Equipment
Note: Biosafety level 2 (BSL 2) is required for culturing M. bovis BCG
Software
Procedure
Representative data
Figure 1. Free and bound cAMP in M. smegmatis. Free (white) and bound (gray) cAMP levels were measured from log phase cultures of M. smegmatis for both intracellular and extracellular fractions. The sum of free and bound levels represent the total cAMP. Mean and Standard Error of the Mean (SEM) were plotted for three biological replicates using GraphPad Prism 5. SEM was calculated by dividing the standard deviation (SD) by the square root of N, where N is the number of independent determinations. SD = , where is the sample mean.
Notes
Recipes
Acknowledgments
This protocol is adapted from the original method described in Banerjee et al. (2015). A.B. is supported by Senior Research Fellowship from the Council of Scientific & Industrial Research, Government of India. S.S.V. is a recipient of a J.C. Bose National Fellowship from the Department of Science & Technology, Government of India. This work was supported by the Department of Biotechnology, Government of India.
References
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