Quantification of 3-ketodihydrosphingosine using HPLC-ESI-MS/MS to study SPT activity in yeast Saccharomyces cerevisiae.

Abstract:

:Serine palmitoyltransferase (SPT) catalyzes the rate-limiting step of condensation of L-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (3KDS). Here, we report a HPLC-ESI-MS/MS method to directly quantify 3KDS generated by SPT. With this technique, we were able to detect 3KDS at a level comparable to that of dihydrosphingosine in yeast Saccharomyces cerevisiae An in vitro SPT assay measuring the incorporation of deuterated serine into deuterated 3KDS was developed. The results show that SPT kinetics in response to palmitoyl-CoA fit into an allosteric sigmoidal model, suggesting the existence of more than one palmitoyl-CoA binding site on yeast SPT and positive cooperativity between them. Myriocin inhibition of yeast SPT activity was also investigated and we report here, for the first time, an estimated myriocin Ki for yeast SPT of approximately 10 nM. Lastly, we investigated the fate of serine α-proton during SPT reaction. We provide additional evidence to support the proposed mechanism of SPT catalytic activity in regard to proton exchange between the intermediate NH3+ base formed on the active Lys residue with surrounding water. These findings establish the current method as a powerful tool with significant resolution and quantitative power to study SPT activity.

journal_name

J Lipid Res

authors

Ren J,Snider J,Airola MV,Zhong A,Rana NA,Obeid LM,Hannun YA

doi

10.1194/jlr.D078535

subject

Has Abstract

pub_date

2018-01-01 00:00:00

pages

162-170

issue

1

eissn

0022-2275

issn

1539-7262

pii

jlr.D078535

journal_volume

59

pub_type

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