Electrospray Mass Spectrometry 10:0 Acyl Coenzyme A


Reverse Phase HPLC 10:0 Acyl Coenzyme A

References:

• Hanada, K. (2003)
  Serine Palmitoyltransferase, A Key Enzyme of Sphingolipid Metabolism
  The first step in the biosynthesis of sphingolipids is the condensation of serine and palmitoyl CoA, a reaction catalyzed by serine palmitoyltransferase (SPT) to produce 3-ketodihydrosphingosine (KDS). This review focuses on recent advances in the biochemistry and molecular biology of SPT. SPT belongs to a family of pyridoxal 5'-phosphate (PLP)-dependent alpha-oxoamine synthases (POAS). Mammalian SPT is a heterodimer of 53-kDa LCB1 and 63-kDa LCB2 subunits, both of which are bound to the endoplasmic reticulum (ER) most likely with the type I topology, whereas other members of the POAS family are soluble homodimer enzymes. LCB2 appears to be unstable unless it is associated with LCB1. Potent inhibitors of SPT structurally resemble an intermediate in a probable multistep reaction mechanism for SPT. Although SPT is a housekeeping enzyme, its activity is regulated transcriptionally and post-transcriptionally, and its up-regulation is suggested to play a role in apoptosis induced by certain types of stress. Specific missense mutations in the human LCB1 gene cause hereditary sensory neuropathy type I, an autosomal dominantly inherited disease, and these mutations confer dominant-negative effects on SPT activity.
  Biochim Biophys Acta. 1632(1-3):16-30.
  
  
• Lewin, T.M., N.M. Schwerbrock, D.P. Lee, and R.A. Coleman. (2004)
  Identification of A New Glycerol-3-phosphate Acyltransferase Isoenzyme
  mtGPAT2, in Mitochondria
  Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate limiting step of glycerolipid synthesis. Two distinct GPAT isoenzymes had been identified in mammalian tissues, an N-ethylmaleimide (NEM)-sensitive isoform in the endoplasmic reticulum membrane (microsomal GPAT) and an NEM-resistant form in the outer mitochondrial membrane (mtGPAT). Although only mtGPAT has been cloned, the microsomal and mitochondrial GPAT isoforms can be distinguished because they differ in acyl-CoA substrate preference, sensitivity to inhibition by dihydroxyacetone phosphate (DHAP) and polymixin B, temperature sensitivity, and ability to be activated by acetone. The preponderance of evidence supports a role for mtGPAT in synthesizing the precursors for triacylglycerol synthesis. In mtGPAT-/- mice, PCR enotyping and Northern analysis showed successful knockout of mtGPAT, however, we detected a novel NEM-sensitive GPAT activity in mitochondrial fractions and an anti-mtGPAT immunoreactive protein in liver mitochondria, but not in microsomes. Rigorous analysis using 2-dimensional gel electrophoresis revealed that the anti-mtGPAT immunoreactive proteins in wildtype and mtGPAT-/- liver mitochondria have different isoelectric points.
  These results suggested the presence of a second GPAT in liver mitochondria from mtGPAT-/- mice. Characterization of this GPAT activity in liver from mtGPAT null mice showed that, unlike the mtGPAT activity in wildtype samples, activity in mtGPAT knockout mitochondria did not prefer palmitoyl-CoA, was sensitive to inactivation by NEM, was inhibited by DHAP and polymixin B, was temperature sensitive, and was not activated by acetone. We conclude that a novel GPAT (mtGPAT2) with antigenic epitopes similar to those of mtGPAT is detectable in mitochondria from the livers of mtGPAT-/- mice.
  J Biol Chem. Jan 14 [Epub ahead of print]

Additional References:

1. Hanada, K. (2003)
   Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism.
   Biochim Biophys Acta. 1632(1-3):16-30.
   [PubMed]
   
2. Lewin, T.M., N.M. Schwerbrock, D.P. Lee, and R.A. Coleman. (2004)
   Identification of a new glycerol-3-phosphate acyltransferase isoenzyme, mtGPAT2, in mitochondria
   J Biol Chem. 279:13488-95.
   [PubMed]
   
3. Yamada, J., Y. Kuramochi, M. Takagi, and T. Suga. (2004)
   Expression of acyl-CoA hydrolase in the developing mouse brain
   Neurosci Lett. 355(1-2): 89-92.
   [PubMed]
   
4. Morillas M, Gomez-Puertas P, Roca R, Serra D, Asins G, Valencia A, Hegardt FG. (2004)
   Structural model of carnitine palmitoyltransferase I based on
   the carnitine acetyltransferase crystal.
   Biochem J. 2004 May 1;379(Pt 3):777-84.
   [PubMed]
   
5. Manning Fox, J.E., C.G. Nichols, and P.E. Light. (2003)
   Activation of ATP-sensitive potassium channels by acyl CoAs
   involves multiple PIP2-interacting residues.
   Mol Endocrinol. 2004 Mar;18(3):679-86. Epub 2003 Dec 23.
   [PubMed]