ISSN:
0173-0835
Keywords:
Preparative isotachophoresis
;
Golgi apparatus
;
Sphingomyelin synthesis
;
Phosphatidylcholine:ceramide phosphocholine transferase
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
A new displacement electrophoresis technique, termed free-solution isotachophoresis (FS-ITP) was used for the analysis of sphingolipid metabolism in Golgi subfractions. The discontinuous electrolyte system enables tissue-derived membrane vesicles to be separated and purified due to their polarity patterns in a mobility gradient. In this study total Golgi apparatus obtained from rat liver by discontinuous density gradient centrifugation was subfractionated by preparative FS-ITP, yielding enzymatically active cis-, medial-, and trans-Golgi subfractions. These membrane vesicles were assayed by the following established enzyme marker activities: NADH cytochrome c reductase (cis-Golgi), NADP phosphatase (medial-Golgi), and thiamine pyrophosphatase (trans-Golgi). The activity of phosphatidylcholine:ceramide phosphocholine transferase, a sphingomyelin synthesizing enzyme, is attributed to the cis- and medial-Golgi-derived subfractions. Analysis of Golgi lipids revealed a decline in membranous ceramide along the cis- to trans-Golgi polarity axis. Furthermore, significant amounts of newly synthesized sphingomyelin and diacylglycerol are transferred from the medial/cis- to the trans-Golgi compartment. The FS-ITP system is well suited for micropreparative experimental applications, as demonstrated by studies on phosphatidylcholin:exeramide phosphocholine transferase activity in Golgi membrane vesicles of rat liver obtained by FS-ITP.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150190720
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