Preparation of Golgi subfractions with free-solution isotachophoresis: analysis of sphingomyelin synthesis in Golgi subfractions from rat liver.
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 phosphatidylcholine:ceramide phosphocholine transferase activity in Golgi membrane vesicles of rat liver obtained by FS-ITP.
Hofmeister
R
R
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany.
Böttcher
A
A
Schmitz
G
G
D000818:Animals; D002518:Ceramides / Q000032:analysis; D002784:Cholesterol / Q000032:analysis; D002788:Cholesterol Esters / Q000032:analysis; D004586:Electrophoresis / Q000379:methods*; D019075:Electrophoresis, Capillary; D005260:Female; D006056:Golgi Apparatus / Q000737:chemistry* / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012996:Solutions; D013109:Sphingomyelins / Q000032:analysis / Q000096:biosynthesis*; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000032:analysis
D002518:Ceramides; D002788:Cholesterol Esters; D012996:Solutions; D013109:Sphingomyelins; D002784:Cholesterol; D017855:Transferases (Other Substituted Phosphate Groups); C070186:phosphatidylcholine-ceramide phosphocholine transferase