Biochemical, kinetic and cytochemical approaches resolve Golgi subcompartments of IgM-secreting rat myeloma cells.
The rat myeloma cells chosen for study (IR202) are highly specialized toward the synthesis and secretion of immunoglobulin M (IgM). In [35S]methionine pulse-chase protocols the half-time for secretion of newly synthesized [35S]Ig at 37 degrees C is approximately 2 1/2 h. No degradation of [35S]Ig was detected in such experiments. Pulse-chase experiments with [3H]galactose show that addition of this terminal sugar occurs only approximately 2 min before discharge. The intracellular pool of Ig bearing mature oligosaccharides is therefore very small. Incubation at 20 degrees C stops secretion of the [35S]- and [3H]Ig. We describe a subcellular fractionation protocol for these cells which results in the recovery of a total microsomal fraction by gel filtration. This fraction includes approximately 1/4 of the galactosyltransferase and uridine diphosphatase (UDPase) of the homogenate. By employing two cytological Golgi markers (an "overosmicatable material" and UDPase), galactosyltransferase activity and [35S]methionine and [3H]galactose pulse-chase protocols with the chase at 15 degrees C we document the partial resolution of Golgi subcompartments in isopycnic sucrose gradients used to subfractionate the total microsomal fraction. Electron microscopic and enzymologic examination of the fractions resolved by these gradients confirm that rough microsomes are well separated from Golgi membranes and that the fractions most highly enriched in galactosyltransferase activity have a protein-based specific activity approximately 10 times that of the total microsomal fraction. These studies, therefore, form the basis for an analysis of the composition of the membranes of the Golgi Complex and document the location of proximal Golgi elements, as defined by cytological criteria, in isopycnic gradients.
Gupta
D K
DK
Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106.
Tartakoff
A M
AM
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D006056:Golgi Apparatus / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure*; D006651:Histocytochemistry; D007075:Immunoglobulin M / Q000032:analysis / Q000378:metabolism* / Q000493:pharmacokinetics; D008854:Microscopy, Electron; D008861:Microsomes / Q000032:analysis / Q000648:ultrastructure; D009101:Multiple Myeloma / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D009844:Oligosaccharides / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
D007075:Immunoglobulin M; D009844:Oligosaccharides