Complex subcellular distributions of enzymatic markers in intestinal epithelial cells

Abstract

Current procedures for isolating intestinal epithelial cell surface and intracellular membranes are based on the assumption that each organelle is marked by some unique constituent. This assumption seemed inconsistent with the dynamic picture of subcellular organization emerging from studies of membrane turnover and recycling. Therefore, we have designed an alternative fractionation which is independent of a priori marker assignments. We subjected mucosal homogenates to a sequence of separations based on sedimentation coefficient, equilibrium density, and partitioning in aqueous polymer two-phase systems. The resulting distributions of protein and enzymatic markers define a total of 17 physically and biochemically distinct membrane populations. Among these are: basal-lateral membranes, with Na,K-ATPase enriched 21-fold; brush-border membranes, with alkaline phosphatase enriched as much as 38-fold; two populations apparently derived from the endoplasmic reticulum; a series of five populations believed to have been derived from the Golgi complex; and a series of five acid phosphatase-rich populations which we cannot identify unequivocally. Each of the five enzymatic markers we have followed is associated with a multiplicity of membrane populations. Basallateral, endoplasmic reticulum, and Golgi membranes contain alkaline phosphatase at the same specific activity as the initial homogenate. Similarly, Na,K-ATPase appears to be associated with Golgi, endoplasmic reticulum, and brush-border membranes at specific activities two- to seven-fold that of the initial homogenate.

Laterobasal membranes from intestinal epithelial cells: Isolation free of intracellular membrane contaminants

“…Although this could reflect actual residence of the galactosyl transferase or aryl esterase markers in the LBM, we obtained an LBM/ER-Golgi purification ratio of 15, an observation more consistent with the concept that the appreciable copurification of LBM and ERGolgi in previous reports represented cross-contamination. In the most recent study of Mircheff and coworkers in 1985 where a simplified differential centrifugation scheme was used, the enrichment of LBM was 15-fold and the recovery greater than 25% [11]; yet, as estimated from LBM/ER-Golgi ratios (Table 3), cross-contamination with ER-Golgi was still appreciable. Our new method provides LBM at reasonable yield that is purified at least fivefold greater relative to ER-Golgi than achieved by any previously detailed enterocyte subcellular membrane fractionation technique (Table 3).…”

“…The fact that LBM copurifies with ER-Golgi in conventional enterocyte subcellular fractionation procedures [2,21], has made it difficult and even treacherous to examine the kinetics and structural dynamics of intracellular membrane protein assembly that is initiated in ERGolgi and eventually terminates in preferential insertion into one or the other of the surface membranes. Established methods for intestinal subcellular fractionation, utilizing equilibrium centrifugation with discontinuous or linear gradients, have produced appreciable copurification or crosscontamination of ER-Golgi and laterobasal membranes [2,11,21]. Although galactosyl transferase is highly concentrated in Golgi membranes and is generally considered to be a suitable marker for these membranes, it is possible that some of the apparent contamination of LBM with Golgi may be related to actual residence of a portion of the enterocyte's galactosyl transferase in the LBM [11,21].…”

“…Established methods for intestinal subcellular fractionation, utilizing equilibrium centrifugation with discontinuous or linear gradients, have produced appreciable copurification or crosscontamination of ER-Golgi and laterobasal membranes [2,11,21]. Although galactosyl transferase is highly concentrated in Golgi membranes and is generally considered to be a suitable marker for these membranes, it is possible that some of the apparent contamination of LBM with Golgi may be related to actual residence of a portion of the enterocyte's galactosyl transferase in the LBM [11,21]. Also, recent immunocytochemical studies have localized some galactosyl transferase to the BB [17].…”

“…Although counter-current distribution may allow further separation of the ER-Golgi from LBM [11], this technique requires specialized expensive equipment not generally available. We report here a simplified method for isolating highly purified LBM, uncontaminated by ER or Golgi membranes, that can be readily prepared from either rat small intestinal scrapings or dispersed enterocytes.…”

Cell surface and Golgi pools of beta-1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation.

“…These two membrane markers segregated cleanly from one another during fractionation (Fig. 6), occupying relative positions characteristic for plasma membranes and Golgi membranes, respectively (7,9,21,31,32). Furthermore, the utility of alkaline phosphatase as a surface marker for uninduced and differentiated cells was demonstrated by a coincident distribution of 1251 and alkaline phosphatase after lodobead-catalyzed surface iodination (data not shown).…”