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A K Mircheff

Publications and source records attributed to A K Mircheff.

87 records · Page 5Linked to original sources
PubMed1983

Empirical strategy for analytical fractionation of epithelial cells.

Despite the importance of cell fractionation methods in studies of epithelial transport mechanisms and of a variety of subcellular processes, current practices in cell fractionation have a number of shortcomings. Most cell fractionation studies depend on biochemical markers, but they provide little independent confirmation of the initial assumptions that markers are uniquely associated with particular subcellular structures and that they are uniformly distributed over the surfaces with which they are associated. Moreover, it is generally difficult and time-consuming to design new membrane isolation procedures. After reviewing the analytical nature of physical separation procedures, I suggest an empirical approach to cell fractionation that is both general and comprehensive. This approach uses physical separation procedures to generate spatial distributions of particles in which position is related to such physical properties as sedimentation coefficient, density, cholesterol content, surface charge, and coefficient of partitioning in aqueous polymer two-phase systems. Determination of the frequency distributions of biochemical markers permits detection of separate populations of particles even when the populations lack unique markers. This process requires no a priori assumptions about the subcellular localizations of particles.

Alkaline Phosphatase↗
PubMed1983

Basal-lateral and intracellular membrane populations of rat exorbital lacrimal gland.

With the goal of isolating and identifying plasma membrane vesicle populations from epithelial cells of the rat exorbital lacrimal gland, we have designed an analytical fractionation of homogenates of the gland parenchyma. This fractionation utilizes separation procedures based on three independent physical properties of subcellular particles: sedimentation coefficient, density, and density after interaction of membrane cholesterol with digitonin. A commonly accepted marker for basal-lateral membranes, Na-K-ATPase, is associated with at least two physically distinct membrane populations. One population can be identified as basal-lateral membrane fragments on the basis of its fractional and specific contents of Na-K-ATPase; it accounts for 50% of the total Na-K-ATPase activity, enriched 29-fold with respect to the initial homogenate. With these values we calculate that the sample of basal membranes has been purified 60-fold with respect to the initial homogenate. The remaining Na-K-ATPase activity appears to be associated, at three- to fivefold lower specific activities, with intracellular membrane populations. We speculate that these populations have been derived from the Golgi complex.

Alkaline Phosphatase↗
PubMed1983

Resolution of apical and basal-lateral membrane populations from rat exorbital gland.

We performed analytical fractionation studies with the goal of isolating basal-lateral and apical membrane vesicles from epithelial cells of rat exorbital lacrimal gland. A density region designated window II contained elements of three physically and biochemically distinct membrane populations. These were resolved by countercurrent distribution in an aqueous polymer two-phase system. One population contained 50% of the NADPH-cytochrome c reductase activity recovered from window II and appeared to be of intracellular origin. A second population contained 70% of the recovered Na-K-ATPase, maximally enriched 42-fold with respect to the initial homogenate. This population was the major locus of alanine transport activity, roughly 85% of which was mediated by systems believed to be characteristic of epithelial cell basal-lateral membranes. It also contained portions of the alkaline phosphatase, galactosyltransferase, acid phosphatase, and NADPH-cytochrome c reductase activities. A third population accounted for 33% of the recovered alkaline phosphatase and was a secondary locus of Na-alanine transport activity, 45% of which could be attributed to systems believed to be characteristic of epithelial cell apical membranes.

Alanine↗
PubMed1982

Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles.

We have confirmed previous demonstrations of sodium gradient-stimulated transport of L-alanine, phenylalanine, proline, and beta-alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these 14C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability of D-glucose to inhibit tracer uptake. L-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the 14C-L-alanine uptake could be inhibited by proline and beta-alanine, while lysine and MeAIB were no more effective than D-glucose. Roughly 50% of the 14C-phenylalanine uptake could be inhibited by proline and beta-alanine; lysine was as effective as proline and beta-alanine, and the effects of pairs of these amino acids at 50 mM each were not cumulative. MeAIB was no more effective than D-glucose. We conclude that three pathways mediate the uptake of neutral L, alpha-amino acids. One system is inaccessible to lysine, proline, and beta-alanine. The second system carries a major fraction of the L-alanine flux; it is sensitive to proline and beta-alanine, but not to lysine. The third system carries half the 14C-phenylalanine flux, and it is sensitive to proline, lysine, and beta-alanine. Since the neutral, L, alpha-amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classical A system, and since all of the L-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classical ASC system. L-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective than D-glucose in inhibiting lysine uptake, while proline and beta-alanine appear to inhibit a component of the lysine flux. We conclude that the 14C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, beta-alanine, and L-alanine, and one which is inhibited by L-alanine and phenylalanine but inaccessible to proline, beta-alanine, and MeAIB. Fluxes of 14C-proline and 14C-MeAIB are completely inhibited by L-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit 14C-beta-alanine uptake. However, beta-alanine inhibits only 38% of the 14C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports beta-alanine.

Amino Acids↗
PubMed1981

Transport properties of intestinal basolateral membranes.

Techniques for the isolation and study of basolateral membrane vesicles from the intestinal epithelium have afforded new insights into the mechanisms of intestinal absorption. First, we have confirmed the hypothesis that the second stage of glucose transport involves facilitated diffusion. Second, we have shown that the major system for translocation of neutral amino acids across the basolateral membrane is the classical "L" system. Third, we have established that basolateral membranes contain sodium-dependent transport systems that may be useful in the supply of essential amino acids to the epithelium from the blood. And, finally, our studies of the basolateral (Na + K)-ATPase have clarified the role of this enzyme in sodium absorption.

4-Chloromercuribenzenesulfonate↗
PubMed1980

Sugar uptake by intestinal basolateral membrane vesicles.

A high yield of membrane vesicles was prepared from the basolateral surface of rat intestinal cells using an N2 cavitation bomb and density gradient centrifugation. The membranes were enriched 10-fold and were free of significatn contamination by brush border membranes and mitochondria. The rate of D-E114C]glucose and L-E13H]glucose uptake into the vesicle was measured using a rapid filtration technique. D-Glucose equilibrated within the vesicles with a half-time 1/25th that for L-glucose. The stereospecific uptake exhibited saturation kinetics with a Km of approx. 44 mM and a V of approx. 110 nmol . mg-1 min-1 at 10 degrees C. The activation energy for the process was 14 kcal . mol-1 below 15 degrees C and it approached 3 kcal . mol-1 above 22 degrees C. Carrier-mediated uptake was eliminated in the presence of 1 mM HgCl2 and 0.5 mM phloretin. The rate of transport was unaffected by the absence or presence of sodium concentration gradients. Competition studies demonstrated that all sugars with the D-glucose pyranose ring chair conformation shared the transport system, and that, with the possible exception of the -OH group at carbon No. 1, there were no specific requirements for an equatorial -OH group at any position in the pyranose ring. In the case of alpha-methyl-D-glucoside its inability to share the D-glucose transport system may be due to steric hindrance posed by the -OCH3 group rather than by a specific requirement for a free hydroxyl group at the position in the ring. It is concluded that sugars are transported across the basolateral membrane of the intestinal epithelium by a facilitated diffusion system reminiscent of that in human red blood cells.

Animals↗
PubMed1979

Highly purified basal lateral plasma membranes from rat duodenum. Physical criteria for purity.

Preparations of intestinal epithelial cell basal lateral plasma membranes were analyzed with free flow electrophoresis and density perturbation with digitonin. The initial basal lateral membrane preparations were obtained by equilibrium density gradient centrifugation after two different schemes of homogenization and differential sedimentation (A.K. Mircheff, C.H. van Os, and E.M. Wright. 1978. Membr. Biochem. 1:177, and A.K. Mircheff, S.D. Hanna, M.W. Walling, and E.M. Wright. 1979. Prep. Biochem. 9:33. In these preparations, Na,K-ATPase, a marker for the basal lateral mambrane, was purified 16- to 18-fold over the initial homogenate. The preparations were also enriched in NADPH-cytochrome c reductase, alkaline phosphatase, acid phosphatase, and galactosyltransferase. Both free-flow electrophoresis, which separates on the basis of surface charge, and density perturbation with digitonin, which depends on a specific interaction of digitonin with cholesterol-rich membranes, resolved the preparation into three populations of particles. The major population, which represented basal lateral membranes purified 20- to 32-fold with respect to the initial homogenate, contained Na,K-ATPase, alkaline phosphatase, adenylate cyclase, and acid phosphatase. A second population was defined by its content of NADPH-cytochrome c reductase, and the third was defined by its content of galactosyltransferase. Guanylate cyclase appeared to be partitioned between the Na,K-ATPase-rich and NADPH-cytochrome c reductase-rich populations. Galactosyltransferase is also present in fractions which contain the Na,K-ATPase-rich membranes, but the present data cannot exclude the possibility of spillover by the adjacent, galactosyltransferase-rich population. This work emphasizes the importance of multiple, physical criteria for purity in the isolation of subcellular components.

Animals↗
PubMed1979

Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium.

The alkaline phosphatase present on isolated brush border and basal lateral membranes of rat duodenal epithelium were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6--9.8, similar substrate km's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin of papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000--150,000, and both enzymes show an Rf value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The Vmax of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 mumoles/mg-h as opposed to 14 mumoles/mg-h. The differences in Vmax are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.

Alkaline Phosphatase↗
PubMed1979

Large scale, analytical method for isolating basal lateral plasma membranes from rat duodenum.

A procedure is described for obtaining large amounts of basal lateral plasma membranes from the rat duodenal epithelium. The yield is approximately 50%, and the purification factor is 18; preparations from 25 rats routinely contain 100 mg of protein. The procedure depends on mild homogenization with a nitrogen cavitation bomb, followed by removal of brush borders by sedimentation in a weak centrifugal field. Basal lateral membranes in the resulting supernatant are partially purified by differential centrifugation in a medium which approximates their equilibrium density, and then further purified by equilibrium density gradient centrifugation in a high capacity zonal rotor. Brush border membranes may be isolated from the 450 x g pellet. Since both brush border and basal lateral membranes may be isolated from the same homogenate, this preparative procedure is suitable for such analytical purposes as determinations of distribution of enzyme activities between the two surfaces of the epithelium. The large scale of the isolation procedure makes it an appropriate starting point for purification of specific basal lateral membrane components.

Animals↗
PubMed1978

Subcellular distribution of nucleotide cyclases in rat intestinal epithelium.

The subcellular distributions of adenylate cyclase and guanylate cyclase were determined for the mature enterocyte from the rat duodenum. Brush-border and basolateral membranes were prepared from isolated cells by an analytical isolation procedure, and multiple linear regression analysis was used to obtain a quantitative estimate of the distribution of recovered cyclase activities between the brush borders and basolateral membranes. Adenylate cyclase was largely confined to the basolateral surface of the epithelium, whereas guanylate cyclase was found on the brush-border and basolateral membrane fractions in the ratio 2.4:1. There was no evidence for the presence of nucleotide cyclases in the cytosol. Guanylate cyclase in both the brush-border and basolateral membranes was stimulated by epinephrine, insulin, and Triton X-100, but not by carbachol. Adenylate cyclase was not influenced by epinephrine, but was markedly stimulated by NaF and vasoactive intestinal peptide. These results are discussed in relation to the effects of hormones on transport across the small intestine.

Adenylyl Cyclases↗
PubMed1978

Preparative scale isolation of basal-lateral plasma membranes from rat intestinal epithelial cells.

A simple, efficient procedure is described for the preparative scale isolation of basal-lateral membranes from the rat intestinal epithelium. The intestinal mucosa was mildly homogenized and soluble protein and RNA were separated from the homogenate by differential centrifugation. The basal-lateral membranes were then separated from nuclei, mitochondria, and brush border membranes by differential centrifugation in a medium close to the equilibrium density of the basal-lateral membranes. Final purification of the basal-lateral membranes was achieved on a linear density gradient in a high-capacity zonal rotor. The final product (usually at least 40 mg protein) represented a 34% yield of basal-lateral membranes purified 18-fold with respect to protein, 26-fold with respect to brush border membranes, and 53-fold with respect to mitochondria.

Animals↗
PubMed1977

Distribution of bicarbonate-stimulated ATPase in rat intestinal epithelium.

This study reports on the distribution of bicarbonate-stimulated ATPase in rat intestinal epithelial cells. Brush-border membranes and basolateral membranes were separated from each other and from mitochondrial and other intracellular membranes by differential and density gradient centrifugation. Bicarbonate-sensitive ATPase activity followed the mitochondrial marker succinic dehydrogenase closely throughout all the centrifugation steps. The low HCO3--ATPase activity in purified brush-border and basolateral plasma membranes could be accounted for quantitatively by the small mitochondrial contamination. Consequently, there are no grounds for postulating that this enzyme has a direct role in H+ or HCO3- transport across the rat small intestine.

Adenosine Triphosphatases↗
PubMed1976

Analytical isolation of plasma membranes of intestinal epithelial cells: identification of Na, K-ATPase rich membranes and the distribution of enzyme activities.

A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and core material. Sucrase specific activity in the purified brush border plasma membranes was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochrome c reductase, nonspecific esterase, beta-glucuronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity. Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent 35S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein. Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.

Acid Phosphatase↗