Lipid fluidity and composition of intestinal microvillus membranes isolated from rats of different ages

The lipid composition and fluidity of microvillus (luminal) membranes isolated from the small intestines of Fisher 344 rats aged 6, 17, and 117 weeks were compared. Lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, was significantly greater in rats aged 6 weeks as compared to 17 or 117 weeks. A lipid thermotropic transition was observed at 17.5 +/- 1.3 degrees C in the membranes of the youngest group, approx. 5-6 degrees C lower than that of the older animals. The differences in lipid composition which account for the higher fluidity of the youngest preparations include a decreased cholesterol/phospholipid molar ratio in both the proximal and distal halves of the small intestine and, in the proximal half alone, increases in the lipid/protein ratio and double bond index. The foregoing reduction in cholesterol/phospholipid ratio derives mainly from a higher content of total phospholipid, and the increment in double bond index results from an increase in arachidonic acid residues. The results demonstrate an age-dependent decrease in fluidity of intestinal microvillus membranes in the early post-weaning period in the rat. This pattern was unlike that of the microvillus membrane p-nitrophenylphosphatase, whose specific activity declined progressively in the older age groups.     

Rat intestinal microvillus membranes. Purification and biochemical characterization

1. A technique is described for the removal of subcellular contaminants from intact rat intestinal brush borders, and for the subsequent separation of a microvillus membrane fraction from a fibrillar residue. 2. Increments in invertase activity, microscopic homogeneity and low nucleic acid content indicate that the microvillus plasma membrane has been extensively purified. Multiple membrane preparations have been shown to be highly reproducible with respect to their invertase specific activity, cholesterol content and phospholipid content. Alkaline phosphatase, leucine aminopeptidase, Mg(2+)- and Ca(2+)-dependent adenosine triphosphatase and seven separate disaccharidases were shown to be predominantly confined to the membrane fraction. 3. The fibrillar fraction has been shown to contain approximately 30% of the total protein of purified brush borders, plus most of the residual nucleic acid contaminant. No evidence was found for the localization of any specific enzyme in this fraction.     

Cholesterol modulates alkaline phosphatase activity of rat intestinal microvillus membranes

Experiments were conducted, using a nonspecific lipid transfer protein, to vary the cholesterol/phospholipid molar ratio of rat proximal small intestinal microvillus membranes in order to assess the possible role of cholesterol in modulating enzymatic activities of this plasma membrane. Cholesterol/phospholipid molar ratios from 0.71 to 1.30 were produced from a normal value of 1.05 by incubation with the transfer protein and an excess of either phosphatidylcholine or cholesterol/phosphatidylcholine liposomes for 60 min at 37 degrees C. Cholesterol loading or depletion of the membranes was accompanied by a decrease or increase, respectively, in their lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the lipid-soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Increasing the cholesterol/phospholipid molar ratio also decreased alkaline phosphatase specific activity by approximately 20-30%, whereas decreasing this ratio increased this enzymatic activity by 20-30%. Sucrase, maltase, and lactase specific activities were not affected in these same preparations. Since the changes in alkaline phosphatase activity could be secondary to alterations in fluidity, cholesterol, or both, additional experiments were performed using benzyl alcohol, a known fluidizer. Benzyl alcohol (25 mM) restored the fluidity of cholesterol-enriched preparations to control levels, did not change the cholesterol/phospholipid molar ratio, and failed to alter alkaline phosphatase activity. These findings, therefore, indicate that alterations in the cholesterol content and cholesterol/phospholipid molar ratio of microvillus membranes can modulate alkaline phosphatase but not sucrase, maltase, or lactase activities. Moreover, membrane fluidity does not appear to be an important physiological regulator of these enzymatic activities.     

Interaction of retinol and intestinal microvillus membranes studied by fluorescence polarization

The interaction of retinol and microvillus membranes prepared from the duodenum, jejunum and ileum of the rat can be studied readily by fluorescence and fluorescence polarization. A characteristic pattern of increased retinol anisotropy in membranes from more distal segments is demonstrated. Studies with trypsin indicate that the membrane proteins influence retinol fluorescence intensity and anisotropy.     

Functional interactions of lipids and proteins in rat intestinal microvillus membranes.

Interactions of lipids and proteins in isolated rat intestinal microvillus membranes were examined by studying the temperature dependence of enzyme activities and of D-glucose transport in relation to the membrane lipid thermotropic transition observed by fluorescence polarization (26 +/- 2 degrees C) and differential scanning calorimetry (23--39 degrees C). Two groups of activities were defined. Enzymes of the first group, comprising lactase, maltase, sucrase, leucine aminopeptidase, and gamma-glutamyl transpeptidase, all yielded a single slope on the Arrhenius plot in the range 10--40 degrees C and did not appear to experience functionally the effects of the lipid thermotropic transition. Each activity of the second group, comprising calcium- and magnesium-dependent adenosine triphosphatases, p-nitrophenylphosphatase, and D-glucose transport, showed a change in the slope of the Arrhenius plot in the range 25--30 degrees C, corresponding to the lower region of the lipid transition. The terms "extrinsic" and "intrinsic" activities could be applied to these groups. Delipidation of the particulate p-nitrophenylphosphatase removed the discontinuity in the Arrhenius plot. Subsequent relipidation with a variety of lipids restored a break point, but the temperature corresponded to the original discontinuity (25--29 degrees C) rather than to the phase transition temperature of the exogenous lipid added.     

Studies on the binding of iron by rabbit intestinal microvillus membranes.

1. 59Fe binding by microvillus membranes purified from rabbit intestine was studied by means of a microfiltration procedure. 2. Binding activity from ferrous ascorbate chelates was 100-fold greater than from ferric chelates of citrate and nitrilotriacetate. Dual-label experiments indicated dissociation of iron complexes before binding to the membranes. 3. Binding was inhibited at low incubation temperatures and was optimal at neutral pH. 4. Binding activity was reduced in ileal preparations when compared with membranes prepared from proximal intestine. 5. Initial binding velocity followed saturation kinetics over the range 45-450 microM-iron: it was weakly inhibited in the presence of excess Co2+ and V3+. 6. The data provide additional evidence for high-affinity iron-binding sites on the intestinal microvillus membrane and indicate properties that may reflect the functional significance of the binding step in the absorption pathway for iron.     

Membrane lipids can modulate guanylate cyclase activity of rat intestinal microvillus membranes

Studies of the temperature dependence (10-40 degrees C) of guanylate cyclase in rat intestinal microbillus membranes reveal a change in energy of activation (slope of the Arrhenius plot) at 30 +/- 1 degree C. The break point temperature corresponds to the lipid thermotropic transition in these membranes previously characterized by differential scanning calorimetry (range: 23-39 degrees C; peak temperature, 31 degrees C). The break point temperature for guanylate cyclase also corresponds to that of a number of other microbillus membrane enzymes and of D-glucose transport. These activities are defined as "intrinsic" membrane activities by this operational criterion. Treatment with the nonionic detergent Lubrol WX increased the guanylate cyclase activity 4- to 8-fold and removed the discontinuity in the Arrhenius plot.     

Glutathione-degrading enzymes of microvillus membranes

Microvillus membranes from rat kidney, jejunum, and epididymis have been purified by the Ca precipitation method. The membranes exhibit enrichment in specific activities of gamma-glutamyl transpeptidase, aminopeptidase M, and a dipeptidase. The latter has been characterized and shown to be the principal activity responsible for the hydrolysis of S derivatives of Cys-Gly (including cystinyl-bis-glycine (Cys-bis-Gly) and 5-hydroxy-6-S-cysteinylglycyl-1-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene D4)). A method is described for the simultaneous purification of papain-solubilized forms of the three enzymes from renal microvilli. Dipeptidase (Mr = 105,000) appears to be a zinc metalloprotein composed of two Mr = 50,000 subunits. The enzyme is severalfold more effective in the hydrolysis of dipeptides than aminopeptidase M. Dipeptidase, in contrast to aminopeptidase M, is inhibited by thiol compounds; Cys-Gly, in particular, is a potent inhibitor (Ki = 20 microM). The inhibition of dipeptidase by thiols has been employed to probe the relative significance of dipeptidase and aminopeptidase M in the metabolism of glutathione and its derivatives at the membrane surface.     

Differences in composition and fluidity of intestinal microvillus membrane vesicles prepared by different methods

Multiple methods have been developed to isolate the intestinal microvillus membrane and facilitate the study of its composition and function. Variations in membrane composition and fluidity may result from different preparative techniques. This study shows that the use of MgCl2 and/or KSCN in vesicle preparation alters phospholipid and protein composition of the membrane compared to CaCl2 precipitation. The use of MgCl2 in membrane preparation increased phosphatidylethanolamine and decreased phosphatidylinositol content. The use of KSCN in membrane preparation decreased the protein content. The structural changes seen with the use MgCl2 alone are accompanied by an increase in both static and dynamic membrane fluidity. These results suggest that different methods of membrane vesicle preparation affect membrane phospholipid and protein content as well as membrane fluidity.     

Tannic acid characterized membranes of animal cells

Summary Tannic acid affects one face of some cytoplasmic membranes causing them to appear thin in electron micrographs.Trans vesicles of Golgi apparatus, dictyosome-like-structures, headcaps and aerosomes of germ cells, and certain lysosomes all have membranes that appear thin after tannic acid fixation and, in addition, are all characterized as being acid phosphatase positive. Thus, thin membranes appear functionally related and to be associated with cellular components that have lysosome or lysosome-like character.     

Establishment of tight junctions between cells from different animal species and different sealing capacities

Summary Epithelial cells establish tight junctions (TJs) that offer an ample range of transepithelial electrical resistances (TER), in adjustment to physiological requirements. In the present work, we demonstrate that cells from different animal origins, co-cultured in monolayers, can make sealed TJs, suggesting that this structure has a basic universal structure. TJs cannot be established, however, if one of the partners does not normally express TJs, indicating that each neighbor has to contribute its moiety. Furthermore, we observe that clones of the same cell line, with widely different values of TER, do not differ, in the number and length of their junctional trands, suggesting that the difference is due to their ability to express ionic channels traversing their strands. The value of TER achieved in mixed monolayers of cells of the same or different lines is the one that may be expected by taking into account the proportion of each type in the mixture and adding in parallel the electrical resistance that they exhibit in pure monolayers. Therefore, epithelial TJs appear to behave as parallel resistances.