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.     

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.     

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.     

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.     

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.     

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.     

Embryonic hemoglobins of different animal species

Summary The existence of embryonic hemoglobins is demonstrated in sheep-, calf and pig embryos. The occurrence and disappearance of these hemoglobins is quantitatively determined by cellulose acetate gel electrophoresis; hemoglobins as well as the globin chains, dissociated in 8 molar urea were quantitated. Sedimentation and diffusion experiments in the analytical ultracentrifuge revealed a S₂₀ of 4.3 and a D₂₀ of 6.6 for the examined hemoglobins. Therefore it is concluded that all hemoglobins occurring at different stages of embryonic and fetal development consist of 4 polypeptide chains with a total molecular weight of 66,000. The subsequent formation of the different polypeptide chains during ontogenesis is shown: At first only -chains are formed as demonstrated by the existence of Hb Gower I, consisting of four identical -chains. Subsequently the -chain appears, which leads to Hb Gower 2 (₂₂). The third polypeptide chain formed during the ontogenesis the -chain results finally in the appearance of HbF.In addition the existence of a HbF pig is demonstrated by the fingerprint technique.     

Two trans-sialidase forms with different sialic acid transfer and sialidase activities from Trypanosoma congolense

Trypanosomes express an enzyme called trans-sialidase (TS), which enables the parasites to transfer sialic acids from the environment onto trypanosomal surface molecules. Here we describe the purification and characterization of two TS forms from the African trypanosome Trypanosoma congolense. The purification of the two TS forms using a combination of anion exchange chromatography, isoelectric focusing, gel filtration, and subsequently, antibody affinity chromatography resulted, in both cases, in the isolation of a 90-kDa monomer on SDS-PAGE, which was identified as trans-sialidase using micro-sequencing. Monoclonal antibody 7/23, which bound and partially inhibited TS activity, was found in both cases to bind to a 90-kDa protein. Both TS forms possessed sialidase and transfer activity, but markedly differed in their activity ratios. The TS form with a high transfer-to-sialidase activity ratio, referred to as TS-form 1, possessed a pI of pH 4-5 and a molecular mass of 350-600 kDa. In contrast, the form with a low transfer-to-sialidase activity ratio, referred to as TS-form 2, exhibited a pI of pH 5-6.5 and a molecular mass of 130-180 kDa. Both TS forms were not significantly inhibited by known sialidase inhibitors and revealed no significant differences in donor and acceptor substrate specificities; however, TS-form 1 utilized various acceptor substrates with a higher catalytic efficiency. Interestingly, glutamic acid-alanine-rich protein, the surface glycoprotein, was co-purified with TS-form 1 suggesting an association between both proteins.