Life Sciences, Vol . 24, pp . 669-678 Printed in the U .S .A .
Pergamon Press
PURIFICATION OF INTESTINAL BRUSH BORDER MEMBRANE VESICLES BY THE USE OF CONTROLLED-PORE GLASS-BEADS COLUMN Kazuaki Ohsawa, Asako Kano and Takeshi Hoshi Department of Physiology, Faculty of Medicine University of Tokyo, Tokyo 113, Japan (Received in final form January 12, 1979) Summary A simple rapid method for obtaining highly purified and efficiently transporting intestinal brush border membrane vesicles was developed. The new method consists of two major steps : Ca-treatment of the homogenate of the scraped epithelium (rabbit ileum) and the subsequent chromatography of the supernatant of the Ca-treated homogenate through a controlledpore glass-beads column . The fraction showing the peak value of the optical density at 280 nm and two subsequent fractions were identified as those containing purified brush border membrane vesicles as judged from the activities of the marker enzymes (sucrase and alkaline phosphatase) and the rate of D-glucose uptake . Whole procedures could be completed in about 90 min. Specific activities of sucrase and alkaline phosphatase increased to 35 .5 and 34 times, respectively, while Na,KATPase activity decreased to one tenth as compared with the initial homogenate . Overshoot uptake of D-glucose in the presence of a NaSCN gradient showed a peak at 1-1 .5 min after the start of incubation, when it reached 10-40 nmoles/mg protein . Electron microscopic examination revealed that the highly purified vesicles had fairly homogeneous size, the average diameter being about 80 nm . Various methods have been published thus far for the isolation of highly purified intestinal brush border membranes, e .g ., hypotonic EDTA treatment followed by density gradient centrifugation (1), Tris-disruption procedure (2), free-flow electrophoresis (3), or the Ca-precipitation method (4,5) . For the study of transport, shortness of time required for whole preparation procedures and preservation of high transport function are needed . With this regard, the Ca-precipitation method modified by Kessler et al . (5) seems, at least at present, superior to the other methods, but the vesicles obtained by their method seem to be still inhomogeneous . In the present study, a further purification of vesicles obtained by the Ca-precipitation method was attempted without prolonging the time of preparation much . For this purpose, a controlled-pore glass-beads column chromatography (6) has been introduced, which enabled us to obtain much more homogeneous, highly purified and efficiently transporting brush border membrane vesicles . Such purified vesicles had an average diameter of 80 nm .
0300-9653/79/080669-09$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd .
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Materials and Methods Preparation of a "crude" brush border membrane fraction : Female rabbits, weighing from 1 .5-3 .0 Kg B .W ., were sacrificed by a blow on the neck, and the ileum was removed immediately. The gut was opened by a longitudinal cut and the mucosal surface was rinsed with ice-cold buffer solution (100 mM mannitol, 10 mM HEPES/Tris, pH 7 .4) . After wiping off the rinsing solution and excess mucus on the mucosal surface, the epithelial cells were gently scraped off with a slide glass . Usually, one to 1 .5 g mass could be obtained from one animal . The scraped material was suspended in 10 ml buffer solution containing 50 mM mannitol and 2 mM Tris/HC1 (pH 7 .4), and then homogenized in a blaring blendor at the maximum speed for 2 min . To this homogenate, powdered CaC12 was added to the final concentration of 10 mM . The homogenate was allowed to stand in ice for 15 min, thereafter it was centrifuged at 2,000 x ~ for 10 min. Large particles, such as nuclei and mitochondria are known to be precipitated by such a Ca-treatment followed by a low-speed centrifugation (4,5) . The supernatant, referred to as S1 here, was then centrifuged at 45,000 x ~ for 30 min . The pellet thus obtained is referred to as P2, which usually amounted about 0.3 g (wet .w) . Chromatography by using a controlled-pore glass-beads (CPG) column : The pellet, P2, was resuspended in 7 ml solution of 100 mM mannitol, 10 mM HEPES/ Tris (pH 7.4), and homogenized with a Potter-Elvehjem homogenizer. The homo genate was then chromatographed with a CPG-column . A glass column, 1 .6 cm in inner diameter and 100 cm long, was packed with controlled-pore glass-beads, (Electro-Nucleonics, Inc., pore diameter 300 nm) . Elution was carried out with the same solution as that used for resuspension . The flow rate was kept constant at 6 ml/min . The elution fluid was degassed with an evacuator before use. The eluent from the column was monitored for its W(280 nm) optical density with a W monitor (Hitachi spectrophotometer No . 124) . The void volume of the column, V0, and internal pore salt volume, Vi, were 35 and 45 ml, respectively . Calibration of the system was carried out with polystyrene latex beads of two different diameters, 87 and 106 nm . The effluent was fractionated every 4 ml and each fraction was numbered serially . Enz e assa s and other anal ical rocedures : As the marker enzymes of the brush bo er me raves, bot sucrase an al al ne phosphatase activities were determined . The former was assayed by the glucose-oxidase method (7), the latter by the method of Bessey et al . (8) . Isomaltase and maltase activities were also examined by incubating samples with the appropriate substrate and determining the glucose released by the glucose-oxidase method . As the marker of the basolateral membrane, Na,K-ATPase activity was measured (9,10), and, as the marker of the cytosol, lactate dehydrogenase was assayed (11) . ATP content was also determined by the luciferin-luciferase method (12) . Protein concentration was determined by Lowry's method (13) by using bovine serum albumin as a standard . Glucose uptake experiments : The uptake of D-glucose was measured with a Millipore-filtration technique in a manner similar to that described by Hopfer et al . ~(14) . Each fraction was centrifuged at 45,000 x & and the pellet was resuspended in 0.5 ml buffer solution (100 mM mannitol, HEPES/Tris 1 mM, pH 7 .5) . An aliquot of the preparation (protein amount 40-100 Ug) was incubated in 4-fold volume of the incubation solution containing 1 mM D-glucose, 1 .0 ~aCi /ml D-[14 C(U)] glucose, 100 mM mannitol, 1 mM HEPES/Tris (pH 7 .5), 4 uCi/ml D-(1-3H(N)] mannitol, and 100 mM NaSCN . The incubation was carried out at 20 ° C, and the uptake reaction was terminated by dilution of the reaction medium
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10-fold with ice-cold stop solution (150 mM NaCl plus 1 mM HEPES/Tris pH 7 .5) and immediate collection on a Millipore filter (Sartorius membrane filter, 11305, 0.6 um) . The filters were rinsed with 5 ml stop solution, then put into The radioactivities were counted minivials containing 3 ml Bray's solution . in a liquid scintillation counter (Beckman CPM-100 or Packard Tri Carb 2425) . When the uptake in the absence of a Na gradient was examined, 100 mM NaSCN in the incubation medium was replaced by 100 mM KC1, and 150 mM KC1 solution containing 1 mM HEPES/Tris was used as the stop solution . Electron microsco :The pellets of the fractions showing enriched sucrase an alkaline phosphatase activities were fixed with Millonig fixation reagent (15) and post-fixed with 1 $ Os04 for 1 hr each at 4°C . After dehydrat ion, the pellets were embedded in Epoxy resin . The thin sections were stained with uranylacetate and lead citrate (16) and examined with an electron microscope (Akashi 5-500) . SDS-polyacrylamide slab gel electrophoresis : The pellets of fractions to be examined were solubilized in a solution containing 58 mM Tris/HC1 (pH 6 .8), 71 mM nercapotoethanol and 2 $ sodium-dodecylsulfate (SDS) at 95 °C for 3 min. The slab gel system used and the conditions of the electrophoresis were the same as those described by Kassler et al . (5) . Fine discrimination of Relative mobilities of protein bands was carried out by using a densitometer . protein subunits were calculated by comparing with those of marker proteins of known molecular weight ; trypsin inhibitor (21,500), RNA polymerase a (39,000), bovine serum albumin (68,000), RNA polymerase ß (155,000) and ß' (165,000) . Chemicals and other materials : All chemicals used for the experiments were o the hig est g=ade available. Bovine serum albumin, glucose-oxidase reagent and tris-aminomethane sulfonate (Tris) were purchased from Sips Chemical All radioCo ., uniform latex styrene butadiene particles from Dow Chemical Co . . active compounds were obtained from New England Nuclear (Boston, Mass .), marker proteins for slab gel electrophoresis from Boehringer Mannheim (~bH Biochemica . Results 1 . Identification of fractions containing purified brush border membrane vesicles by enzyme assays . The tracing of optical density (OD) at 280 nm usually began to rise when the external elution volume reached 80 ml (Fraction 20) and formed a peak at the fraction 23 (elution volume 92 ml) . Thereafter, it declined smoothly as the eluted volume further increased (Fig . lA) . The protein concentration determined by Lowry's method changed almost in parallel with the OD curve . Alkaline phosphatase and sucrase activities changed in parallel with each other and their peak values were seen one or two fractions later than the protein or OD peak (Fig . 1B) . In contrast, Na,K-ATPase activity appeared in fractions earlier than the protein peak and formed a peak in the fraction immediately before the fraction of the protein peak . Little activity of Na,K-ATPase was seen in the fractions which had very high activities of sucrase and alkaline phosphatase (Fig . 1C) . Lactate dehydrogenase was absent in the fractions of enriched sucrase and alkaline phosphatase . From these enzyme patterns, the fraction of the OD peak and subsequent two or three fractions were taken as those containing purified brush border membrane vesicles . The data from enzyme assays for the fractions containing purified brush border membrane are summarized in Table 1, where protein concentration, specific activities and relative enrichment factors are shown for each purifi cation step : the initial homogenate, the supernatant Sl, the pellet of the
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Fig. 1 An example of changes in the optical density (OD2g0), protein concentration and activities of various marker enzymes with increase in fraction number after a single run of the CPG column chromatography . A : Optical density and protein concentration (mg/ml), B: sucrase and alkaline phosphatase (tiimoles of substrate hydrolysed per min per mg protein), C : Na,K-ATPase ~uuoles of Pi produced per min per mg protein) . supernatant of Ca-treated homogenate P2, and the final -brush border membrane vesicle preparations (final BBM) . As seen in the table, the specific activities of sucrase and alkaline phosphatase were elevated by about 35 times and 34 times in the final preparations, respectively, as compared with those of the initial homogenate . Avery low Na,K-ATPase activity was seen in these fractions, but the enrichment factor was only 0.08 . As compared with the data reported by Kassler et al . (1978), the enrichment factors for the brush border membrane enzymes were about two times higher, whereas that for the basolateral membrane marker enzyme was much lower, about one twentieth . These indicate that the degree of purification is at least two-fold higher in the present method than that of Kassler et al . (5) and comparable with that of free-flow electrophoretic separation (3) . In some experiments, a second increase in sucrase activity was observed in the later fractions (Fractions 28-32, the data not shown) . In such fractions, protein concentrations were very low, so that the specific activity of this enzyme was very high ; 200-300 times higher than that in the initial homogenate . As the proteins could not be precipitated by the centrifugation at 45,000 x ~ for 60 min, the observed activity may be due to solubilized sucrase. Na-dependent D-glucose uptake was not seen in these fractions .
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TABLE 1 Specific Activities of Enzymes during the Preparation of Brush Border Membrane Vesicles . Specific activity
Amount of protein ($)
Sucrase
Alkaline phosphatase
Na,K-ATPase
Lactate dehydrogenase
100
1 .3+0 .5
1 .04*0.5
17 .113 .4
1 .68
Homogenate $1
64*5 .4
17 .719 .3
2 .2711 .2
35 .71.4 .1
1 .68
P2
1212 .1
30 .819 .3
12 .410 .5
43 .2112.0
0 .73
Final BBM vesicle
0 .8310.05 46 .115 .7
35 .415 .8
1 .3610 .5
0 .03
34 .0116.4
0.0810 .06
0.02
Enrichment factor
35 .518 .5
Specific activity is given in hmoles/min/mg protein. Figures are given as meanlS .E . Number of determinations was 4 except for lactate dehydrogenase . The final BBM vesicles are Fraction 23 or 24 . The enrichment factor is the ratio of the specific activities in the final BBM vesicles and the initial homogenate . 2 . Identification by D-~ucose uptake D-glucose uptake was examined in the presence or absence of a NaSCN gradient at the start of incubation . A marked Na-dependent overshoot uptake was seen only in the fractions which had both high sucrase and high alkaline phos phatase activities . An example of the time course of D-glucose uptake was shown in Fig. 2 . The characteristics of uptake were essentially the same as those reported by previous investigators (3,5,14,17) . In the presence of a NaSCN gradient, the peak of overshoot uptake was seen usually 1-1 .5 min after the start of incubation, when it reached more than 10 nmoles/mg protein . In the absence of Na, e .g . in the presence of a KC1 or KSCN gradient, there was no overshoot and the time course of D-glucose uptake was almost the same as that of D-mannitol uptake . Smaller but distinct overshoot uptake of D-glucose was observed also in the later fractions, but the peak value of uptake rapidly decreased as the fraction number increased (Fig . 3) . The value of peak uptake of D-glucose was about two times larger than that obtained by Kessler et al . (5) and this is in good agreement with the 2-fold increase in purification as judged from enzyme data . In contrast, the peak uptake observed in the present study was much higher than that reported by Hopfer et al . (14), Murer and Hopfer (17), and Murer et al . (3), who prepared the BBM vesicles by repeated centrifugations or the free-flow electrophoresis . This suggests that Ca-precipitation and rapid separation of BBM vesicles by CPG-column is very effective in preserving the transport activity as compared with other separation methods .
674
Purification of the Brush Border Membranes
m
s
â
Vol . 24, No . 8,
1a
0
ô E o. 0
O
0 m n Y
m û
2
i g
4
i 10
i 8
12
Time ( min 1
14
18
Fig. 2 Typical example of time course of D-glucose uptake by purified BHM vesicles in the presence of a NaSCN gradient (open circles) . Time course of D-mannitol uptake by the same prepa ration is shown for the sake of comparison (closed circles) . 50c E u c
0.2Wm Filter
O.gymFihv
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C _~ rA c
O Ô Y
~ .E a ~ 20 Y 0 b n Ec e ô
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Fraction number Fig . 3 Comparison of D-glucose uptake during the first one minute among various fractions. For the fractions of larger fraction numbers (Fractions 28-35), the membrane filter of smaller diameter (0 .2 um) was used since the average diameter of particles contained was expected to become smaller as fraction number increased.
1979
Vol . 24, No . 8,
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3 . Electron microscopic observation Electron microscopic examinations of the pellets of the fractions which had high sucrase activities revealed that all the materials were in vesicular forms of fairly uniform sizes though some were spherical and some others ellip soidal . Three-layer membrane structure was clearly seen in most vesicles . No coagulated mass was seen . The results of measurements of diameters of vesicles are summarized in Fig . 4 . The mean values of diameter were 81, 78, 56 and 41 nm for the fractions 23, 24, 25 and 26, respectively . Thus, the size of vesicle became smaller as eluted volume increased . This indicates that the CPG-column chromatography effectively separates vesicles having different diameters . The vesicles which had the highest sucrase activity and D-glucose transport activity were those having diameter of 78f5 (meantS .D .) nm .
30
~:o
Fraction number
23
24
26 I`-Z~ 90 105 1Y0 135 Diameter 1 nm i
1-~.-~ ._ 1 . 30 "5 90 75
i
Fig. 4 Distribution of diameter of the vesicles in four different fractions (Fractions 23-26) . The diameter was measured on the ultra-thin section electron micrograph of the pellet of each fraction . In cases of ellipsoidal structures, the largest width in the horizontal direction was measured . Dotted vertical lines indicate the mean values of diameter .
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4 . SDS-polyac rylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis of the purified BHM preparations yielded about 40 bands of protein subunits, their molecular weight ranging from 10,000 to 200,000 . The number of protein bands was determined by densito metry (Fig . 5) . The electrophoresic pattern was quite similar to those obtained by other authors (4,5) .
Mdeculsr weight 185000
I "-155000
39000
21500
Fig . 5 The pattern of SDS-polyacrylamide gel electrophoresis of a purified BBM vesicle preparation . 8 $ polyacrylamide gel slab was used . The molecular weights calibrated with the standard proteins are indicated between the traced diagram and the densitometric curve. Discussion Most current methods of preparation of a certain membrane system from a cell homogenate are laborious and time-consuming because they require repeated ultracentrifugation in combination with density gradient centrifugation or the free-flow electrophoresis . As to the BHM of intestinal epithelial cells, Kessler et al . (5) developed a very simple method of purification by modifying the Ca-precipitation method of Schmitz et al . (4) . However, the BBM vesicles that they finally obtained seem still to be inhomogeneous . The present method includes the same Ca-precipitation procedure, but the subsequent chromatography by the use of controlled-pore glass-beads (CPG) column enabled us to obtain, within one and a half hour, much more uniform vesicles which had significantly higher transport activity and higher marker enzyme activities as compared with those obtained by Kessler et al . (Table 1 and Figs . 2 and 3) . CPG-chromatography possesses favorable properties which are suitable for isolating and purifying certain macromolecules and biological materials such as viruses (6) . The glass is inert to practically all chemicals, and hig~ pres-
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sure can be applied without any change of column bed or pore size . Welldefined pore size with its narrow distribution which is independent of the presence of elution fluid is quite useful for the application of the theory of chromatographic fractionation of a mixture of particles of different sizes (6) . Also, the CPG-column can be easily prepared and regenerated, and the time required for a single run of chromatography is very short (usually only about The sur15-20 min) . The surface of glass is known to be negatively charged . face of the BBM vesicles is also negatively charged as evidenced by electroTherefore, binding of vesicles to glass by electric force phoretic mobility . can be neglected and only molecular sieving or size-separation process is considered to take place in the present CPG-chromatography . The results of electron microscopic examination of pellets of fractions 23-26 revealed that the vesicles of different sizes were effectively separated and fractionated by this procedure . The results of enzyme assays and glucose uptake experiments indicate that the best preparation of the BBM vesicles which have very high transport activity Since the microvilli in vivo have relatively uniform diameters around 80 nm . have uniform diameters about 100 nm, it is easily anticipated that the BBM tends to form vesicles of about 80 nm in diameter when the cells are homogenized . In the present method, we adopted the Ca-precipitation method in the first This method has been shown to step in order to obtain crude BBM preparation . be effective in precipitating the granular components (nuclei and mitochondria) and other intracellular membrane systems (4,5) . This was confirmed by the presCalcium ent study as indicated by the data of enzyme assays for P2 (Table 1) . ion has been speculated to be effective in preserving the membrane permeability characteristics or transport function (4,5), but our additional observations disclosed that the transport or permeability properties of the BBM was independent of the presence of calcium . The details of the experimental results in this regard will be described in a subsequent paper.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 .
D. Miller and R. K. Crane, Anal . Biochem . 2, 284-286 (1961) A. Eichholz and R. K. Crane, J. Cell Biol .26, 687-692 (1965) H. Murex, U. Hopfen, E . Kinne-Sai~ran and R.Kinne, Biochim. Biophys. Acta 345, 170-179 (1974) .Schmitz, H . Presier, D . Maestrocci, B. K . Ghosh, J. J . Cerda and R. K. J Crane, Biochim. Biophys . Acta 323, 98-112 (1973) M . Kess erb Acuto, C . Storelli, H . Murex, M . Murex and G. Semenza, Biochim . Biophys . Acta 506, 136-154 (1978) W . Haller, Nature 206, 693-696 (1965) M . Messer and A. Dahlgvist, Anal . Biochem. 14, 376-392 (1965) A . 0 . Bessey, 0. H. Lowry and J . Brock, _J . Biol . Chem . _164, 321-329 (1946) J . P . Quigley and G. S . Gotterer, Biochim. Biophys . Acta 173, 456-468 (1969) J . B . Martin and M. Doty, Ann. Chemist . 21, 965-967 (1949 P . G . Cabaud and F . Wroblewski, Am . J . Clin . Path . 30, 234-236 (1958) P . E . Stanley and S. G. Williams,Anâl . Biochem . 29,381-392 (1969) . Randall, _J . Biol . Chem . 0 . H . Lowry, N . J . Rosebrouch, A. L, FarmR. J 193, 265-275 (1951) .Hopfen, K . Nelson, J. Perrotto and K. J. Isselbacher, _J . Biol . Chem . U 248, 25-32 (1973) .Millonig, _J . Appl . Phys . _32, 1637 (1961) G J. H. Luft, J Bio h s . Biochem. Cytol . 9, 409-412 (1961) H. Murex andlJ . Ho , Proc . Nat . Acad .ici . USA 71, 484-488 (1974)
Pergamon Press
PURIFICATION OF INTESTINAL BRUSH BORDER MEMBRANE VESICLES BY THE USE OF CONTROLLED-PORE GLASS-BEADS COLUMN Kazuaki Ohsawa, Asako Kano and Takeshi Hoshi Department of Physiology, Faculty of Medicine University of Tokyo, Tokyo 113, Japan (Received in final form January 12, 1979) Summary A simple rapid method for obtaining highly purified and efficiently transporting intestinal brush border membrane vesicles was developed. The new method consists of two major steps : Ca-treatment of the homogenate of the scraped epithelium (rabbit ileum) and the subsequent chromatography of the supernatant of the Ca-treated homogenate through a controlledpore glass-beads column . The fraction showing the peak value of the optical density at 280 nm and two subsequent fractions were identified as those containing purified brush border membrane vesicles as judged from the activities of the marker enzymes (sucrase and alkaline phosphatase) and the rate of D-glucose uptake . Whole procedures could be completed in about 90 min. Specific activities of sucrase and alkaline phosphatase increased to 35 .5 and 34 times, respectively, while Na,KATPase activity decreased to one tenth as compared with the initial homogenate . Overshoot uptake of D-glucose in the presence of a NaSCN gradient showed a peak at 1-1 .5 min after the start of incubation, when it reached 10-40 nmoles/mg protein . Electron microscopic examination revealed that the highly purified vesicles had fairly homogeneous size, the average diameter being about 80 nm . Various methods have been published thus far for the isolation of highly purified intestinal brush border membranes, e .g ., hypotonic EDTA treatment followed by density gradient centrifugation (1), Tris-disruption procedure (2), free-flow electrophoresis (3), or the Ca-precipitation method (4,5) . For the study of transport, shortness of time required for whole preparation procedures and preservation of high transport function are needed . With this regard, the Ca-precipitation method modified by Kessler et al . (5) seems, at least at present, superior to the other methods, but the vesicles obtained by their method seem to be still inhomogeneous . In the present study, a further purification of vesicles obtained by the Ca-precipitation method was attempted without prolonging the time of preparation much . For this purpose, a controlled-pore glass-beads column chromatography (6) has been introduced, which enabled us to obtain much more homogeneous, highly purified and efficiently transporting brush border membrane vesicles . Such purified vesicles had an average diameter of 80 nm .
0300-9653/79/080669-09$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd .
67 0
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Vol . 24, No . 8,
1979
Materials and Methods Preparation of a "crude" brush border membrane fraction : Female rabbits, weighing from 1 .5-3 .0 Kg B .W ., were sacrificed by a blow on the neck, and the ileum was removed immediately. The gut was opened by a longitudinal cut and the mucosal surface was rinsed with ice-cold buffer solution (100 mM mannitol, 10 mM HEPES/Tris, pH 7 .4) . After wiping off the rinsing solution and excess mucus on the mucosal surface, the epithelial cells were gently scraped off with a slide glass . Usually, one to 1 .5 g mass could be obtained from one animal . The scraped material was suspended in 10 ml buffer solution containing 50 mM mannitol and 2 mM Tris/HC1 (pH 7 .4), and then homogenized in a blaring blendor at the maximum speed for 2 min . To this homogenate, powdered CaC12 was added to the final concentration of 10 mM . The homogenate was allowed to stand in ice for 15 min, thereafter it was centrifuged at 2,000 x ~ for 10 min. Large particles, such as nuclei and mitochondria are known to be precipitated by such a Ca-treatment followed by a low-speed centrifugation (4,5) . The supernatant, referred to as S1 here, was then centrifuged at 45,000 x ~ for 30 min . The pellet thus obtained is referred to as P2, which usually amounted about 0.3 g (wet .w) . Chromatography by using a controlled-pore glass-beads (CPG) column : The pellet, P2, was resuspended in 7 ml solution of 100 mM mannitol, 10 mM HEPES/ Tris (pH 7.4), and homogenized with a Potter-Elvehjem homogenizer. The homo genate was then chromatographed with a CPG-column . A glass column, 1 .6 cm in inner diameter and 100 cm long, was packed with controlled-pore glass-beads, (Electro-Nucleonics, Inc., pore diameter 300 nm) . Elution was carried out with the same solution as that used for resuspension . The flow rate was kept constant at 6 ml/min . The elution fluid was degassed with an evacuator before use. The eluent from the column was monitored for its W(280 nm) optical density with a W monitor (Hitachi spectrophotometer No . 124) . The void volume of the column, V0, and internal pore salt volume, Vi, were 35 and 45 ml, respectively . Calibration of the system was carried out with polystyrene latex beads of two different diameters, 87 and 106 nm . The effluent was fractionated every 4 ml and each fraction was numbered serially . Enz e assa s and other anal ical rocedures : As the marker enzymes of the brush bo er me raves, bot sucrase an al al ne phosphatase activities were determined . The former was assayed by the glucose-oxidase method (7), the latter by the method of Bessey et al . (8) . Isomaltase and maltase activities were also examined by incubating samples with the appropriate substrate and determining the glucose released by the glucose-oxidase method . As the marker of the basolateral membrane, Na,K-ATPase activity was measured (9,10), and, as the marker of the cytosol, lactate dehydrogenase was assayed (11) . ATP content was also determined by the luciferin-luciferase method (12) . Protein concentration was determined by Lowry's method (13) by using bovine serum albumin as a standard . Glucose uptake experiments : The uptake of D-glucose was measured with a Millipore-filtration technique in a manner similar to that described by Hopfer et al . ~(14) . Each fraction was centrifuged at 45,000 x & and the pellet was resuspended in 0.5 ml buffer solution (100 mM mannitol, HEPES/Tris 1 mM, pH 7 .5) . An aliquot of the preparation (protein amount 40-100 Ug) was incubated in 4-fold volume of the incubation solution containing 1 mM D-glucose, 1 .0 ~aCi /ml D-[14 C(U)] glucose, 100 mM mannitol, 1 mM HEPES/Tris (pH 7 .5), 4 uCi/ml D-(1-3H(N)] mannitol, and 100 mM NaSCN . The incubation was carried out at 20 ° C, and the uptake reaction was terminated by dilution of the reaction medium
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10-fold with ice-cold stop solution (150 mM NaCl plus 1 mM HEPES/Tris pH 7 .5) and immediate collection on a Millipore filter (Sartorius membrane filter, 11305, 0.6 um) . The filters were rinsed with 5 ml stop solution, then put into The radioactivities were counted minivials containing 3 ml Bray's solution . in a liquid scintillation counter (Beckman CPM-100 or Packard Tri Carb 2425) . When the uptake in the absence of a Na gradient was examined, 100 mM NaSCN in the incubation medium was replaced by 100 mM KC1, and 150 mM KC1 solution containing 1 mM HEPES/Tris was used as the stop solution . Electron microsco :The pellets of the fractions showing enriched sucrase an alkaline phosphatase activities were fixed with Millonig fixation reagent (15) and post-fixed with 1 $ Os04 for 1 hr each at 4°C . After dehydrat ion, the pellets were embedded in Epoxy resin . The thin sections were stained with uranylacetate and lead citrate (16) and examined with an electron microscope (Akashi 5-500) . SDS-polyacrylamide slab gel electrophoresis : The pellets of fractions to be examined were solubilized in a solution containing 58 mM Tris/HC1 (pH 6 .8), 71 mM nercapotoethanol and 2 $ sodium-dodecylsulfate (SDS) at 95 °C for 3 min. The slab gel system used and the conditions of the electrophoresis were the same as those described by Kassler et al . (5) . Fine discrimination of Relative mobilities of protein bands was carried out by using a densitometer . protein subunits were calculated by comparing with those of marker proteins of known molecular weight ; trypsin inhibitor (21,500), RNA polymerase a (39,000), bovine serum albumin (68,000), RNA polymerase ß (155,000) and ß' (165,000) . Chemicals and other materials : All chemicals used for the experiments were o the hig est g=ade available. Bovine serum albumin, glucose-oxidase reagent and tris-aminomethane sulfonate (Tris) were purchased from Sips Chemical All radioCo ., uniform latex styrene butadiene particles from Dow Chemical Co . . active compounds were obtained from New England Nuclear (Boston, Mass .), marker proteins for slab gel electrophoresis from Boehringer Mannheim (~bH Biochemica . Results 1 . Identification of fractions containing purified brush border membrane vesicles by enzyme assays . The tracing of optical density (OD) at 280 nm usually began to rise when the external elution volume reached 80 ml (Fraction 20) and formed a peak at the fraction 23 (elution volume 92 ml) . Thereafter, it declined smoothly as the eluted volume further increased (Fig . lA) . The protein concentration determined by Lowry's method changed almost in parallel with the OD curve . Alkaline phosphatase and sucrase activities changed in parallel with each other and their peak values were seen one or two fractions later than the protein or OD peak (Fig . 1B) . In contrast, Na,K-ATPase activity appeared in fractions earlier than the protein peak and formed a peak in the fraction immediately before the fraction of the protein peak . Little activity of Na,K-ATPase was seen in the fractions which had very high activities of sucrase and alkaline phosphatase (Fig . 1C) . Lactate dehydrogenase was absent in the fractions of enriched sucrase and alkaline phosphatase . From these enzyme patterns, the fraction of the OD peak and subsequent two or three fractions were taken as those containing purified brush border membrane vesicles . The data from enzyme assays for the fractions containing purified brush border membrane are summarized in Table 1, where protein concentration, specific activities and relative enrichment factors are shown for each purifi cation step : the initial homogenate, the supernatant Sl, the pellet of the
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Vol . 24, No . 8, 1979
S 6 â i
Fig. 1 An example of changes in the optical density (OD2g0), protein concentration and activities of various marker enzymes with increase in fraction number after a single run of the CPG column chromatography . A : Optical density and protein concentration (mg/ml), B: sucrase and alkaline phosphatase (tiimoles of substrate hydrolysed per min per mg protein), C : Na,K-ATPase ~uuoles of Pi produced per min per mg protein) . supernatant of Ca-treated homogenate P2, and the final -brush border membrane vesicle preparations (final BBM) . As seen in the table, the specific activities of sucrase and alkaline phosphatase were elevated by about 35 times and 34 times in the final preparations, respectively, as compared with those of the initial homogenate . Avery low Na,K-ATPase activity was seen in these fractions, but the enrichment factor was only 0.08 . As compared with the data reported by Kassler et al . (1978), the enrichment factors for the brush border membrane enzymes were about two times higher, whereas that for the basolateral membrane marker enzyme was much lower, about one twentieth . These indicate that the degree of purification is at least two-fold higher in the present method than that of Kassler et al . (5) and comparable with that of free-flow electrophoretic separation (3) . In some experiments, a second increase in sucrase activity was observed in the later fractions (Fractions 28-32, the data not shown) . In such fractions, protein concentrations were very low, so that the specific activity of this enzyme was very high ; 200-300 times higher than that in the initial homogenate . As the proteins could not be precipitated by the centrifugation at 45,000 x ~ for 60 min, the observed activity may be due to solubilized sucrase. Na-dependent D-glucose uptake was not seen in these fractions .
Vol . 24, No . 8,
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Purification of the Brush Border Membranes
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TABLE 1 Specific Activities of Enzymes during the Preparation of Brush Border Membrane Vesicles . Specific activity
Amount of protein ($)
Sucrase
Alkaline phosphatase
Na,K-ATPase
Lactate dehydrogenase
100
1 .3+0 .5
1 .04*0.5
17 .113 .4
1 .68
Homogenate $1
64*5 .4
17 .719 .3
2 .2711 .2
35 .71.4 .1
1 .68
P2
1212 .1
30 .819 .3
12 .410 .5
43 .2112.0
0 .73
Final BBM vesicle
0 .8310.05 46 .115 .7
35 .415 .8
1 .3610 .5
0 .03
34 .0116.4
0.0810 .06
0.02
Enrichment factor
35 .518 .5
Specific activity is given in hmoles/min/mg protein. Figures are given as meanlS .E . Number of determinations was 4 except for lactate dehydrogenase . The final BBM vesicles are Fraction 23 or 24 . The enrichment factor is the ratio of the specific activities in the final BBM vesicles and the initial homogenate . 2 . Identification by D-~ucose uptake D-glucose uptake was examined in the presence or absence of a NaSCN gradient at the start of incubation . A marked Na-dependent overshoot uptake was seen only in the fractions which had both high sucrase and high alkaline phos phatase activities . An example of the time course of D-glucose uptake was shown in Fig. 2 . The characteristics of uptake were essentially the same as those reported by previous investigators (3,5,14,17) . In the presence of a NaSCN gradient, the peak of overshoot uptake was seen usually 1-1 .5 min after the start of incubation, when it reached more than 10 nmoles/mg protein . In the absence of Na, e .g . in the presence of a KC1 or KSCN gradient, there was no overshoot and the time course of D-glucose uptake was almost the same as that of D-mannitol uptake . Smaller but distinct overshoot uptake of D-glucose was observed also in the later fractions, but the peak value of uptake rapidly decreased as the fraction number increased (Fig . 3) . The value of peak uptake of D-glucose was about two times larger than that obtained by Kessler et al . (5) and this is in good agreement with the 2-fold increase in purification as judged from enzyme data . In contrast, the peak uptake observed in the present study was much higher than that reported by Hopfer et al . (14), Murer and Hopfer (17), and Murer et al . (3), who prepared the BBM vesicles by repeated centrifugations or the free-flow electrophoresis . This suggests that Ca-precipitation and rapid separation of BBM vesicles by CPG-column is very effective in preserving the transport activity as compared with other separation methods .
674
Purification of the Brush Border Membranes
m
s
â
Vol . 24, No . 8,
1a
0
ô E o. 0
O
0 m n Y
m û
2
i g
4
i 10
i 8
12
Time ( min 1
14
18
Fig. 2 Typical example of time course of D-glucose uptake by purified BHM vesicles in the presence of a NaSCN gradient (open circles) . Time course of D-mannitol uptake by the same prepa ration is shown for the sake of comparison (closed circles) . 50c E u c
0.2Wm Filter
O.gymFihv
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Fraction number Fig . 3 Comparison of D-glucose uptake during the first one minute among various fractions. For the fractions of larger fraction numbers (Fractions 28-35), the membrane filter of smaller diameter (0 .2 um) was used since the average diameter of particles contained was expected to become smaller as fraction number increased.
1979
Vol . 24, No . 8,
Purification of the Brush Border Membranes
1979
675
3 . Electron microscopic observation Electron microscopic examinations of the pellets of the fractions which had high sucrase activities revealed that all the materials were in vesicular forms of fairly uniform sizes though some were spherical and some others ellip soidal . Three-layer membrane structure was clearly seen in most vesicles . No coagulated mass was seen . The results of measurements of diameters of vesicles are summarized in Fig . 4 . The mean values of diameter were 81, 78, 56 and 41 nm for the fractions 23, 24, 25 and 26, respectively . Thus, the size of vesicle became smaller as eluted volume increased . This indicates that the CPG-column chromatography effectively separates vesicles having different diameters . The vesicles which had the highest sucrase activity and D-glucose transport activity were those having diameter of 78f5 (meantS .D .) nm .
30
~:o
Fraction number
23
24
26 I`-Z~ 90 105 1Y0 135 Diameter 1 nm i
1-~.-~ ._ 1 . 30 "5 90 75
i
Fig. 4 Distribution of diameter of the vesicles in four different fractions (Fractions 23-26) . The diameter was measured on the ultra-thin section electron micrograph of the pellet of each fraction . In cases of ellipsoidal structures, the largest width in the horizontal direction was measured . Dotted vertical lines indicate the mean values of diameter .
676
Purification of the Brush Border Membranes
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1979
4 . SDS-polyac rylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis of the purified BHM preparations yielded about 40 bands of protein subunits, their molecular weight ranging from 10,000 to 200,000 . The number of protein bands was determined by densito metry (Fig . 5) . The electrophoresic pattern was quite similar to those obtained by other authors (4,5) .
Mdeculsr weight 185000
I "-155000
39000
21500
Fig . 5 The pattern of SDS-polyacrylamide gel electrophoresis of a purified BBM vesicle preparation . 8 $ polyacrylamide gel slab was used . The molecular weights calibrated with the standard proteins are indicated between the traced diagram and the densitometric curve. Discussion Most current methods of preparation of a certain membrane system from a cell homogenate are laborious and time-consuming because they require repeated ultracentrifugation in combination with density gradient centrifugation or the free-flow electrophoresis . As to the BHM of intestinal epithelial cells, Kessler et al . (5) developed a very simple method of purification by modifying the Ca-precipitation method of Schmitz et al . (4) . However, the BBM vesicles that they finally obtained seem still to be inhomogeneous . The present method includes the same Ca-precipitation procedure, but the subsequent chromatography by the use of controlled-pore glass-beads (CPG) column enabled us to obtain, within one and a half hour, much more uniform vesicles which had significantly higher transport activity and higher marker enzyme activities as compared with those obtained by Kessler et al . (Table 1 and Figs . 2 and 3) . CPG-chromatography possesses favorable properties which are suitable for isolating and purifying certain macromolecules and biological materials such as viruses (6) . The glass is inert to practically all chemicals, and hig~ pres-
Vol, 24, No . 8,
1979
Purification of the Brush Border Membranes
677
sure can be applied without any change of column bed or pore size . Welldefined pore size with its narrow distribution which is independent of the presence of elution fluid is quite useful for the application of the theory of chromatographic fractionation of a mixture of particles of different sizes (6) . Also, the CPG-column can be easily prepared and regenerated, and the time required for a single run of chromatography is very short (usually only about The sur15-20 min) . The surface of glass is known to be negatively charged . face of the BBM vesicles is also negatively charged as evidenced by electroTherefore, binding of vesicles to glass by electric force phoretic mobility . can be neglected and only molecular sieving or size-separation process is considered to take place in the present CPG-chromatography . The results of electron microscopic examination of pellets of fractions 23-26 revealed that the vesicles of different sizes were effectively separated and fractionated by this procedure . The results of enzyme assays and glucose uptake experiments indicate that the best preparation of the BBM vesicles which have very high transport activity Since the microvilli in vivo have relatively uniform diameters around 80 nm . have uniform diameters about 100 nm, it is easily anticipated that the BBM tends to form vesicles of about 80 nm in diameter when the cells are homogenized . In the present method, we adopted the Ca-precipitation method in the first This method has been shown to step in order to obtain crude BBM preparation . be effective in precipitating the granular components (nuclei and mitochondria) and other intracellular membrane systems (4,5) . This was confirmed by the presCalcium ent study as indicated by the data of enzyme assays for P2 (Table 1) . ion has been speculated to be effective in preserving the membrane permeability characteristics or transport function (4,5), but our additional observations disclosed that the transport or permeability properties of the BBM was independent of the presence of calcium . The details of the experimental results in this regard will be described in a subsequent paper.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 .
D. Miller and R. K. Crane, Anal . Biochem . 2, 284-286 (1961) A. Eichholz and R. K. Crane, J. Cell Biol .26, 687-692 (1965) H. Murex, U. Hopfen, E . Kinne-Sai~ran and R.Kinne, Biochim. Biophys. Acta 345, 170-179 (1974) .Schmitz, H . Presier, D . Maestrocci, B. K . Ghosh, J. J . Cerda and R. K. J Crane, Biochim. Biophys . Acta 323, 98-112 (1973) M . Kess erb Acuto, C . Storelli, H . Murex, M . Murex and G. Semenza, Biochim . Biophys . Acta 506, 136-154 (1978) W . Haller, Nature 206, 693-696 (1965) M . Messer and A. Dahlgvist, Anal . Biochem. 14, 376-392 (1965) A . 0 . Bessey, 0. H. Lowry and J . Brock, _J . Biol . Chem . _164, 321-329 (1946) J . P . Quigley and G. S . Gotterer, Biochim. Biophys . Acta 173, 456-468 (1969) J . B . Martin and M. Doty, Ann. Chemist . 21, 965-967 (1949 P . G . Cabaud and F . Wroblewski, Am . J . Clin . Path . 30, 234-236 (1958) P . E . Stanley and S. G. Williams,Anâl . Biochem . 29,381-392 (1969) . Randall, _J . Biol . Chem . 0 . H . Lowry, N . J . Rosebrouch, A. L, FarmR. J 193, 265-275 (1951) .Hopfen, K . Nelson, J. Perrotto and K. J. Isselbacher, _J . Biol . Chem . U 248, 25-32 (1973) .Millonig, _J . Appl . Phys . _32, 1637 (1961) G J. H. Luft, J Bio h s . Biochem. Cytol . 9, 409-412 (1961) H. Murex andlJ . Ho , Proc . Nat . Acad .ici . USA 71, 484-488 (1974)
