Summary
Kinetic data in (brush-border) membrane vesicles which rely on the validity of the initial rate assumption for their interpretation and depend on tracer flux studies using the rapid filtration technique for their experimental measurement have been limited to some extent by the absence of techniques that would allow for real-time data analysis. In this paper, we report on our successful design of a fast sampling, rapid filtration apparatus (FSRFA) which seems to fill up this technical gap since showing the following characteristics: (i) rapid injection (5 msec) and mixing (less than 100 msec) of small amounts of vesicles (10–40 μl) with an incubation medium (0.2–1.0 ml); (ii) fast (20 to 80 msec depending on the sample volume) and multiple (up to 18 samples at a maximal rate of 4/sec) sampling of the uptake mixture followed by rapid quenching in the stop solution (approximately 5 msec) according to a predetermined time schedule (any time combination from 0.25 to 9999 sec); and (iii) fast, automated, and sampling-synchronized filtration and washings of the quenched uptake medium (only 15–20 sec are necessary for the first filtration followed by two washings and extra filtrations). As demonstrated using adult human jejunal brush-border membrane vesicles and Na+-d-glucose cotransport as models, the FSRFA accurately reproduces the manual aspects of the rapid filtration technique while allowing for very precise initial rate determinations. Moreover, the FSRFA has also been designed to provide as much versatility as possible and, in its present version, allows for a very precise control of the incubation temperature and also permits a few efflux protocols to be performed. Finally, its modular design, which separates the fast sampling unit from the rapid filtration device, should help in extending its use to fields other than transport measurement.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aronson, P.S. 1981. Identifying secondary active solute transport in epithelia.Am. J. Physiol. 240:F1-F11
Berteloot, A. 1984. Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles.Biochim. Biophys. Acta 775:129–140
Berteloot, A. 1986. Membrane potential dependency of glutamic acid transport in rabbit jejunal brush-border membrane vesicles: K+ and H+ effects.Biochim. Biophys. Acta 857:180–188
Berteloot, A., Semenza, G. 1990. Advantages and limitations of vesicles for the characterization and the kinetic analysis of transport systems.In: Methods in Enzymology. Biomembranes. Part W. Cellular and Subcellular Transport: Epithelial Cells. Vol. 192. pp. 409–437. S. Fleischer and B. Fleischer, editors. Academic, Orlando, FL
Berteloot, A., Vidal, H., Larue, M.J., Van De Werwe, G. 1991. Rapid kinetics of liver microsomal glucose-6-phosphatase: I. Evidence for tight-coupling between glucose-6-phosphate transport and phosphohydrolase activity.J. Biol. Chem. (in press)
Bluett, M.K., Abumrad, N.N., Arab, N., Ghishan, F.K. 1986. Aboral changes ind-glucose transport by human intestinal brush-border membrane vesicles.Biochem. J. 237:229–234
Busse, D. 1978. Transport ofl-arginine in brush border vesicles derived from rabbit kidney cortex.Arch. Biochem. Biophys. 191:551–560
Champeil, P., Guillain, F. 1986. Rapid filtration study of the phosphorylation-dependent dissociation of calcium from transport sites of purified sarcoplasmic reticulum ATPase and ATP modulation of the catalytic cycle.Biochemistry 25:7023–7033
Dorando, F.C., Crane, R.K. 1984. Studies of the kinetics of Na+ gradient-coupled glucose transport as found in brush-border membrane vesicles from rabbit jejunum.Biochim. Biophys. Acta 772:273–287
Dupont, Y. 1984. A rapid-filtration technique for membrane fragments or immobilized enzymes: Measurements of substrate binding or ion fluxes with a few-millisecond time resolution.Anal. Biochem. 142:504–510
Forbush, B., III 1984. An apparatus for rapid kinetics analysis of isotopic efflux from membrane vesicles and of ligand dissociation from membrane proteins.Anal. Biochem. 140:495–505
Gasco, O.D., Knowles, A.F., Shertzer, H.G., Suolinna, E.M., Racker, E. 1976. The use of ion-exchange resins for studying ion transport in biological systems.Anal. Biochem. 72:57–65
Grunhagen, H.H. 1980. Fast tracer efflux from membrane vesicles: Investigation by controlled elution.Anal. Biochem. 109:18–26
Harig, J.M., Barry, J.A., Rajendran, V.M., Soergel, K.H., Ramaswamy, K. 1989.d-glucose andl-leucine transport by human intestinal brush border membrane vesicles.Am. J. Physiol. 256:G618-G623
Hopfer, U., Nelson, K., Perrotto, J., Isselbacher, K.J. 1973. Glucose transport in isolated brush-border membrane from rat small intestine.J. Biol. Chem. 248:25–32
Kessler, M., Tannenbaum, V., Tannenbaum, C. 1978. A simple apparatus for performing short-time (1–2 seconds) uptake measurements in small volumes: its application tod-glucose transport studies in brush border vesicles from rabbit jejunum and ileum.Biochim. Biophys. Acta 509:348–359
Kunst, A., Draeger, B., Ziegenhorn, J. 1984. UV-methods with hexokinase and glucose-6-phosphate dehydrogenase.In: Methods of Enzymatic Analysis. (3rd ed.) Vol. 6, pp. 163–172. Metabolites 1: Carbohydrates. H.U., Bergmeyer, editor. Verlag Chemie, Weinheim
Lever, J.E. 1980. The use of membrane vesicles in transport studies.CRC Crit. Rev. Biochem. 7:187–246
Lucke, H., Berner, W., Menge, H., Murer, H. 1978. Sugar transport by brush border membrane vesicles isolated from human small intestine.Pfluegers Arch. 373:243–248
Malo, C. 1988. Kinetic evidence for heterogeneity in Na+-d-glucose cotransport systems in the normal human fetal small intestine.Biochim. Biophys. Acta 938:181–188
Malo, C., Berteloot, A. 1991. Analysis of kinetic data in transport studies: New insights from kinetic studies of Na+-d-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus.J. Membrane Biol. 122:
Murer, H., Biber, J., Gmaj, P., Stieger, B. 1984. Cellular mechanisms in epithelial transport: Advantages and disadvantages of studies with vesicles.Mol. Physiol. 6:55–82
Murer, H., Gmaj, P. 1986. Transport studies in plasma membrane vesicles isolated from renal cortex.Kidney Int. 30:171–186
Otsu, K., Kinsella, J., Sacktor, B., Froehlich, J.P. 1989. Transient state kinetic evidence for an oligomer in the mechanism of Na+−H+ exchange.Proc. Natl. Acad. Sci. USA 86:4818–4822
Paraschos, A., Gonzales-Ros, J.M., Martinez-Carrion, M. 1983. Absorption filtration. A tool for the measurement of ion tracer flux in native membranes and reconstituted lipid vesicles.Biochim. Biophys. Acta 733:223–233
Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J., Crane, R.K. 1973. Purification of the human intestinal brush border membrane.Biochim. Biophys. Acta 323:98–112
Semenza, G., Kessler, M., Hosang, M., Weber, J., Schmidt, U. 1984. Biochemistry of the Na+,d-glucose cotransporter of the small-intestinal brush-border membrane. The state of the art in 1984.Biochim. Biophys. Acta 779:343–379
Torrent-Quetglas, M. 1985. Oil-stop method as an alternative to filtration for transport studies on plasma membrane vesicles. A preliminary report.Rev. Esp. Fisiol. 41:177–182
Turner, R.J. 1983. Quantative studies of cotransport systems: Models and vesicles.J. Membrane Biol. 76:1–15
Wierzbicki, W., Berteloot, A., Roy, G. 1990. Presteady-state kinetics and carrier-mediated transport: A theoretical analysis.J. Membrane Biol. 117:11–27
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Berteloot, A., Malo, C., Breton, S. et al. Fast sampling, rapid filtration apparatus: Principal characteristics and validation from studies ofd-glucose transport in human jejunal brush-border membrane vesicles. J. Membrain Biol. 122, 111–125 (1991). https://doi.org/10.1007/BF01872635
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01872635