Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1968 Jan;106(2):381–390. doi: 10.1042/bj1060381

Rat intestinal microvillus membranes. Purification and biochemical characterization

G G Forstner 1,*, S M Sabesin 1, K J Isselbacher 1
PMCID: PMC1198514  PMID: 4295331

Abstract

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, Mg2+- and Ca2+-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.

Full text

PDF
381

Images in this article

386-1PLATE 1
on p.386-1
386-2PLATE 2
on p.386-2

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  2. CROFT D. N., LUBRAN M. THE ESTIMATION OF DEOXYRIBONUCLEIC ACID IN THE PRESENCE OF SIALIC ACID: APPLICATION TO ANALYSIS OF HUMAN GASTRIC WASHINGS. Biochem J. 1965 Jun;95:612–620. doi: 10.1042/bj0950612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. DAHLQVIST A. METHOD FOR ASSAY OF INTESTINAL DISACCHARIDASES. Anal Biochem. 1964 Jan;7:18–25. doi: 10.1016/0003-2697(64)90115-0. [DOI] [PubMed] [Google Scholar]
  4. DISCHE Z. Qualitative and quantitative colorimetric determination of heptoses. J Biol Chem. 1953 Oct;204(2):983–997. [PubMed] [Google Scholar]
  5. Eichholz A. Structural and functional organization of the brush border of intestinal epithelial cells. 3. Enzymic activities and chemical composition of various fractions of tris-disrupted brush borders. Biochim Biophys Acta. 1967 Jul 3;135(3):475–482. doi: 10.1016/0005-2736(67)90037-5. [DOI] [PubMed] [Google Scholar]
  6. FIELD J. B., EPSTEIN S., EGAN T. STUDIES IN GLYCOGEN STORAGE DISEASES. I. INTESTINAL GLUCOSE-6-PHOSPHATASE ACTIVITY IN PATIENTS WITH VON GIERKE'S DISEASE AND THEIR PARENTS. J Clin Invest. 1965 Jul;44:1240–1247. doi: 10.1172/JCI105230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. FLECK A., MUNRO H. N. The precision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochim Biophys Acta. 1962 May 14;55:571–583. doi: 10.1016/0006-3002(62)90836-3. [DOI] [PubMed] [Google Scholar]
  8. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  9. Forstner G. G., Riley E. M., Daniels S. J., Isselbacher K. J. Demonstration of glyceride synthesis by brush borders of intestinal epithelial cells. Biochem Biophys Res Commun. 1965 Oct 8;21(1):83–88. doi: 10.1016/0006-291x(65)90430-4. [DOI] [PubMed] [Google Scholar]
  10. HARRISON D. D., WEBSTER H. AN IMPROVED METHOD FOR THE ISOLATION OF BRUSH BORDERS FROM THE RAT INTESTINE. Biochim Biophys Acta. 1964 Dec 9;93:662–664. doi: 10.1016/0304-4165(64)90354-x. [DOI] [PubMed] [Google Scholar]
  11. HOLT J. H., MILLER D. The localization of phosphomonoesterase and aminopeptidase in brush borders isolated from intestinal epithelial cells. Biochim Biophys Acta. 1962 Apr 9;58:239–243. doi: 10.1016/0006-3002(62)91004-1. [DOI] [PubMed] [Google Scholar]
  12. HUEBSCHER G., WEST G. R. SPECIFIC ASSAYS OF SOME PHOSPHATASES IN SUBCELLULAR FRACTIONS OF SMALL INTESTINAL MUCOSA. Nature. 1965 Feb 20;205:799–800. doi: 10.1038/205799a0. [DOI] [PubMed] [Google Scholar]
  13. Heimberg M., Weinstein I., Dishmon G., Fried M. Lipoprotein lipid transport by livers from normal and CCl-4-poisoned animals. Am J Physiol. 1965 Nov;209(5):1053–1060. doi: 10.1152/ajplegacy.1965.209.5.1053. [DOI] [PubMed] [Google Scholar]
  14. Hübscher G., West G. R., Brindley D. N. Studies on the fractionation of mucosal homogenates from the small intestine. Biochem J. 1965 Dec;97(3):629–642. doi: 10.1042/bj0970629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnson C. F. Disaccharidase: localization in hamster intestine brush borders. Science. 1967 Mar 31;155(3770):1670–1672. doi: 10.1126/science.155.3770.1670. [DOI] [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. MCDOUGAL D. B., Jr, LITTLE K. D., CRANE R. K. Studies on the me hanism of intestinal absorption of sugars. IV. Localization of galactose concenhrations within the intestinal wall during active transport, in vitro. Biochim Biophys Acta. 1960 Dec 18;45:483–489. doi: 10.1016/0006-3002(60)91484-0. [DOI] [PubMed] [Google Scholar]
  18. MILLER D., CRANE R. K. The digestive function of the epithelium of the small intestine. I. An intracellular locus of disaccharide and sugar phosphate ester hydrolysis. Biochim Biophys Acta. 1961 Sep 16;52:281–293. doi: 10.1016/0006-3002(61)90677-1. [DOI] [PubMed] [Google Scholar]
  19. MILLINGTON P. F. COMPARISON OF THE THICKNESSES OF THE LATERAL WALL MEMBRANE AND THE MICROVILLUS MEMBRANE OF INTESTINAL EPITHELIAL CELLS FROM RAT AND MOUSE. J Cell Biol. 1964 Mar;20:514–517. doi: 10.1083/jcb.20.3.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. MILLINGTON P. F., FINEAN J. B. Electron microscope studies of the structure of the microvilli on principal epithelial cells of rat jejunum after treatment in hypo- and hypertonic saline. J Cell Biol. 1962 Jul;14:125–139. doi: 10.1083/jcb.14.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. NACHLAS M. M., MONIS B., ROSENBATT D., SELIGMAN A. M. Improvement in the histochemical localization of leucine aminopeptidase with a new substrate, L-leucyl-4-methoxy-2-naphthylamide. J Biophys Biochem Cytol. 1960 Apr;7:261–264. doi: 10.1083/jcb.7.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. NEWEY H., PARSONS B. J., SMYTH D. H. The site of action of phlorrhizin in inhibiting intestinal absorption of glucose. J Physiol. 1959 Oct;148:83–92. doi: 10.1113/jphysiol.1959.sp006274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. PALAY S. L., KARLIN L. J. An electron microscopic study of the intestinal villus. I. The fasting animal. J Biophys Biochem Cytol. 1959 May 25;5(3):363–372. doi: 10.1083/jcb.5.3.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. PHILIPSON L., BENGTSSON S., BRISHAMMAR S., SVENNERHOLM L., ZETTERQVIST O. PURIFICATION AND CHEMICAL ANALYSIS OF THE ERYTHROCYTE RECEPTOR FOR HEMAGGLUTINATING ENTEROVIRUSES. Virology. 1964 Apr;22:580–590. doi: 10.1016/0042-6822(64)90080-7. [DOI] [PubMed] [Google Scholar]
  25. PHILIPSON L., ZETTERQVIST O. THE PRESENCE OF DNA IN HUMAN ERYTHROCYTE MEMBRANES. Biochim Biophys Acta. 1964 Sep 11;91:171–173. doi: 10.1016/0926-6550(64)90185-9. [DOI] [PubMed] [Google Scholar]
  26. PORTEOUS J. W., CLARK B. THE ISOLATION AND CHARACTERIZATION OF SUBCELLULAR COMPONENTS OF THE EPITHELIAL CELLS OF RABBIT SMALL INTESTINE. Biochem J. 1965 Jul;96:159–171. doi: 10.1042/bj0960159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. PUCHTLER H., LEBLOND C. P. Histochemical analysis of cell membranes and associated structures as seen in the intestinal epithelium. Am J Anat. 1958 Jan;102(1):1–31. doi: 10.1002/aja.1001020102. [DOI] [PubMed] [Google Scholar]
  28. VENABLE J. H., COGGESHALL R. A SIMPLIFIED LEAD CITRATE STAIN FOR USE IN ELECTRON MICROSCOPY. J Cell Biol. 1965 May;25:407–408. doi: 10.1083/jcb.25.2.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wins P., Schoffeniels E. Studies on red-cell ghost ATPase systems: properties of a (Mg2+ + Ca2+)-dependent ATPase. Biochim Biophys Acta. 1966 Jul 13;120(3):341–350. doi: 10.1016/0926-6585(66)90301-3. [DOI] [PubMed] [Google Scholar]
  30. ZLATKIS A., ZAK B., BOYLE A. J. A new method for the direct determination of serum cholesterol. J Lab Clin Med. 1953 Mar;41(3):486–492. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES