Abstract
A technique is described for the isolation of a plasma-membrane fraction from the rat intestinal epithelial cell which is distinct from the microvillus membrane of that cell. The isolated fraction contains only about 0.2% of the sucrase activity in the original homogenate and negligible quantities of nuclear and mitochondrial membrane markers. It contains 12% of the total Na+,K+-dependent adenosine triphosphatase and 7% of the alkaline phosphatase, with significant increments in specific activity of these enzymes. Multiple membrane preparations were highly reproducible with respect to the specific activities of the markers studied. The small intestine of one rat yields material containing about 1.3mg of protein. In addition an assay is described suitable for determining 5′-nucleotidase in the small intestine.
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- AUTILIO L. A., NORTON W. T., TERRY R. D. THE PREPARATION AND SOME PROPERTIES OF PURIFIED MYELIN FROM THE CENTRAL NERVOUS SYSTEM. J Neurochem. 1964 Jan;11:17–27. doi: 10.1111/j.1471-4159.1964.tb06719.x. [DOI] [PubMed] [Google Scholar]
- Avruch J., Wallach D. F. Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochim Biophys Acta. 1971 Apr 13;233(2):334–347. doi: 10.1016/0005-2736(71)90331-2. [DOI] [PubMed] [Google Scholar]
- BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
- Barry R. J. Electrical changes in relation to transport. Br Med Bull. 1967 Sep;23(3):266–269. doi: 10.1093/oxfordjournals.bmb.a070569. [DOI] [PubMed] [Google Scholar]
- Belfield A., Ellis G., Goldberg D. M. A specific colorimetric 5'-nucleotidase assay utilizing the Berthelot reaction. Clin Chem. 1970 May;16(5):396–401. [PubMed] [Google Scholar]
- Berg G. G., Chapman B. The sodium and potassium activated ATPase of intestinal epithelium. I. Location of enzymatic activity in the cell. J Cell Physiol. 1965 Jun;65(3):361–372. doi: 10.1002/jcp.1030650309. [DOI] [PubMed] [Google Scholar]
- Berman H. M., Gram W., Spirtes M. A. An improved, reproducible method opreparing rat liver plasma cell membranes in buffered isotonic sucrose. Biochim Biophys Acta. 1969 Jun 3;183(1):10–18. doi: 10.1016/0005-2736(69)90124-2. [DOI] [PubMed] [Google Scholar]
- Bramhall S., Noack N., Wu M., Loewenberg J. R. A simple colorimetric method for determination of protein. Anal Biochem. 1969 Oct 1;31(1):146–148. doi: 10.1016/0003-2697(69)90251-6. [DOI] [PubMed] [Google Scholar]
- CLARK S. L., Jr The localization of alkaline phosphatase in tissues of mice, using the electron microscope. Am J Anat. 1961 Jul;109:57–83. doi: 10.1002/aja.1001090106. [DOI] [PubMed] [Google Scholar]
- Clark M. L., Lanz H. C., Senior J. R. Enzymatic distinction of rat intestinal cell brush border and endoplasmic reticular membranes. Biochim Biophys Acta. 1969 Jun 3;183(1):233–235. doi: 10.1016/0005-2736(69)90147-3. [DOI] [PubMed] [Google Scholar]
- 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]
- EMMELOT P., BOS C. J., BENEDETTI E. L., RUEMKE P. STUDIES ON PLASMA MEMBRANES. I. CHEMICAL COMPOSITION AND ENZYME CONTENT OF PLASMA MEMBRANES ISOLATED FROM RAT LIVER. Biochim Biophys Acta. 1964 Jul 15;90:126–145. doi: 10.1016/0304-4165(64)90125-4. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Forstner G. G., Sabesin S. M., Isselbacher K. J. Rat intestinal microvillus membranes. Purification and biochemical characterization. Biochem J. 1968 Jan;106(2):381–390. doi: 10.1042/bj1060381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Forstner G. G., Tanaka K., Isselbacher K. J. Lipid composition of the isolated rat intestinal microvillus membrane. Biochem J. 1968 Aug;109(1):51–59. doi: 10.1042/bj1090051. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fujita M., Matsui H., Nagano K., Nakao M. Asymmetric distribution of ouabain-sensitive ATPase activity in rat intestinal mucosa. Biochim Biophys Acta. 1971 Apr 13;233(2):404–408. doi: 10.1016/0005-2736(71)90337-3. [DOI] [PubMed] [Google Scholar]
- GINSBURG V., HERS H. G. On the conversion of fructose to glucose by guinea pig intestine. Biochim Biophys Acta. 1960 Mar 11;38:427–434. doi: 10.1016/0006-3002(60)91278-6. [DOI] [PubMed] [Google Scholar]
- Glynn I. M. Membrane adenosine triphosphatase and cation transport. Br Med Bull. 1968 May;24(2):165–169. doi: 10.1093/oxfordjournals.bmb.a070620. [DOI] [PubMed] [Google Scholar]
- 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]
- KOEFOED-JOHNSEN V., USSING H. H. The nature of the frog skin potential. Acta Physiol Scand. 1958 Jun 2;42(3-4):298–308. doi: 10.1111/j.1748-1716.1958.tb01563.x. [DOI] [PubMed] [Google Scholar]
- Korn E. D. Current concepts of membrane structure and function. Fed Proc. 1969 Jan-Feb;28(1):6–11. [PubMed] [Google Scholar]
- Latner A. L., Parsons M., Skillen A. W. Isoelectric focusing of alkaline phosphatases from human kidney and calf intestine. Enzymologia. 1971 Jan 31;40(1):1–7. [PubMed] [Google Scholar]
- Le Pecq J. B., Paoletti C. A new fluorometric method for RNA and DNA determination. Anal Biochem. 1966 Oct;17(1):100–107. doi: 10.1016/0003-2697(66)90012-1. [DOI] [PubMed] [Google Scholar]
- Lygre D. G., Nordlie R. C. Phosphohydrolase and phosphotransferase activities of intestinal glucose 6-phosphatase. Biochemistry. 1968 Sep;7(9):3219–3226. doi: 10.1021/bi00849a026. [DOI] [PubMed] [Google Scholar]
- 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]
- OTSUKA S., KOBAYASHI Y. RADIOISOTOPIC ASSAY FOR MONOAMINE OXIDASE DETERMINATIONS IN HUMAN PLASMA. Biochem Pharmacol. 1964 Jul;13:995–1006. doi: 10.1016/0006-2952(64)90096-6. [DOI] [PubMed] [Google Scholar]
- PENNINGTON R. J. Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J. 1961 Sep;80:649–654. doi: 10.1042/bj0800649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Quigley J. P., Gotterer G. S. Distribution of (Na+-K+)-stimulated ATPase activity in rat intestinal mucosa. Biochim Biophys Acta. 1969 Apr;173(3):456–468. doi: 10.1016/0005-2736(69)90010-8. [DOI] [PubMed] [Google Scholar]
- Rosenberg I. H., Rosenberg L. E. Localization and characterization of adenosine triphosphatase in guinea pig intestinal epithelium. Comp Biochem Physiol. 1968 Mar;24(3):975–985. doi: 10.1016/0010-406x(68)90809-8. [DOI] [PubMed] [Google Scholar]
- Schachter D., Kowarski S., Finkelstein J. D., Ma R. I. Tissue concentration differences during active transport of calcium by intestine. Am J Physiol. 1966 Nov;211(5):1131–1136. doi: 10.1152/ajplegacy.1966.211.5.1131. [DOI] [PubMed] [Google Scholar]
- Skipski V. P., Barclay M., Archibald F. M., Terebus-Kekish O., Reichman E. S., Good J. J. Lipid composition of rat liver cell membranes. Life Sci. 1965 Sep;4(17):1673–1680. doi: 10.1016/0024-3205(65)90213-4. [DOI] [PubMed] [Google Scholar]
- Stein Y., Widnell C., Stein O. Acylation of lysophosphatides by plasma membrane fractions of rat liver. J Cell Biol. 1968 Oct;39(1):185–192. doi: 10.1083/jcb.39.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stern B. K. Some biochemical properties of suspensions of intestinal epithelial cells. Gastroenterology. 1966 Nov;51(5):855–867. [PubMed] [Google Scholar]
- TAYLOR C. B. Cation-stimulation of an ATPase system from the intestinal mucosa of the guinea-pig. Biochim Biophys Acta. 1962 Jul 2;60:437–440. doi: 10.1016/0006-3002(62)90429-8. [DOI] [PubMed] [Google Scholar]
- Trier J. S. Structure of the mucosa of the small intestine as it relates to intestinal function. Fed Proc. 1967 Sep;26(5):1391–1404. [PubMed] [Google Scholar]
- 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]




