Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1966 Sep 1;30(3):519–530. doi: 10.1083/jcb.30.3.519

DISTRIBUTION OF NEWLY SYNTHESIZED AMYLASE IN MICROSOMAL SUBFRACTIONS OF GUINEA PIG PANCREAS

P Siekevitz 1, G E Palade 1
PMCID: PMC2107018  PMID: 5971004

Abstract

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).1 Amylase appeared in the following positions in the gradient: (a) a light region which contained ∼35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained ∼10% of enzymic activity in a sharp peak but which had very little accompanying OD260 absorption; (c) a pellet at the bottom of the centrifuge tube which contained ∼20% of the enzymic activity. After 5 to 20 min' in vivo labeling with leucine-1-C14, radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized ∼80% of the total amylase in the fraction from the heavy region of the gradient, but only ∼20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Aronson A. Membrane-bound messenger RNA and polysomes in sporulating bacteria. J Mol Biol. 1965 Aug;13(1):92–104. doi: 10.1016/s0022-2836(65)80082-1. [DOI] [PubMed] [Google Scholar]
  2. BECKER Y., PENMAN S., DARNELL J. E. A CYTOPLASMIC PARTICULATE INVOLVED IN POLIOVIRUS SYNTHESIS. Virology. 1963 Oct;21:274–276. doi: 10.1016/0042-6822(63)90271-x. [DOI] [PubMed] [Google Scholar]
  3. Campbell P. N., Cooper C., Hicks M. Studies on the role of the morphological constituents of the microsome fraction from rat liver in protein synthesis. Biochem J. 1964 Aug;92(2):225–234. doi: 10.1042/bj0920225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. GIERER A. Function of aggregated reticulocyte ribosomes in protein synthesis. J Mol Biol. 1963 Feb;6:148–157. doi: 10.1016/s0022-2836(63)80131-x. [DOI] [PubMed] [Google Scholar]
  5. HENDLER R. W. A model for protein synthesis. Nature. 1962 Mar 3;193:821–823. doi: 10.1038/193821a0. [DOI] [PubMed] [Google Scholar]
  6. HENDLER R. W., BANFIELD W. G., TANI J., KUFFEL ON THE CYTOLOGICAL UNIT FOR PROTEIN SYNTHESIS IN VIVO IN E. COLI. III. ELECTRON MICROSCOPIC AND ULTRACENTRIFUGAL EXAMINATION OF INTACT CELLS AND FRACTIONS. Biochim Biophys Acta. 1964 Feb 17;80:307–314. [PubMed] [Google Scholar]
  7. HENDLER R. W., TANI J. ON THE CYTOLOGICAL UNIT FOR PROTEIN SYNTHESIS IN VIVO IN E. COLI. II. STUDIES WITH INTACT CELLS OF TYPE B. Biochim Biophys Acta. 1964 Feb 17;80:294–306. doi: 10.1016/0926-6550(64)90101-x. [DOI] [PubMed] [Google Scholar]
  8. HENSHAW E. C., BOJARSKI T. B., HIATT H. H. PROTEIN SYNTHESIS BY FREE AND BOUND RAT LIVER RIBOSOMES IN VIVO AND IN VITRO. J Mol Biol. 1963 Aug;7:122–129. doi: 10.1016/s0022-2836(63)80041-8. [DOI] [PubMed] [Google Scholar]
  9. HOWELL R. R., LOEB J. N., TOMKINS G. M. CHARACTERIZATION OF RIBOSOMAL AGGREGATES ISOLATED FROM LIVER. Proc Natl Acad Sci U S A. 1964 Nov;52:1241–1248. doi: 10.1073/pnas.52.5.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. HUNTER G. D., BROOKES P., CRATHORN A. R., BUTLER J. A. Intermediate reactions in protein synthesis by the isolated cytoplasmic-membrane fraction of Bacillus megaterium. Biochem J. 1959 Oct;73:369–376. doi: 10.1042/bj0730369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. HUNTER G. D., GODSON G. N. Later stages of protein synthesis and the role of phospholipids in the process. Nature. 1961 Jan 14;189:140–141. doi: 10.1038/189140a0. [DOI] [PubMed] [Google Scholar]
  12. Jamieson J. D., Palade G. E. Role of the Golgi complex in the intracellular transport of secretory proteins. Proc Natl Acad Sci U S A. 1966 Feb;55(2):424–431. doi: 10.1073/pnas.55.2.424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LOYTER A., SCHRAMM M. The glycogen-amylase complex as a means of obtaining highly purified alpha-amylases. Biochim Biophys Acta. 1962 Dec 4;65:200–206. doi: 10.1016/0006-3002(62)91039-9. [DOI] [PubMed] [Google Scholar]
  14. Marks P. A., Burka E. R., Schlessinger D. PROTEIN SYNTHESIS IN ERYTHROID CELLS, I. RETICULOCYTE RIBOSOMES ACTIVE IN STIMULATING AMINO ACID INCORPORATION. Proc Natl Acad Sci U S A. 1962 Dec;48(12):2163–2171. doi: 10.1073/pnas.48.12.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Morgan C., Rosenkranz H. S., Chan B., Rose H. M. Electron microscopy of magnesium-depleted bacteria. J Bacteriol. 1966 Feb;91(2):891–895. doi: 10.1128/jb.91.2.891-895.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. PALADE G. E., SIEKEVITZ P. Pancreatic microsomes; an integrated morphological and biochemical study. J Biophys Biochem Cytol. 1956 Nov 25;2(6):671–690. doi: 10.1083/jcb.2.6.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. PENMAN S., BECKER Y., DARNELL J. E. A CYTOPLASMIC STRUCTURE INVOLVED IN THE SYNTHESIS AND ASSEMBLY OF POLIOVIRUS COMPONENTS. J Mol Biol. 1964 Apr;8:541–555. doi: 10.1016/s0022-2836(64)80010-3. [DOI] [PubMed] [Google Scholar]
  18. PETERS T., Jr The biosynthesis of rat serum albumin. II. Intracellular phenomena in the secretion of newly formed albumin. J Biol Chem. 1962 Apr;237:1186–1189. [PubMed] [Google Scholar]
  19. Redman C. M., Siekevitz P., Palade G. E. Synthesis and transfer of amylase in pigeon pancreatic micromosomes. J Biol Chem. 1966 Mar 10;241(5):1150–1158. [PubMed] [Google Scholar]
  20. SIEKEVITZ P., PALADE G. E. A cytochemical study on the pancreas of the guinea pig. 5. In vivo incorporation of leucine-1-C14 into the chymotrypsinogen of various cell fractions. J Biophys Biochem Cytol. 1960 Jul;7:619–630. doi: 10.1083/jcb.7.4.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. SIEKEVITZ P., PALADE G. E. A cytochemical study on the pancreas of the guinea pig. 6. Release of enzymes and ribonucleic acid from ribonucleoprotein particles. J Biophys Biochem Cytol. 1960 Jul;7:631–644. doi: 10.1083/jcb.7.4.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. SIEKEVITZ P., PALADE G. E. Cytochemical study on the pancreas of the guinea pig. VII. Effects of spermine on ribosomes. J Cell Biol. 1962 May;13:217–232. doi: 10.1083/jcb.13.2.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. TANI J., HENDLER R. W. ON THE CYTOLOGICAL UNIT FOR PROTEIN SYNTHESIS IN VIVO IN E. COLI. I. STUDIES WITH SPHEROPLASTS OF TYPE K-12. Biochim Biophys Acta. 1964 Feb 17;80:279–293. doi: 10.1016/0926-6550(64)90100-8. [DOI] [PubMed] [Google Scholar]
  24. TOMASZ A., JAMIESON J. D., OTTOLENGHI E. THE FINE STRUCTURE OF DIPLOCOCCUS PNEUMONIAE. J Cell Biol. 1964 Aug;22:453–467. doi: 10.1083/jcb.22.2.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. WARNER J. R., KNOPF P. M., RICH A. A multiple ribosomal structure in protein synthesis. Proc Natl Acad Sci U S A. 1963 Jan 15;49:122–129. doi: 10.1073/pnas.49.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. WEBB T. E., BLOBEL G., POTTER V. R. POLYRIBOSOMES IN RAT TISSUES. I. A STUDY OF IN VIVO PATTERNS IN LIVER AND HEPATOMAS. Cancer Res. 1964 Aug;24:1229–1237. [PubMed] [Google Scholar]
  27. WETTSTEIN F. O., STAEHELIN T., NOLL H. Ribosomal aggregate engaged in protein synthesis: characterization of the ergosome. Nature. 1963 Feb 2;197:430–435. doi: 10.1038/197430a0. [DOI] [PubMed] [Google Scholar]
  28. Warner J. R., Rich A., Hall C. E. Electron Microscope Studies of Ribosomal Clusters Synthesizing Hemoglobin. Science. 1962 Dec 28;138(3548):1399–1403. doi: 10.1126/science.138.3548.1399. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES