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. 1976 Sep 15;158(3):513–527. doi: 10.1042/bj1580513a

Neuronal N-acetyl-beta-D-glucosaminidase. Evidence for its biosynthesis in vitro.

J A Khawaja, O Z Sellinger
PMCID: PMC1164005  PMID: 985446

Abstract

Neuronal cell bodies, isolated in bulk from 8-day-old rat cerebral cortices, were incubated in the presence of a 3H-labelled amino acid mixture, and subcellular fractions isolated by differential centrifugation. The particulate fractions were frozen/thawed in 0.20 M-sucrose/0.1 M-KCl [Selling et al. (1973) Biochim. Biophys. Acta 315, 128-146] and the profiles of acid-insoluble radioactivity and N-acetyl-beta-D-glucosaminidase (glucosaminidase) activity compared in the resulting non-sedimentable fractions by DEAE-cellulose chromatography and cellulose acetate electrophoresis. Radioactivity and glucosaminidase activity co-migrated to a significant extent. Electrophoresis revealed that after 1 min of incubation 42% of the radioactivity of the non-sedimentable microsomal fraction after freezing and thawing co-migrated with an intensely fluorescent band of glucosaminidase activity. Since the pellet fraction obtained on freezing/thawing the microsomal fraction contained up to 75% of the RNA, 95% of the radioactivity and 45% of the glucosaminidase, a detailed study of the association between its radioactivity and nascent glucosaminidase activity was undertaken. After 1 and 2 min of incubation, followed by centrifugation of the microsomal pellet on 35-60% (w/v) sucrose density gradients, radioactivity and glucosaminidase activity exhibited parallel profiles in the region of heavy polyribosomes and at the top of the gradient which contains spontaneously released nascent polypeptide chains. DEAE-cellulose chromatography of these chains revealed glucosaminidase A to be the principal nascent glucosaminidase component, with glucosaminidases B and C as minor peaks. After 2 min of incubation, all of the glucosaminidase components appeared labelled, and glucosaminidase A exhibited two distinct sub-components. The pattern of glucosaminidase labelling in the soluble and microsomal fractions suggested that newly formed glucosaminidase molecules traverse both the cellular sap and the lumen of the endoplasmic reticulum. Only glucosaminidase A reacted specifically with concanavalin A and radioactive glucosaminidase A could be successfully regenerated by treatment with alpha-methyl-D-glucoside. Glucosaminidase A and a substantial portion of the radioactivity associating with it could be readily converted into glucosaminidase B by re-chromatography on DEAE-cellulose and by reaction of the concanavalin A-glucosaminidase A complex with methyl glucosides.

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Selected References

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

  1. Beutler E., Kuhl W. Subunit structure of human hexosaminidase verified: interconvertibility of hexosaminidase isozymes. Nature. 1975 Nov 20;258(5532):262–264. doi: 10.1038/258262a0. [DOI] [PubMed] [Google Scholar]
  2. Bishayee S., Bachhawat B. K. ConA-sepharose affinity chromatography: a study on the glycoprotein nature of brain acid hydrolases. Neurobiology. 1974;4(1):48–56. [PubMed] [Google Scholar]
  3. Blomstrand C., Hamberger A. Protein turnover in cell-enriched fractions from rabbit brain. J Neurochem. 1969 Sep;16(9):1401–1407. doi: 10.1111/j.1471-4159.1969.tb05992.x. [DOI] [PubMed] [Google Scholar]
  4. Cioli D., Lennox E. S. Purification and characterization of nascent chains from immunoglobulin producing cells. Biochemistry. 1973 Aug 14;12(17):3203–3210. doi: 10.1021/bi00741a010. [DOI] [PubMed] [Google Scholar]
  5. Fleck A., Begg D. The estimation of ribonucleic acid using ultraviolet absorption measurements. Biochim Biophys Acta. 1965 Nov 8;108(3):333–339. doi: 10.1016/0005-2787(65)90025-0. [DOI] [PubMed] [Google Scholar]
  6. Goldstone A., Koenig H. Autolysis of glycoproteins in rat kidney lysosomes in vitro. Effects on the isoelectric focusing behaviour of glycoproteins, arylsulphatase and beta-glucuronidase. Biochem J. 1974 Aug;141(2):527–535. doi: 10.1042/bj1410527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goldstone A., Koenig H. Physicochemical modifications of lysosomal hydrolases during intracellular transport. Biochem J. 1973 Feb;132(2):267–282. doi: 10.1042/bj1320267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hamberger A., Blomstrand C., Yanagihara T. Subcellular distribution of radioactivity in neuronal and glial-enriched fractions after incorporation of [3H]leucine in vivo and in vitro. J Neurochem. 1971 Aug;18(8):1469–1478. doi: 10.1111/j.1471-4159.1971.tb00009.x. [DOI] [PubMed] [Google Scholar]
  9. Hamlin J., Zabin I. -Galactosidase: immunological activity of ribosome-bound, growing polypeptide chains. Proc Natl Acad Sci U S A. 1972 Feb;69(2):412–416. doi: 10.1073/pnas.69.2.412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Harzer K., Sandhoff K. Age-dependent variations of the human N-acetyl- -D-hexosaminidases. J Neurochem. 1971 Nov;18(11):2041–2050. doi: 10.1111/j.1471-4159.1971.tb05063.x. [DOI] [PubMed] [Google Scholar]
  11. Hemminki K. Incorporation of 3 H-leucine into insoluble proteins of neuronal and glial cell fractions in vitro. Acta Chem Scand. 1973;27(1):336–344. doi: 10.3891/acta.chem.scand.27-0336. [DOI] [PubMed] [Google Scholar]
  12. Hooghwinkel G. J., Veltkamp W. A., Overdijk B., Lisman J. J. Electrophoretic separation of -N-acetylhexosaminidases of human and bovine brain and liver and of Tay-Sachs brain tissue. Hoppe Seylers Z Physiol Chem. 1972 May;353(5):839–841. [PubMed] [Google Scholar]
  13. Idoyaga-Vargas V., Santiago J. C., Petiet P. D., Sellinger O. Z. The early post-natal development of the neuronal lysosome. J Neurochem. 1972 Nov;19(11):2533–2544. doi: 10.1111/j.1471-4159.1972.tb01312.x. [DOI] [PubMed] [Google Scholar]
  14. Johnson D. E., Sellinger O. Z. Age-dependent utilization of phenylalanine for the synthesis of neuronal and glial proteins. Neurobiology. 1973;3(2):113–123. [PubMed] [Google Scholar]
  15. Johnson D. E., Sellinger O. Z. Protein synthesis in neurons and glial cells of the developing rat brain: an in vivo study. J Neurochem. 1971 Aug;18(8):1445–1460. doi: 10.1111/j.1471-4159.1971.tb00007.x. [DOI] [PubMed] [Google Scholar]
  16. Johnson D. E., Sellinger O. Z. Synthesis of soluble neuronal proteins in vivo. Age-dependent differences in the incorporation of leucine and phenylalanine. Brain Res. 1973 May 17;54:129–142. doi: 10.1016/0006-8993(73)90039-5. [DOI] [PubMed] [Google Scholar]
  17. Klibansky C. Separation of N-acetyl- -D-hexosaminamidase-isoenzymes from human brain and leukocytes by cellulose acetate paper electrophoresis: a simple procedure for the diagnosis of Tay-Sachs disease. Isr J Med Sci. 1971 Sep;7(9):1086–1089. [PubMed] [Google Scholar]
  18. Kohl H. H., Sellinger O. Z. Protein synthesis in neuronal perikarya isolated from cerebral cortex of the immature rat. J Neurochem. 1972 Mar;19(3):699–711. doi: 10.1111/j.1471-4159.1972.tb01385.x. [DOI] [PubMed] [Google Scholar]
  19. Kreibich G., Sabatini D. D. Selective release of content from microsomal vesicles without membrane disassembly. II. Electrophoretic and immunological characterization of microsomal subfractions. J Cell Biol. 1974 Jun;61(3):789–807. doi: 10.1083/jcb.61.3.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Li S. C., Li Y. T. Studies on the glycosidases of jack bean meal. 3. Crystallization and properties of beta-N-acetylhexosaminidase. J Biol Chem. 1970 Oct 10;245(19):5153–5160. [PubMed] [Google Scholar]
  22. Murthy M. R. Free and membrane-bound ribosomes of rat cerebral cortex. Protein synthesis in vivo and in vitro. J Biol Chem. 1972 Mar 25;247(6):1936–1943. [PubMed] [Google Scholar]
  23. Redman C. M., Cherian M. G. The secretory pathways of rat serum glycoproteins and albumin. Localization of newly formed proteins within the endoplasmic reticulum. J Cell Biol. 1972 Feb;52(2):231–245. doi: 10.1083/jcb.52.2.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Redman C. M., Grab D. J., Irukulla R. The intracellular pathway of newly formed rat liver catalase. Arch Biochem Biophys. 1972 Oct;152(2):496–501. doi: 10.1016/0003-9861(72)90244-5. [DOI] [PubMed] [Google Scholar]
  25. Robinson D., Jordan T. W., Horsburgh T. The N-acetyl- -D-hexosaminidases of calf and human brain. J Neurochem. 1972 Aug;19(8):1975–1985. doi: 10.1111/j.1471-4159.1972.tb01487.x. [DOI] [PubMed] [Google Scholar]
  26. Robinson D. Multiple forms of glycosidases in the normal and pathological states. Enzyme. 1974;18(1):114–135. doi: 10.1159/000459418. [DOI] [PubMed] [Google Scholar]
  27. Robinson D., Price R. G., Dance N. Separation and properties of beta-galactosidase, beta-glucosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase from rat kidney. Biochem J. 1967 Feb;102(2):525–532. doi: 10.1042/bj1020525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. SELLINGER O. Z., VERSTER F. D. Glutamine synthetase of rat cerebral cortex: intracellular distribution and structural latency. J Biol Chem. 1962 Sep;237:2836–2844. [PubMed] [Google Scholar]
  29. Sakamoto T., Higashi T. Studies on rat liver catalase. VI. Biosynthesis of catalase by free and membrane-bound polysomes. J Biochem. 1973 May;73(5):1083–1088. doi: 10.1093/oxfordjournals.jbchem.a130162. [DOI] [PubMed] [Google Scholar]
  30. Sellinger O. Z., Azcurra J. M., Johnson D. E., Ohlsson W. G., Lodin Z. Independence of protein synthesis and drug uptake in nerve cell bodies and glial cells isolated by a new technique. Nat New Biol. 1971 Apr 21;230(16):253–256. doi: 10.1038/newbio230253a0. [DOI] [PubMed] [Google Scholar]
  31. Sellinger O. Z., Azcurra J. M., Ohlsson W. G., Kohl H. H., Zand R. Neurochemical correlates of drug-induced seizures: selective inhibition of cerebral protein synthesis by methionine sulfoximine. Fed Proc. 1972 Jan-Feb;31(1):160–165. [PubMed] [Google Scholar]
  32. Sellinger O. Z., Ohlsson W. G., Kohl H. H., Zand R. Precursor and age-dependent differences in in vivo rates of protein synthesis in the cerebral cortex. Brain Res. 1971 Nov;34(2):413–417. doi: 10.1016/0006-8993(71)90298-8. [DOI] [PubMed] [Google Scholar]
  33. Sellinger O. Z., Santiago J. C. Cerebellar N-acetyl-beta-D-glucosaminidase: a study of the enzyme in bulk-isolated purkinje and granule cells. Neurobiology. 1975 Mar;5(1):44–51. [PubMed] [Google Scholar]
  34. Sellinger O. Z., Santiago J. C. Lysosomal N-acetyl-beta-D-glucosaminidase: interneuronal differences in activity and molecular forms. Brain Res. 1976 May 28;108(2):431–435. doi: 10.1016/0006-8993(76)90199-2. [DOI] [PubMed] [Google Scholar]
  35. Sellinger O. Z., Santiago J. C., Sands M. A., Furin-Sloat B. N-acetyl-beta-d-glucosaminidase of nerve cells: a developmental study of two molecular components. Biochim Biophys Acta. 1973 Jul 5;315(1):128–146. doi: 10.1016/0005-2744(73)90137-x. [DOI] [PubMed] [Google Scholar]
  36. Seyama Y., Yamakawa T. Multiple components of beta-N-acetylhexosaminidase from equine kidney. Their action on glycolipids and allied oligosaccharides. J Biochem. 1974 Mar;75(3):495–507. doi: 10.1093/oxfordjournals.jbchem.a130418. [DOI] [PubMed] [Google Scholar]
  37. Seyama Y., Yamakawa T. Relationships among the multiple components of beta-N-acetylhexosaminidase from equine kidney. J Biochem. 1974 Apr;75(4):947–950. doi: 10.1093/oxfordjournals.jbchem.a130471. [DOI] [PubMed] [Google Scholar]
  38. Suzuki Y., Jacob J. C., Suzuki K., Kutty K. M., Suzuki K. Gm2-gangliosidosis with total hexosaminidase deficiency. Neurology. 1971 Apr;21(4):313–328. doi: 10.1212/wnl.21.4.313. [DOI] [PubMed] [Google Scholar]
  39. Tallman J. F., Brady R. O., Quirk J. M., Villalba M., Gal A. E. Isolation and relationship of human hexosaminidases. J Biol Chem. 1974 Jun 10;249(11):3489–3499. [PubMed] [Google Scholar]
  40. Tiplady B., Rose S. P. Amino acid incorporation into protein in neuronal cell body and neuropil fractions in vitro. J Neurochem. 1971 Apr;18(4):549–558. doi: 10.1111/j.1471-4159.1971.tb11985.x. [DOI] [PubMed] [Google Scholar]
  41. Uenoyama K., Ono T. Nascent catalase and its messenger RNA on rat liver polyribosomes. J Mol Biol. 1972 Mar 14;65(1):75–89. doi: 10.1016/0022-2836(72)90493-7. [DOI] [PubMed] [Google Scholar]
  42. Young E. P., Ellis R. B., Lake B. D., Patrick A. D. Tay-sachs disease and related disorders: Fractionation of brain N-acetyl-beta-hexosaminidase on DEAE-cellulose. FEBS Lett. 1970 Jul 15;9(1):1–4. doi: 10.1016/0014-5793(70)80295-2. [DOI] [PubMed] [Google Scholar]

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