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Comparison of liver glycosylasparaginases from six vertebrates.

ABSTRACT: Structural and physical properties of glycosylasparaginase (EC 3.5.1.26) from the livers of human, pig, cow, rat, mouse and chicken were compared. The enzyme in all species had a common basic structure of two N-glycosylated subunits of about 24 (alpha) and 20 (beta) kDa joined by non-covalent forces. Subunit-specific antisera against the rat glycosylasparaginase bound specifically and sensitively to the corresponding subunits from all species. Identity of 80% of the amino acids was found between the N-terminal sequences of corresponding pig and rat glycosylasparaginase alpha- and beta-subunits and the deduced sequence from a human glycosylasparaginase cDNA [Fisher, Tollersrud & Aronson (1990) FEBS Lett. 269, 440-444]. The beta-subunit from all three species has an N-terminal threonine reported to be involved in the reaction mechanism for the human enzyme [Kaartinen, Williams, Tomich, Yates, Hood & Mononen (1991) J. Biol. Chem. 266, 5860-5869]. The native enzyme appeared as a heterodimer among the mammals, whereas the chicken enzyme had a greater molecular mass and is probably either a tetramer or a heterodimer bound to an unrelated peptide(s). All glycosylasparaginases were thermostable, requiring temperatures between 65 degrees C and 80 degrees C to be irreversibly inactivated. In addition, they were unusually stable at high pH and remained active in the presence of SDS except at low pH. The pH maximum was between 5.5 and 6 except for the rat and mouse enzymes which had a broad maximum between pH 7 and 8. A number of other properties were observed which also distinguish the enzyme from individual and closely related species.

SUBMITTER: Tollersrud OK

PROVIDER: S-EPMC1130871 | biostudies-other | 1992 Mar

REPOSITORIES: biostudies-other

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Project description:For in vitro culture of plant and animal cells, one of the critical steps is to adjust the initial cell density. A typical example of this is isolated microspore culture, where specific cell densities have been determined for different species. Out of these ranges, microspore growth is not induced, or is severely reduced. A similar situation occurs in many other plant and animal cell culture systems. Traditionally, researchers have used counting chambers (hemacytometers) to calculate cell densities, but little is still known about their technical advantages. In addition, much less information is available about other, alternative methods. In this work, using isolated eggplant microspore cultures and fluorescent beads (fluorospheres) as experimental systems, we performed a comprehensive comparison of six methods to calculate cell densities: (1) a Neubauer improved hemacytometer, (2) an automated cell counter, (3) a manual-counting method, and three flow cytometry methods based on (4) autofluorescence, (5) propidium iodide staining, and (6) side scattered light (SSC).Our results show that from a technical perspective, hemacytometers are the most reasonable option for cell counting, which may explain their widely spread use. Automated cell counters represent a good compromise between precision and affordability, although with limited accuracy. Finally, the methods based on flow cytometry were, by far, the best in terms of reproducibility and agreement between them, but they showed deficient accuracy and precision.Together, our results show a thorough technical evaluation of each counting method, provide unambiguous arguments to decide which one is the most convenient for the particular case of each laboratory, and in general, shed light into the best way to determine cell densities for in vitro cell cultures. They may have an impact in such a practice not only in the context of microspore culture, but also in any other plant cell culture procedure, or in any process involving particle counting.

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Comparison of liver glycosylasparaginases from six vertebrates.

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