Biochemical characteristics and subcellular localizations of rat liver neuraminidase isozymes: a paradox resolved.
A striking discrepancy in the abilities of two analytical approaches (fluorometric and electrophoretic) to detect the effect of a gene, Neu-2, on rat liver neuraminidase phenotypes led us to examine the biochemical and physical properties of the liver isozymes NEU-1 and NEU-2 that might be responsible for this difference. Cell fractionation via Percoll gradient centrifugation revealed NEU-1 activity almost exclusively in the lysosomal cell fraction, while NEU-2 was strictly cytosolic in distribution. The two isozymes were also found to differ in pH activity curves and optima (optima: 4.6-4.8 and 5.4-5.8 for NEU-1 and NEU-2, respectively) and in solubility characteristics (NEU-2 highly soluble; NEU-1 relatively insoluble but solubilized by freezing/thawing). Both isozymes were found to be freeze-thaw stable in crude, whole-cell extracts, but NEU-1 was destabilized in the enriched (partially purified) lysosomal subcellular fraction. Consideration of these properties relative to those described previously for unidentified cytosolic and membrane bound (lysosomal) rat liver neuraminidases (Tulsiani, D. R. P., and Carubelli, R., J. Biol. Chem. 245:1821, 1970) leads us to believe that NEU-2 also is destabilized by partial purification and that NEU-1 and NEU-2 have very different relative abundances within the cell. The biochemical and physical differences between NEU-1 and NEU-2 can account for the discrepant abilities of the fluorometric and electrophoretic approaches to detect the effects of Neu-2. Ways to increase the sensitivity of the fluorometric approach for quantitative assays of specific NEU-1 and NEU-2 activity are discussed.
Samollow
P B
PB
Department of Biology, Lehigh University, Bethlehem, Pennsylvania 18015-3189.
Ford
A L
AL
VandeBerg
J L
JL
D000818:Animals; D002458:Cell Fractionation; D004586:Electrophoresis; D004795:Enzyme Stability; D005260:Female; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D051381:Rats; D012995:Solubility
D007527:Isoenzymes; D009439:Neuraminidase