Samollow P B, Ford A L, VandeBerg J L
Department of Biology, Lehigh University, Bethlehem, Pennsylvania 18015-3189.
Biochem Genet. 1990 Jun;28(5-6):283-98. doi: 10.1007/BF02401418.
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.
两种分析方法（荧光法和电泳法）在检测基因Neu - 2对大鼠肝脏神经氨酸酶表型的影响时，能力上存在显著差异，这促使我们研究肝脏同工酶NEU - 1和NEU - 2的生化和物理特性，这些特性可能是造成这种差异的原因。通过Percoll梯度离心进行细胞分级分离显示，NEU - 1活性几乎完全存在于溶酶体细胞分级中，而NEU - 2在分布上严格位于胞质溶胶中。还发现这两种同工酶在pH活性曲线和最适pH值（NEU - 1和NEU - 2的最适pH值分别为4.6 - 4.8和5.4 - 5.8）以及溶解性特征（NEU - 2高度可溶；NEU - 1相对不溶，但通过冻融可溶解）方面存在差异。在粗制的全细胞提取物中，两种同工酶都被发现对冻融稳定，但在富集的（部分纯化的）溶酶体亚细胞分级中NEU - 1不稳定。将这些特性与先前描述的未鉴定的胞质溶胶和膜结合（溶酶体）大鼠肝脏神经氨酸酶的特性（Tulsiani, D. R. P., and Carubelli, R., J. Biol. Chem. 245:1821, 1970）进行比较，使我们相信NEU - 2也会因部分纯化而不稳定，并且NEU - 1和NEU - 2在细胞内具有非常不同的相对丰度。NEU - 1和NEU - 2之间的生化和物理差异可以解释荧光法和电泳法在检测Neu - 2效应时能力的差异。本文讨论了提高荧光法对特定NEU - 1和NEU - 2活性进行定量测定的灵敏度的方法。
