Labib R S, Calvanico N J, Tomasi T B
Biochim Biophys Acta. 1978 Oct 12;526(2):547-59. doi: 10.1016/0005-2744(78)90145-6.
Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.
在透析培养基中生长的血链球菌ATCC 10556的培养液中发现了细胞外酪蛋白溶解活性。这种活性归因于多种蛋白酶，它们在从羟基磷灰石上洗脱、对酶抑制剂的敏感性、特异性和最适pH值方面存在差异。能将人免疫球蛋白A1裂解为完整Fc和Fab的IgA蛋白酶可以有效地与这些相对非特异性的蛋白酶分离，并通过五步程序以20%的产率纯化至表观均一性。尽管大部分葡聚糖蔗糖酶活性与IgA蛋白酶分离，但最终的IgA蛋白酶制剂中仍残留少量蔗糖酶活性。在pH 9.5的聚丙烯酰胺凝胶电泳中，两种活性都位于该制剂中检测到的单一蛋白条带中。基于放射免疫扩散法开发了一种定量测定IgA蛋白酶的方法，用于定量产生的Fab。该方法用于跟踪纯化过程中的比活性和产率，并表征该酶的一些催化特性。在酶/底物比为1:400（w/w）时，该蛋白酶在37℃过夜孵育中可使IgA发生50%的蛋白水解。最适活性在pH 8.0，在4×10⁻⁴M邻菲啰啉或8×10⁻⁴M乙二胺四乙酸时实现50%的抑制。高达10⁻²M的二异丙基氟磷酸酯、苯甲基磺酰氟、碘乙酸和对氯汞苯甲酸对IgA蛋白酶活性无影响。通过添加Mg²⁺、Mn²⁺或Ca²⁺可以使螯合剂抑制的酶完全重新活化。
