The heterogeneity of histidine decarboxylase from rat gastric mucosa was studied. The partially purified enzyme was fractionated by preparative isoelectric focusing on a flat-gel bed by using narrow pH-range carrier ampholytes and a short focusing time. The activity was resolved, with about 95% recovery, into three forms, designated I, II and III, with pI values of 5.90, 5.60 and 5.35 respectively. These three forms exhibited similar molecular weights, indicating that the forms were not the result of different degrees of polymerization. By preparative refocusing each form refocused as a single peak of enzyme activity with reproducible pI, but a high loss of activity occurred with repeated focusing. Forms I, II and III were purified by the combined use of preparative isoelectric focusing and gel chromatography and other fractionation methods. The active forms could be distinguished by electrophoresis and isoelectric focusing on polyacrylamide gels and displayed protein heterogeneity. These forms were found in the crude extract and in the partially purified preparations in the presence or absence of proteinase inhibitors. Form II had the highest specific activity, but all three forms had the same optimum pH and Km value for histidine.
Isoelectric focusing of human liver alkaline phosphatase
