A search for multi-enzyme complexes of DNA precursor pathways in eukaryotic cells

In order to prepare pure enzyme before gene cloning the proteins were separated by fast protein liquid chromatography and used to prepare monoclonal antibodies to papaya proteinase A and B. Crude spray-dried latex from Powell and Scholefield was dialysed against 1,3-diaminopropane and the enzyme isolated by the method of Goodenough & Owen (1986) using 6 mM-diaminopropane as buffer and a gradient of 4 mM/min of NaCI. The use of an anion-exchange column gave a good separation of the proteins, and fractions containing papaya proteinase A and B were freeze-dried and redissolved in phosphate-buffered saline (0.1 ~-NaC1/0.06 M-phosphate, pH 7.3). Balb/c mice were injected subcutaneously with 50pg of a mixture of the redissolved protein in 200p1 of Freunds complete adjuvant. Six weeks later, after an intravenous booster injection, fusion of the spleen cells and mouse myeloma cells, P3 x 63-Ag 8-653 was obtained. Eight hybridomas were shown to have immunological activity when reacted against mixed papaya proteinase A and B. Western immunoblotting and enzyme-linked immunosorbent assays were as described by Goodenough et al. (1986). Two lines, PAP 8 and PAP 7, were shown to be specific for papaya proteinases A and B respectively. The reaction of PAP 7 and PAP 8 is shown in Fig. 1 and it can be easily seen that there is a specific reaction with the papaya proteinases. When the papaya proteinases are separated by fast protein liquid chromatography the individual members can be prepared in a nearly pure form. The activity of the preparations of proteinases A and B towards casein is the same qualitatively, although A is more active quantitatively. It is conceivable that activity of A is contaminating B and that B only has a lysozymal activity. However, we have determined the M , of B as 28 000 and the PI as > 1 1.1. These are widely different from the properties of most lysozymes. The fact that monoclonal antibodies that are specific for A and B can be prepared so easily indicates that at least some of the quaternary structure of the molecule is quite different in structure. We have used the monoclonal antibodies to purify quantities of A and B for sequencing and crystallographic analysis. It is hoped to be able to also isolate the DNA coding for this interesting family of proteinases.