An aspartic proteinase from Fusarium moniliforme. Purification and general properties.

An acid proteinase from the culture filtrate of Fusarium moniliforme was purified by acetone precipitation, gel filtration and ion-exchange chromatography. The final enzyme preparation was homogeneous on polyacrylamide gel electrophoresis. The apparent molecular weight was estimated to be 38 000, based on sodium dodecyl sulfate gel electrophoresis and an isoelectric point of pH 4.90. The optimum pH was found to be 3.2 when measured with denatured hemoglobin as substrate. The enzyme showed maximum stability in the pH range 3.0-5.5. Pepstatin, 1,2-epoxy-3-(p-nitrophenoxy)propane and diazoacetyl-DL-norleucine methyl ester strongly inhibited the proteinase activity, whereas thiol reagents, serine-type proteinase inhibitors and metal-chelating reagent had no significant effect. The data indicate that the proteinase from F. moniliforme is an aspartic proteinase which possesses milk-clotting activity.

Aspartyl proteinase from cucumber (Cucumis sativus) seeds. Preparation and characteristics.

Aspartyl proteinase (EC 3.4.23) from cucumber seeds was purified by ammonium sulphate fractionation, chromatography on immobilized pepstatin and gel filtration on Sephacryl S-200. The preparation obtained, homogeneous on polyacrylamide-gel electrophoresis in acidic and alkaline media, has a molecular mass of 42,000, pI of 5.2, and shows the highest activity with denatured haemoglobin at pH 3.2. The proteinase is stable in slightly alkaline medium, whereas it is inactivated in acidic medium, especially in the presence of NaCl. The enzyme activity is affected neither by the inhibitors of serine proteinases, sulfhydryl-proteinases and metalloproteinases, nor by divalent metal ions, whereas the enzyme is inactivated by the inhibitors of aspartyl proteinases: 1,2,3-epoxy(p-nitrophenoxy)propane, diazoacetyl-DL-norleucine and pepstatin.

Rat interferons. Homologous and heterologus rat serum activity.

Serum proteins from normal rat serum and induced with Sindbis virus were separated on Sephadex G-100. Interferon activity was studied in a system of homologous REC and heterologous LG cells. Proteins of normal serum as well as induced serum emerged in two peaks separated by a deep saddle. Four hours after induction, the serum gave two peaks with similar interferon activity in both cell systems. After 8-hour induction, two interferons were also obtained, but activity in the heterologous system was much lower than in the homologous system. Electrophoresis in polyacrylamide gel and on paper showed that interferon activity is connected mainly with the alpha1-globulin fraction.

Proteins in mouse embryonic cell (MEC) culture fluids following interferon induction.

A study was made on the proteins and interferon activity in supernatant culture fluids from mouse fibroblast cultures induced with Newcastle disease virus, and in controls. The studied materials showed low protein content, the concentration of proteins depending on the length of time from induction to their collection. Proteins from induced and control cultures were separated on CM-Sephadex C-50 and by electrophoresis in polyacrylamide gel. No difference was found in the character of proteins from control materials and from materials induced 24 hours after infection; the elution profile was the same, and the distribution of electrophoretic fractions was similar. Interferon activity was not accompanied by the appearance of a new protein fraction. However, 5--10 hours after induction differences were noted in electrophoretic mobility and a marked reduction in the number of protein fractions occurred in control and stimulated culture fluids.