Summary: In addition to the cell wall proteinase, subsp. produced significant amounts of a free extracellular proteinase. The free proteinase activity was highest in the late exponential and early stationary phase of growth, whereas the cell wall activity was highest in the last half of the exponential phase. Both proteinase forms had a pH optimum between 4·6 and 5·8, and they behaved similarly upon anion exchange and hydrophobic interaction chromatography, chromatofocusing and gel filtration, indicating that they were related. Purification to homogeneity, as judged by SDS-PAGE, resulted in a 50000-80000-fold increase in the specific activity of the free proteinase. It contained two major protein species (termed pro150 and pro115) with proteinase activity. As judged by SDS-PAGE, the values of pro150 and pro115 were 150000 and 115000, respectively, and by chromatofocusing the isoelectric points were 4·3 and 4·1, respectively. Upon gel filtration, pro150 and pro115 had values of 300000 and 125000, respectively, indicating that pro150 was a dimer and pro115 a monomer. Pro115 was an autodegradation product of pro150. Other distinct autodegradation products had values of 90000 (p90), 53000 (p53), 37000 (p37) and 30000 (p30). These had little if any proteinase activity. Pro115, p90 and p53 had a common N-terminal sequence with that reported for the cell wall proteinase. Judging from its N-terminal sequence and , p30 was derived from the C-terminal half of p53. Cleavage of pro150 to pro115 generated p37.


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