Autocatalytic processing of the streptococcal cysteine protease zymogen: processing mechanism and characterization of the autoproteolytic cleavage sites.

The autocatalytic processing of the streptococcal cysteine protease zymogen (proSCP) to active streptococcal cysteine protease (SCP) was investigated in vitro using purified protein from Streptococcus pyogenes strain B220. It was found that the autocatalytic maturation of the zymogen proceeds through the sequential appearance of at least six intermediates, five of which were characterized through a combination of N-terminal sequencing and MS. Intermediates were identified as resulting from cleavages after Lys26, Asn41, Lys101, Ala112, and Lys118. Time-course studies of the proSCP processing gave a sigmoidal activity profile and indicated that proSCP catalyses its own transformation, mainly via an intermolecular processing mechanism. A similar sequential appearance of intermediates was observed when inactive Cys192Ser proSCP was treated with native, enzymatically active SCP, thus demonstrating that the maturation can exclusively proceed by a bimolecular mechanism. It was shown that proSCP, but not mature SCP, immobilized on a Sepharose resin is capable of liberating itself from the column, indicating that the zymogen is also capable of intramolecular processing. In order to test whether the amino acid sequences at the processing sites could be used for developing new, specific substrates, 3-amino benzoic acid octapeptide derivatives based on all five characterized amino acid sequences from the autoprocessing cleavage sites were synthesized and tested for activity. The 3-amino benzoic acid derivatives have kcat/KM values ranging from 1200 to 7700.M-1.s-1, making them very good endopeptidase substrates for SCP.     

A three-dimensional study of organelle interrelationships in regenerating rat liver. 3. Organelles related to bile.

A number of cell structures are described which show a morphological relationship to the bile canaliculi. Two types of peribiliary vesicles are identified: osmication positive ones occurring between the bile canaliculi and the osmicated immature Golgi cisternae and probably deriving from the latter, and osmication negative ones related to MVB, on which they appear as buds. Small coated vesicles are seen attached to this second type. Large lacunae may originate from MVB, as suggested by the MVB-like internal vesicles they may contain. Some stay in luminal continuity with the bile canaliculi. Canalicular coated vesicles are seen as parts of the canalicular plasma membrane and free in the cytoplasm.