[Limited proteolysis of human albumin and immunoglobulin G by Legionella pneumophila metalloproteinase]. / Ogranichennyi proteoliz al'bumina i immunoglobulina G cheloveka metalloproteinazoi Legionella pneumophila.

Metalloproteinase of Legionella pneumophila is the major extracellular proteinase of this bacterial species which splits human immunoglobulin G in the hinge region to form the (Fab')2 fragment. This fragment is relatively stable and undergoes further proteolysis at a slow rate. The c' fragment is unstable and is apparently split down to fragments CH2 and CH3. The metalloproteinase splits human serum albumin down to products having lower molecular masses. Another bacterial metalloproteinase, thermolysin, produces a similar effect, although at a slower rate.

[The effect of Legionella pneumophila metalloproteinase on certain human blood proteins. Cleavage of anti(1)-antitrypsin and acid-stable serine proteinase inhibitors]. / Deistvie metalloproteinazy Legionella pneumophila na nekotorye belki syvorotki krovi cheloveka. Rasshcheplenie anti1-antitripsina i kislotostabil'nogo ingibitora serinovykh proteinaz.

Antitryptic activity of human blood serum was decreased after incubation with metalloproteinase from Legionella pneumophila. The enzymatic activity depends on the time of incubation as well as on the ratio between the enzyme content and blood serum total protein. Cross immunoelectrophoresis, involving monospecific rabbit antiserum towards the alpha 1-antitrypsin, demonstrated highly effective hydrolysis of alpha 1-antitrypsin by the metalloproteinase. As shown by polyacrylamide gel electrophoresis the metalloproteinase hydrolyzed acid stable inhibitor of serine proteinases into fragments with distinct loss of the inhibitor activity. Thermolysine hydrolyzed similarly the proteins studied but at a lower rate. The metalloproteinase from L. pneumophila appears to be mainly responsible for production of utilizable components from protein substrates involved in vital activity of the bacteria. It may not be excluded that the enzyme is able to impair some host protective mechanisms.

[The properties of HIV protease synthesized in E. coli cells]. / Izuchenie svoistv proteazy VICh, sintezirovannoi v kletkakh bakterii E. coli.

A hybrid plasmid pPR6 was constructed containing BgII-EcoRI fragment of the pol region of HIV (strain IIIB) genome which determined the synthesis of virus-specific protease. Extracts of E. coli DN5/pPR6 bacteria provided for specific hydrolysis of hybrid protein p165 (the N-terminus of which is presented by complete beta-galactosidase and the C-terminus by duplicated area of virus-specific precursor p55 containing a site for virus-specific protease located at the border of proteins p17 and p24) with formation of products having molecular weights of 19, 42, 28, 23, and 19 kD. Polypeptides 119K, 23K, and 19K are products of complete hydrolysis, and 42K a result of partial cleavage. The kinetics of hydrolysis in relation to pH values of the reaction mixture was analysed. It is suggested that the reported system of HIV protease activity determination be used for screening of potential inhibitors of this enzyme.

[Cytolytic activity of Legionella pneumophila]. / Priroda tsitoliticheskoi aktivnosti Legionella pneumophila.

The properties of cytolysin and metalloproteinase purified by different methods have been studied. The physico-chemical properties of these proteins, including their molecular weight, immunodiffusion patterns, the degree of inhibition by EDTA and diethyl pyrocarbonate, amino acid composition, cytolytic and proteolytic activity, have proved to be similar. We have come to the conclusion that cytolysin and metalloproteinase have similar composition and metalloproteinase activity determines the cytolytic and necrotic activity of the above-mentioned cytolysin.

[Extracellular metalloproteinase from Legionella pneumophila]. / Vnekletochnaia metalloproteinaza Legionella pneumophila.

Using ion-exchange chromatography on QAE-Sephadex A-50, affinity chromatography on DNP-hexamethylenediamine-Sepharose and gramicidin S-Sepharose and gel filtration, a metalloproteinase was isolated from the cultural fluid of L. pneumophila (strain Philadelphia-1) grown for 20 hours. The enzyme was purified 1606-fold with a 31% yield. The enzyme has a Mr of 38,000, pI approximately 4.0 and optimum of proteolytic activity at pH 6.0-7.0, 55 degrees C. The proteinase is the most stable within the pH range of 6.0-9.0. The enzyme contains one atom of zinc per molecule. The amino acid composition of metalloproteinase is close to that of thermolysin and is characterized by a high methionine content--17 residues out of 348. In the B-chain of oxidized bovine insulin the enzyme hydrolyzes the bonds precedent to the amino groups of leucine, phenylalanine and tyrosine. The enzyme is inhibited by chelating agents--Na2-EDTA and o-phenanthroline as well as by diethylpyrocarbonate. The serine and thiol proteinase inhibitors do not influence the enzyme activity. Under the given conditions of cultivation metalloproteinase is the major endopeptidase produced by L. pneumophila. Thus, the proteolytic system of Legionelles is characterized by the combination of metalloproteinase and the earlier described phenylalanine aminopeptidase.

Proteinases of Legionella: phenylalanineaminopeptidase of L. pneumophila.

Phenylalanineaminopeptidase was isolated and purified from the culture filtrate of Legionella pneumophila by affinity chromatography on O-tert-butyl-L-threonyl-L-phenylalanyl-L-prolylglycyl-aminosilo chrom and by gel-filtration; a 401-fold purification with a yield of 18% was achieved. The enzyme was a metalloenzyme with a molecular weight of 35000 and a pI of 5.8. It was stable at pH 7-9 and had an activity optimum in the range of pH 8-9.5 with L-phenylalanine p-nitroanilide as substrate. Enzyme activity was highest towards the latter compound, substantially lower towards L-leucine p-nitroanilide and only marginal towards other p-nitroanilides. Besides phenylalanineaminopeptidase, a metalloproteinase and a serine proteinase were also detected in L. pneumophila culture filtrate.