Enzymatic Defects in Three Genetic Classes of Serine-Requiring Mutants of Bacillus pumilus

Serine-requiring mutants of Bacillus pumilus NRRL B-3275 have been divided into three groups based on the position of the mutant loci on the linkage map of this organism. Representatives of each group were found deficient in enzymatic activities that constitute the phosphorylated pathway for serine biosynthesis. The evidence suggests that the genes coding for the enzymes of the phosphorylated pathway of serine biosynthesis are not clustered in B. pumilus.     

Enzymoblotting: a method for localizing proteinases and their zymogens using para-nitroanilide substrates after agarose gel electrophoresis and transfer to nitrocellulose

A method--enzymoblotting--was developed for localizing various enzymes after electrophoretic separation, transfer to nitrocellulose, and incubation with specific substrates. As an application, the proteinases porcine trypsin (EC 3.4.21.4), bovine chymotrypsin (EC 3.4.21.1), porcine elastase (EC 3.4.22.11), and their zymogen forms from porcine pancreas homogenate were analyzed utilizing specific p-nitroanilide substrates. After agarose gel electrophoresis, transfer of the separated proteinases to a nitrocellulose membrane was performed by capillary diffusion for 30 min. After air-drying of the nitrocellulose membrane, it was incubated in the appropriate substrate solution for 60 min. N-alpha-Benzoyl-DL-arginine-para-nitroanilide HCl was used as a substrate for trypsin, N-benzoyl-L-tyrosine-para-nitroanilide and succinyl-L-phenylalanine-para-nitroanilide for chymotrypsin, and N-succinyl-L-alanyl-L-alanyl-L-alanine-para-nitroanilide for elastase. p-Nitroaniline, the product thus obtained, was diazotized with N-(1-naphthyl)ethylenediamine to a red azo dye, visible at the site of the proteinases on the nitrocellulose membrane. The results could be preserved at -18 degrees C. Zymogen forms of the pancreas proteinases were detected in a similar manner. They were converted to active proteinases in situ on the nitrocellulose membrane after preincubating the nitrocellulose membrane in the activation enzymes enteropeptidase or trypsin.     

Studies on the interaction between leukocyte elastase, antileukoproteinase and the plasma proteinase inhibitors alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The dominating inhibitor of leukocyte elastase in human respiratory tract secretions is a low molecular mass inhibitor, designated antileukoproteinase. An equimolar antileukoproteinase-elastase complex was produced and subjected to gel filtration after differing time intervals and was found to be stable. On addition to human serum, however, elastase dissociated from antileukoproteinase and formed a complex with alpha 1-proteinase inhibitor. A small amount of elastase was also found bound to alpha 2-macroglobulin. Antileukoproteinase was capable of inhibiting elastase bound to alpha 2-macroglobulin. This inhibition was more complete and more rapid when the alpha 2-macroglobulin-elastase complex was in a molar ratio of 1:1 than in a ratio of 1:2.     

Immunoreactive trypsins in sera from dogs before and after induction of experimental pancreatitis

Radioimmunoassays for anionic and cationic dog trypsins are described. Characterization of the immunoreactivities in sera from fasting dogs demonstrated the presence of the two proenzymes only. Fasting sera from 10 dogs contained anionic and cationic trypsinogen in concentrations between 17-110 micrograms/l and 7-19 micrograms/l, respectively. Induction of experimental pancreatitis in dogs was accompanied by a large increase of immunoreactive anionic and cationic trypsins in the circulation. During the progress of the pancreatitis, immunoreactive trypsin with larger molecular weight than trypsinogen appeared. This high molecular weight immunoreactive trypsin was not seen in serum after intravenous injection of pancreatic juice in dogs. The high molecular weight immunoreactive trypsin probably consists of trypsin in complex with protease inhibitors. In vitro studies showed that the immunoreactivity of trypsin decreased considerably after binding to alpha 1-antitrypsin or alpha-macroglobulins.     

Studies on the interaction of Trasylol (aprotinin) with trypsin-pancreatic secretory trypsin inhibitor (PSTI) complexes and with alpha 2-macroglobulin-trypsin-PSTI-complexes

An investigation was performed to study the interaction of Trasylol with both trypsin-pancreatic secretory trypsin inhibitor (PSTI) and alpha 2-macroglobulin (alpha 2-M)-trypsin-PSTI complexes. Trasylol was readily able to displace immunogenic PSTI from a complex with trypsin in vitro. A similar scale of displacement of PSTI by Trasylol from alpha 2-M-trypsin-PSTI complexes could not be demonstrated. Using complexes manufactured in vitro with 125I-labelled PSTI, we found that only a small percentage of the PSTI label could be liberated, even when presented with amounts of Trasylol in a 10-molar excess to the PSTI.     

Release of dog polymorphonuclear leukocyte cathepsin G, normally and in endotoxin and pancreatitic shock. Isolation and partial characterization of dog polymorphonuclear leukocyte cathepsin G

Dog polymorphonuclear leukocyte cathepsin G was isolated from a granule extract using a two-step procedure including affinity chromatography on a Trasylol-Sepharose gel and ion-exchange chromatography on a CM 52 column. 22 of the first 24 N-terminal amino acids were determined and showed 83% and 71% identity to those of human and rat cathepsin G, respectively. Total amino-acid composition demonstrated the basic nature of the protein. In an SDS/polyacrylamide-gel electrophoresis the protein showed an Mr of 29,400 compared to the Mr of 26,800 calculated from the total amino-acid composition. The enzyme was shown to form complexes with alpha 1 alpha 2-macroglobulin and alpha 1-proteinase inhibitor. A specific enzyme-linked immunosorbent assay was developed for the determination of cathepsin G/alpha 1-proteinase inhibitor complex in dog plasma and tissue fluids. The mean concentration of cathepsin G in normal dog plasma was determined to be 38 micrograms/l, measured as cathepsin G/alpha 1-proteinase inhibitor complex. When active dog cathepsin G was added to normal dog plasma in vitro, approximately 56% could be measured by the assay. Slow intravenous infusion of a lethal dose of endotoxin in dogs was followed by a marked drop in white blood cell count and thrombocytes and a simultaneous rapid increase in plasma cathepsin G concentration, reaching a maximum level of 150 micrograms/l. Bile-induced experimental pancreatitis in dogs was accompanied by successive increase in cathepsin G levels in plasma as well as in peritoneal exudates, reaching a maximum level of about 300 micrograms/l in plasma and 18 mg/l in the exudates during the late stages of disease.     

Lactoperoxidase, a dithionite ion dismutase

The dithionite ion is catalytically disproportionated by lactoperoxidase with Km = 0.36 mM in 100 mM glycine HCl pH 3.0. The products formed are thiosulfate and hydrogensulfite ions. The rate of reaction is considerably increased at low pH with a pKa at 3-3.5 possibly indicating the involvement of a carboxyl group. The reaction is competitively inhibited by hydrogensulfite, Ki = 5.5 mM in 100 mM glycine HCl pH 3.50. Four different spectral forms of reduced lactoperoxidase appear during the reaction. The first two forms are found during the lag phase of the reaction. The third form, which is interpreted as a ternary complex, exists under the dismutation phase. After exhaustion of the substrate a visible spectrum similar to that of lactoperoxidase H2O2 compound III appears. A mechanistic model for the lactoperoxidase dismutation of the dithionite ion is proposed and discussed.