Purification and characterization of the Pasteurella haemolytica 35 kilodalton periplasmic iron-regulated protein.

Pasteurella haemolytica serovar A1 is the causative agent of acute fibrinohemorrhagic pneumonia also known as shipping fever. Many pathogens, including P. haemolytica, survive in their respective hosts through the up-regulation of an iron acquisition system. In this study we identified, purified and characterized a 35-kDa periplasmic iron-regulated protein. The N-terminal sequence of the iron-regulated protein ANEVNVYSYRQP YLIEPMLK was identical to the deduced amino acid sequence of the ferric binding protein, FbpA, of P. haemolytica. Growth of P. haemolytica in a synthetic medium (RPMI-1640), without iron and supplemented with 50 gM 2,2' dipyridyl, facilitated the expression, isolation and purification of the native P. haemolytica FbpA. The protein was purified to homogeneity by using ammonium sulfate precipitation followed by CM-Sepharose ion exchange chromatography. SDS-PAGE showed a single band with a molecular weight of 35,369. Isoelectric focusing showed multiple bands with pIs of 5.5, 5.6, 5.8, and one major band with pI of 6.4. The molecular weight obtained by electrospray mass spectrometry was 35,822. Equilibrium velocity ultracentrifugation established that the protein existed as a monomer under native conditions with an apparent molecular weight of 33,795. Analysis of secondary structure of FbpA by circular dichroism showed 42.1% alpha helical structure. This protein is the second periplasmic iron-regulated protein described for P. haemolytica.

Effects of proteolytic enzymes on mammary tumor growth: in vitro and in vivo suppression and cellular death of tumor cells.

Proteolytic enzymes may have potential value in the prophylaxis of malignant tumor development. C3H/HEJ mice, used for their ability to produce spontaneous mammary tumors, were injected intraperitoneally (IP) with proteolytic enzyme hydrolysate at a dosage range of 0.038 to 0.462 mg/gm body weight. The injections were given every other day, once a day for six months. The pathology results showed suppression of growth, and necrosis (and in some cases encapsulation) of the mammary tumors in C3H/HEJ mice. Concurrently, SP 2/0-AG 14 cells grown in the presence of 0.25 mg enzyme/ml to 3.75 mg enzyme/ml of proteolytic enzymes, showed little cellular deterioration when the dosage range remained below 1 mg enzyme/ml. When dosage ranges were greater than 1 mg enzyme/ml, cellular necrosis occurred within three days of the addition of the proteolytic enzymes. These results demonstrate that the proteolytic enzymes used in these experiments were beneficial in preventing tumor development and prolonging survival of C3H/HEJ mice when used in the appropriate concentration range. A portion of these results were presented elsewhere (2nd Int. Biotechnol. Expo; Oct. 1989; San Francisco).