Partial purification of murine B cell stimulatory factor (BSF)-1

BSF-1 was partially purified from serum-free culture supernatants of cells of the EL-4 thymoma line, which had been induced 48 hr earlier with 4 beta-phorbol-12 beta-myristate-12 alpha-acetate (PMA). BSF-1 in 10-liter batches was adsorbed onto and eluted from trimethylsilyl-controlled pore glass beads (TMS-CpG) and then subjected to reverse-phase high-performance liquid chromatography (RP-HPLC). The recovery of BSF-1 activity by TMS-CpG and RP-HPLC ranged from 52 to 55% and 187 to 227%, respectively. The specific activity in units per milligram of protein of partially purified BSF-1 was approximately 2600 times higher than that of the culture supernatant protein. The partially purified BSF-1 had a single isoelectric point of 6.3 and an apparent m.w. between 18,000 and 21,700 when analyzed by isoelectric focusing and gel filtration-HPLC, respectively. The ability to prepare large amounts of partially purified BSF-1 by a rapid and efficient procedure should be of great help in both biochemical and immunologic studies of this lymphokine.     

Interferon-gamma inhibits the action of B cell stimulatory factor (BSF)-1 on resting B cells

B cell stimulatory factor-1 (BSF-1) stimulates resting B cells to increase in volume and prepares these cells to enter the S phase in response to anti-IgM and other B cell mitogens. Interferon-gamma (IFN-gamma) blocks both the volume enlargement and preparation for DNA synthesis caused by BSF-1, although it has little effect on B cells already stimulated by BSF-1. The capacity of IFN-gamma to inhibit the action of BSF-1 on resting B cells suggests a mutual regulatory interaction between these two T cell-derived products.     

BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification,biochemical and molecular characterization

A novel fibrinolytic enzyme, subtilisin BSF1, from a newly isolated Bacillus subtilis A26 was purified, characterized and the gene was isolated and sequenced. The subtilisin BSF1 was purified to homogeneity by five-step procedure with a 4.97-fold increase in specific activity and 6.28% recovery. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and gel filtration. The purified enzyme exhibited high fibrinolytic activity on fibrin agar plates.Interestingly, the enzyme was highly active over a wide range of pH from 7.0 to 12.0, with an optimum at pH 9.0. The relative activities at pH 10.0 and 11.0 were 97.8% and 85.2% of that at pH 9.0. The optimum temperature for enzyme activity was 60 °C. The activity of subtilisin BSF1 was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The N-terminal amino acid sequence of the first 11 amino acids (aa) of the purified fibrinolytic enzyme was AQSVPYGISQI.The bsf1 gene encoding the subtilisin BSF1 was isolated and its DNA sequence was determined. The bsf1 gene consisted of 1146 bp encoding a pre-pro-protein of 381 amino acids organized into a signal peptide (29 aa), a pro-peptide (77 aa) and a mature domain (275 aa). The deduced amino acids sequence of the mature enzyme (BSF1) differs from those of nattokinase from B. subtilis natto and subtilisin DFE from Bacillus amyloliquefaciens DC-4 by 5 and 39 amino acids, respectively.     

Detection of receptors for murine B cell stimulatory factor 1 (BSF1): presence of functional receptors on CBA/N splenic B cells

The presence of receptors specific for murine B cell stimulatory factor 1 (BSF1) was demonstrated by utilizing an internally radiolabeled recombinant BSF1. Radiolabeled BSF1 was efficiently produced in Xenopus laevis oocytes injected with a cloned mRNA for BSF1 and 35S-methionine. The labeled BSF1 specifically bound to splenic B cells. A Scatchard analysis indicated the existence of one class of receptor sites. BSF1 receptors were found to be distributed on a wide range of hematopoietic lineage cells, including B cells, T cells, macrophages, and mast cells. B cells from CBA/N mice with the xid gene defect had a similar level of BSF1 binding capacity compared with BALB/c strain B cells, and responded well to insoluble anti-Ig and BSF1 in proliferation assays, indicating that CBA/N B cells express functional BSF1 receptors at normal levels. Pre-B cell lines showed low levels of BSF1 binding, suggesting that cells in the B cell lineage acquire BSF1 responsiveness early in development.     

BSF1 induces membrane protein phosphorylation but not phosphoinositide metabolism, Ca2+ mobilization, protein kinase C translocation, or membrane depolarization in resting murine B lymphocytes

The findings presented in this study provide evidence that BSF1 receptors and mIg transmit signals via dissimilar transduction mechanisms that result in a common biologic response, hyper-Ia expression. Specifically, BSF1-containing supernatant does not induce PtdInsP2 hydrolysis as determined by measurement of PtdOH and InsP3. Additionally, BSF1 does not stimulate Ca2+ mobilization, PKC translocation from cytosol to membrane, or membrane depolarization. All of these metabolic events appear to play a central role in hyper-Ia expression mediated by mIg and are initiated after treatment of resting B cells with anti-Ig antibodies. In vitro phosphorylation studies with partially purified plasma membranes from resting B cells revealed that BSF1 interaction with membrane receptors stimulates a membrane-associated protein kinase that phosphorylates an endogenous protein of 44 KDa. Anti-Ig does not stimulate phosphorylation of the 44 KDa protein, suggesting that it does not activate the membrane-associated protein kinase. This observation provides the first evidence of a signal transduction mechanism associated with BSF1-receptor ligation. It indicates that although BSF1 does not modulate events associated with PKC activation, it may function via activation of a membrane-associated protein kinase. This provides a focal point for further studies directed at elucidating signal transduction resulting from BSF1-receptor interaction.