Growth‐promoting activity in serum‐free medium of kallikreinlike arginylesteropeptidases from rat submaxillary gland

Abstract

The characterization and purification of the growth-promoting activity present in rat submaxillary gland extracts, known to be required for the proliferation of adipose precursor cells in serum-free medium, have been undertaken. Fractionation of the extracts by ion-exchange chromatography, gel filtration, and affinity chromatography on immobilized benzamidine allowed the copurification of the mitogenic activity with two distinct arginylesteropeptidases of apparent molecular weight 25,000; one of these enzymes has been purified to homogeneity and shown to be immunologically related to tonin, a well-characterized kallikreinlike protease from submaxillary gland. The specificity of both enzymes was similar to that of plasma and glandular kallikreins, as indicated by the relative rates of hydrolysis of peptide p-nitroanilide substrates. Prior treatment of the kallikreinlike proteases with phenylmethylsulfonylfluoride or aprotinin abolished completely both mitogenic and arginylesteropeptidase activities, indicating that enzymatic activity was essential for the manifestation of their growth-promoting ability. The kallikreinlike proteases from rat submaxillary gland were able to replace thrombin to support the proliferation of Chinese hamster lung fibroblasts in serum-free medium. These results underline the role of proteases in controlling cell growth and are discussed in light of adipose tissue development.

Proliferation and differentiation of rat adipose precursor cells in chemically defined medium: Differential action of anti‐adipogenic agents

“…Water was added up to 1 ml and the absorbance, proportional to cell number (Gaillard et al, 19841, measured at 625 nm. Variations in optical densities between assays on separate wells did not exceed 4%, as observed in previous studies (Czerucka et al, 1986;Catalioto et al, 1987).…”

“…Male Wistar rats (forty days old) were obtained from IFFA Credo (L'Arbresle, France). Submaxillary gland kallikrein was prepared according to Catalioto et al (1987). Carbaprostacyclin and murine recombinant TNF-a were purchased from Cascade Biochem.…”

Protease Activity in Protein-Free Ns0 Myeloma Cell Cultures

“…Aprotinin has also been found to increase the survival of rat hepatocytes presumably by inhibiting serine proteases located in the plasma membrane Nakamura et al, 1984) and to promote growth of transformed rat embryo fibroblasts, an effect attributed to decreased proteolysis of autocrine growth factors (Cook and Chen, 1988). In contrast, aprotinin has also been found to eliminate the growth-promoting effect of rat submaxillary gland extracts on adipose precursor cells due to inhibition of a kallikrein-like protease (Catalioto et al, 1987).…”

Prostacyclin as a potent effector of adipose-cell differentiation

“…Stock cultures of Ob1771 cells (a subclone of Ob17) [18,19] and 3T3-F442A cells [20] were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum as previously described [10,[18][19][20]. After plating in serum-supplemented medium at a density of 8.5 x I03 cells/cm2 in 24-multiwell Linbro tissue-culture plates (2 cm2/well), cells were extensively washed 20-24 h later with a mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium (1:1, v/v) and subsequently grown to confluence (3 days) in the same mixture as above supplemented with insulin (5 ,ug/ml), transferrin (10 ,tg/ml), a partially purified kallikrein-like activity from rat submaxillary gland (1 ,ug/ ml) [21] and fibroblast growth factor (25 ng/ml) [6].…”