Abstract
Expression of lysosomal cysteine proteinases was studied during fetal calf myoblast-myotube differentiation. Activities of cathepsin B and L, but not cathepsin H, increase during bovine myogenic differentiation. In fetal muscle, cathepsin B and L activities are 2-4-fold orders of magnitude lower than in cultured myoblasts. Active-site titrations of cathepsin B with E-64 nevertheless reveal similar concentrations of active cathepsin B in myoblasts and myotubes, but 5-6-fold lower concentrations in fetal muscle. To specify whether concentrations of cathepsin B are related to levels of cathepsin B transcript, a cDNA clone encoding bovine cathepsin B was isolated and liquid hybridizations were performed with 32P-riboprobes complementary to the mRNA. In agreement with active-site titrations, there is no difference in cathepsin B mRNA levels between cultured myoblasts and myotubes, but lower levels of mRNA are found in fetal muscle. Concentrations of active cathepsin B therefore reflect levels of cathepsin B mRNA. Kinetic studies revealed that the catalytic efficiency (kcat/Km) of cathepsin B is 2-3-fold higher in myotubes than in myoblasts. The increase in cathepsin B activity during calf myoblast-myotube differentiation is thus due to modifications of enzymatic properties, and not of enzyme concentrations. The different catalytic efficiency of cathepsin B in myotubes and myoblasts was related neither to modifications of mRNA size, as revealed by Northern blot analysis, nor to a different Mr of the active enzyme, as revealed by affinity labeling with benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2, but to limited differences in cathepsin B isozymes.
Highlights
From the Unite de Recherches sur 1’Expression des Proteases, SRV Theix, Institut National de la Recherche Agronomique, 63122 Ceyrat France
To specify whether concentrations of cathepsin B a r e related to levelsof cathepsin B transcript, a cDNA clone encoding bovine cathepsin B was isolated and liquid hybridizations were performed with 32P-riboprobescomplementaryto the mRNA
There are several examples of tumor cells exhibiting over-expression and secretion of cathepsin B (Sloane et al, 1987), cathepsin L (Gal and Gottesman, 1986), or cathepsin D (Rochefort et al, 1987).Over-expression of cathepsinis most probably due t o induction of cathepsin gene transcription (Doherty et al, 1985; Cavaill6s et al, 1989), but there is evidence that high levels of secretion from tumorcells are associated with alteration in the secretion pathway (Dong cathepsin B mRNA levels between cultured myoblasts et al, 1989)
Summary
Z-, benzyloxycarbonyl; FPLC, fast was dissolved in buffer D (1mg/ml). Solubilized cells or total protein liquid chromatography; Bzb-e,nzoyl-;. RNA were mixed with 1-2 ng of riboprobe in a total volume of 15 pl methy1)coumarylamide; SDS, sodium dodecyl sulfate; E-64, L-3-car- of buffer D, heated 5 min at 60 "C, and hybridized 18 h at 25 "C. hoxy-trans-2,3-epoxypropionyl-leucylamido-(4-~anidino)butane; RNase A digestion was carried as described by Firestein etal. Northern Blot Analysis-Poly(A)+ mRNA from myoblasts or myotubes was subjected to 1%agarose gel electrophoresis containing formaldehyde and transferred overnight to Hybond N+ membrane (Amersham Corp.) with 20 X SSC (1 X SSC is 0.15 M NaCl, 15 mM trisodium citrate, pH 7.0). The membrane was washed for 1 h at 65 "C in 2 X SSC, 0.5% SDS, and prehybridized for 2 h at 55 "C in 50% formamide containing 5X SSC, 5 X Denhardt's reagent, 50 mM sodium phosphate buffer, pH 6.5, 0.1% SDS, 0.1 mg/ml sonicated salmon sperm DNA, and 10 rg/ml yeast tRNA. Similar results were obtained when, after hybridization, the membrane was treated for 1h at 30 "C with 1pg/ml RNase A in 2 X ssc
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