Expression of lysosomal cathepsin B during calf myoblast-myotube differentiation. Characterization of a cDNA encoding bovine cathepsin B.
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.
Béchet
D M
DM
Unité de Recherches sur l'Expression des Protéases, SRV Theix, Institut National de la Recherche Agronomique, Ceyrat, France.
Ferrara
M J
MJ
Mordier
S B
SB
Roux
M P
MP
Deval
C D
CD
Obled
A
A
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D002454:Cell Differentiation; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D007525:Isoelectric Focusing; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology*; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping
D000345:Affinity Labels; D012333:RNA, Messenger; D004247:DNA; D002401:Cathepsin B