Subject(s)
Budgets , Federal Government , Medicare/economics , Politics , Private Practice/economics , Health Care Reform/economics , Humans , United StatesABSTRACT
Expression of lysosomal cysteine proteinases was studied during fetal calf muscle development. The peptide cleaving activities of cathepsins B and L decreased strongly from 80 to 250 days of fetal age. This decrease in cathepsin activities occurred similarly in three muscles exhibiting different metabolic and contractile properties in adult animals. Cathepsin B from adult or fetal muscle revealed similar enzymatic properties, but presented a five- to sixfold lower concentration in adult muscle as indicated by active-site titration with L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guani din o)butane. During fetal growth, decreases in muscle cathepsin B specific activity and active enzyme concentration were associated with a parallel drop of cathepsin B mRNA levels. Bovine cathepsin B is encoded by two different transcripts resulting from alternative polyadenylation [Mordier, S. B., Béchet, D. M., Roux, M. P., Obled, A., and Ferrara, M. (1995) Eur. J. Biochem. 229, 35-44]. As revealed by ribonuclease protection assays, the two mRNAs encoding cathepsin B declined similarly during fetal muscle growth. This study indicates that lysosomal proteinases in skeletal muscle are under developmental control. The decrease of muscle cathepsins during fetal development appears sufficient to account for the low levels of these enzymes in adult muscles. In fetuses, high activities of lysosomal cysteine proteinases might be important for remodeling muscles during early development.
Subject(s)
Aging/metabolism , Cathepsin B/biosynthesis , Embryonic and Fetal Development , Gene Expression Regulation, Developmental , Muscle, Skeletal/enzymology , Animals , Animals, Newborn , Binding Sites , Cathepsin B/isolation & purification , Cathepsin B/metabolism , Cattle , Chromatography, Ion Exchange , Fetus , Gene Expression Regulation, Enzymologic , Gestational Age , Lysosomes/enzymology , Muscle Development , Muscle, Skeletal/embryology , Muscle, Skeletal/growth & development , RNA, Messenger/biosynthesis , Substrate Specificity , Transcription, GeneticABSTRACT
Cathepsin B is a cysteine proteinase which plays an important role in lysosomal proteolysis. We report here the characterization of a 15-kbp genomic clone of the bovine cathepsin B gene. Bovine preprocathepsin B is encoded by nine exons from translational initiation to stop codon. The last exon is sandwiched between Alu-like short interspersed nuclear elements. Alternate use of polyadenylation sites generates three transcripts encoding bovine cathepsin B. In all tissues tested, 3' end cleavage was found to occur in equal proportion at the first and second polyadenylation site, producing transcripts of 2.6-kb. Polyadenylation at the third polyadenylation site generates a 3.2-kb mRNA, only expressed at low levels and in a developmental and tissue-specific manner. Due to micro-heterogeneities between genomic and cDNA clones, sequence polymorphism was investigated in the trailer region. DNA sequencing of PCR-amplified genomic fragments revealed that, in contrast to the protein-encoding region, genetic variability exists in the 3' untranslated region. Polymorphism in the trailer was confirmed in cathepsin B mRNA by ribonuclease-protection assays. We finally emphasize that while exon-exon boundaries in mature cathepsin B are well conserved from nematodes to mammals, exons also tend to correspond to discrete units of cathepsin B structure.
Subject(s)
Cathepsin B/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle , Chromosome Mapping , Cloning, Molecular , Molecular Sequence Data , Sequence AlignmentABSTRACT
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.
