Isolation and characterization of the chicken cystatin-encoding gene: mapping transcription start point and polyadenylation sites.

A 6.9-kb fragment containing coding sequences for chicken egg white cystatin (CsnEW) was isolated from a chicken genomic library using the CsnEW cDNA as a probe. The gene is approximately 2.4 kb in length; it contains three exons, two introns and two polyadenylation signals. The exon-intron arrangement corresponds exactly with those of other members of the Csn superfamily. The sequence of the 5' flanking region contains two SP1-binding sites and a high G+C content suggestive of a housekeeping gene. All tissues studied express CsnEW mRNA; Northern analysis showed that CsnEW mRNA levels are most abundant in lung and least abundant in liver and spleen. Mapping of the 3' end of the CsnEW mRNA isolated from brain tissue resolved two CsnEW mRNA species. The larger transcript resulting from the use of the second polyadenylation signal was more abundant than the smaller transcript. Determination of the transcription start point (tsp) of CsnEW mRNA by primer extension and RNase protection assays showed that CsnEW mRNA from a number of chicken tissues was approximately 40-50 nucleotides shorter than that predicted from the CsnEW cDNA isolated from chicken oviduct.

Isolation and partial characterisation of ACV synthetase from Cephalosporium acremonium and Streptomyces clavuligerus. Evidence for the presence of phosphopantothenate in ACV synthetase.

delta-(L-alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase was isolated and partially characterised from Cephalosporium acremonium CO728 and Streptomyces clavuligerus. The purification procedure resulted in a 745- and 277-fold increase in specific enzyme activity, respectively. Both enzymes had similar apparent molecular masses of ca. 300 kdaltons by SDS-polyacrylamide electrophoresis, under reducing and denaturing conditions, and in excess of 600 kdaltons in the native state by gel filtration. Attempts to obtain an N-terminal amino acid sequence of ACV synthetase from C. acremonium were unsuccessful, hence internal amino acid sequence data were obtained after tryptic digestion of the protein. Phosphopantothenic acid was shown to be associated with the enzyme from both sources, which suggests the possible involvement of pantothenate as a 'swinging arm' in the formation of the tripeptide ACV.

Steady-state cystatin mRNA levels in chicken tissues in response to estrogen.

A full length cDNA clone coding for the cysteine proteinase inhibitor, chicken cystatin, was used to hybridize to RNA extracted from various tissues of the hen during several well defined stages of the 25 h ovulatory cycle. Measurements were made immediately after egg-laying (ovulation), 6 h prior to ovulation (estrogen surge), and 16 h prior to ovulation (minimum estrogen levels). Cystatin was expressed in all tissues examined, being most abundant in lung, followed by brain, heart, oviduct, pectoral muscle, and liver in decreasing order. There were no significant differences in the cystatin mRNA levels at the three different time points for any of the tissues examined, and no differences in the 0.95 kb size of the message. Pharmacological doses of estradiol administered to immature female chickens decreased the steady-state cystatin mRNA levels as analyzed by Northern blots. There were no differences in the pattern of expression in the different tissues between rooster and hens. Our data suggest that estrogen has no direct effect on the regulation of cystatin expression in chicken tissues.

Chicken egg white cystatin. Molecular cloning, nucleotide sequence, and tissue distribution.

A lambda gt11 chicken oviduct cDNA library was screened with a mixed synthetic oligonucleotide corresponding to amino acid residues 81-90 of chicken egg white cystatin, a cysteine proteinase inhibitor. Two initial cDNA clones of 367 and 431 bases were isolated. Both clones contained coding sequences for cystatin from amino acid residue 82 to the carboxyl end plus 3'-untranslated region and a poly(A)+ tail. The two clones utilized different polyadenylation signals located 55 nucleotides apart. Further screening of the library yielded a full-length cystatin cDNA. Sequence analysis indicated that cystatin contains an NH2-terminal extension of 23 amino acids which is probably a signal sequence. The cystatin cDNA hybridized to an mRNA of approximately 0.95 kilobase and was present in varying amounts in all chicken tissues examined. The highest concentration was found in the lung. Gizzard, brain, and heart contained lesser amounts of cystatin mRNA but considerably higher than oviduct. Among a limited number of embryonic tissues examined, significantly higher levels of the mRNA were found in liver and heart tissues when compared with the corresponding adult tissues. These results suggested that the expression of the chicken cystatin gene is tissue-dependent and under developmental control.

The question of chymase in cultured muscle cells.

An alkaline proteinase, identical to mast cell chymase, has been described by a number of laboratories as being associated with myofibrils extracted from adult rat skeletal muscle tissue. A more recent study has indicated that chymase may be an intrinsic protein in the rat myocyte. The present study of rat myogenic cell lines, using more stringent controls and a probe of more highly defined specificity, supports the view that (i) chymase originates from mast cells of the interstitium and (ii) chymase from mast cells becomes adsorbed to myofibrils of adult muscle during homogenization of this complex tissue.

Neutral proteinases in muscle tissue and cells.

Chymase isolated from rat skeletal muscle tissue was compared with other proteinases in rat muscle that hydrolyse chymase substrates. These enzymes were purified from the 100,000 x g supernatant of hind limb rat muscles homogenized with 0.15 M NaCl-20 mM phosphate buffer, pH 7.2. A metallproteinase (MMP-7-ase) and a serine proteinase (ATN-ase) were partially characterized, and shown to be different from chymase.

Calcium-dependent proteolysis of calcium-binding proteins.

In several myopathic disorders, the internal muscle cell calcium concentration increases significantly as compared to normal muscle cells. We report that in the presence of elevated calcium levels, the calcium-binding proteins troponin C and calmodulin are protected from digestion by the chymotrypsin-like serine proteinase that co-purifies with isolated myofibrils. Degradation of the 67k calcimedin in the presence of calcium shows altered major cleavage fragments while degradation of myosin is unaffected by the presence of calcium. A role for this serine proteinase in muscle-wasting diseases is suggested.

mRNA levels of cathepsins B and D during myogenesis.

Muscle development is characterized by the fusion of myoblasts to form myotubes and the co-ordinate expression of muscle-specific proteins such as actin, myosin and creatine phosphokinase. Our laboratory has been involved in the study of the role of lysosomal proteinases, namely, cathepsins B, H and L and the endogenous cysteine proteinase inhibitor, cystatin, during muscle differentiation in vitro. Specific activities of the cysteine proteinase in chicken primary cultures and a number of rat myogenic lines increased with the degree of myotube formation. This has suggested that lysosomal proteinases play an important role in myogenesis. We have measured the mRNA levels of two of the lysosomal enzymes, cathepsins B and D. This is advantageous because the presence of the endogenous inhibitor, cystatin, masks the levels of the specific activities of the cysteine proteinases present in the cell. RNA was extracted from developing muscle at three stages of development: proliferating myoblasts, confluent cells, and myotubes. Hybridization of RNA extracted from the L6 myogenic cell line with cathepsin B cDNA showed an increase in the level of cathepsin B mRNA. However, in the L8 rat myogenic line the level decreased after fusion. Cathepsin D mRNA levels remained constant throughout differentiation of the L8 cells. This paper also reports on the characterization of lysosomal proteinases of a newly obtained mouse myogenic line, C2.

Activity of lysosomal cysteine proteinase during differentiation of rat skeletal muscle.

Cysteine-proteinase activities were measured in extracts of pre- and post-fusion populations of rat myogenic line L6 cells and in extracts of whole rat muscle. Activities of cathepsins B, L and H were compared. The substrates used included Z-Phe-Arg-NMec (cathepsins B and L), Z-Arg-Arg-NMec (cathepsin B), and Arg-NMec (cathepsin H) (where Z = benzyloxycarbonyl, and NMec = 4-methyl-7-coumarylamide); the enzyme activities were more specifically differentiated by appropriate concentrations of the inhibitors Z-Phe-Phe-CHN2 (CHN2 = diazomethane), bestatin and E-64 [L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane]. These experiments have demonstrated the feasibility of determining the cysteine-proteinase activities of myoblasts from a single (60 mm-diameter) Petri dish, with enzyme concentrations in the range of 5-20 ng/ml. Specific activities of the enzymes in L6 cells increased 2-20-fold after fusion. Concentrations of cysteine proteinases in extracts from cultured myoblasts were two orders of magnitude greater than those in muscle-tissue extracts. Cultured-cell extracts contained endogenous inhibitor(s) to purified rat cathepsins B, L and H.

Identification of the 3'-ends of the two mouse mitochondrial ribosomal RNAs. The 3'-end of 16 S ribosomal RNA contains nucleotides encoded by the gene for transfer RNALeuUUR.

The 3'-termini of the mitochondrial 12 S and 16 S ribosomal RNAs from mouse L cells have been definitively characterized by mobility-shift RNA sequencing, RNase digestion followed by fingerprinting using two-dimensional homochromatography, and precise mapping of RNA-DNA duplexes using nuclease S1. The results have been correlated with the known DNA sequence of the rRNA region. The vast majority of the 12 S rRNA consists of a family of transcripts whose last template-encoded nucleotide corresponds to a position immediately adjacent to the 5' end of the tRNAVal gene in the DNA sequence. These transcripts are oligoadenylated at their 3'-ends with from 1 to about 5 adenylate residues that are not encoded in the DNA sequence. A minor proportion of the 12 S rRNA ends one nucleotide before the 12 S/tRNAVal gene boundary and is also oligoadenylated. In contrast, the 3'-termini of 16 S rRNA have considerably greater heterogeneity, with the genomic location of the last template-encoded nucleotide varying from the nucleotide immediately adjacent to the 5'-end of the tRNALeuUUR gene to any position up to 7 nucleotides downstream within the tRNALeuUUR gene sequence. These various 16 S rRNA transcripts are oligoadenylated to a somewhat greater degree than the 12 S rRNA. The extent of the 16 S rRNA 3' heterogeneity, as compared to the 12 S rRNA, suggests that the 16 S rRNA 3'-termini may be generated by a mechanism involving termination of transcription rather than by processing of a primary transcript. The data are similar to those reported for human mitochondrial rRNA 3'-termini, and support a general role for adenylation of 3'-termini in the termination or processing of mammalian mitochondrial RNAs.

Endogenous cathepsin B inhibitor activity in normal and myopathic red and white skeletal muscle.

Despite extensive biochemical and morphological studied on the degenerative muscle diseases, the primary chemical lesions are still obscure, both in humans and animals. In this report we examine the activities of the lysosomal endoproteinase cathepsin B and its endogenous inhibitor(s) in the red and white skeletal muscles of guinea pigs with nutritional muscular myopathy induced by vitamin E deficiency. We observed a twofold increase (P less than 0.005) in the activity of cathepsin B in the white skeletal muscles of the vitamin E-deficient (E-) animals over that of the normal (N) and control (E+) groups. Assessment of the activity of endogenous cathepsin B inhibitor revealed a one and a half times greater amount of inhibitor in N when compared to E-; this difference in inhibitor activity applied to both red (masseter) and white (medial head, gastrocnemius) muscle. When the specific activity of cathepsin B in the E-tissue was corrected for inhibitor activity, the corrected value was not significantly different from either the E+ or the N tissue.

Muscle proteolytic enzyme activities in diabetic rats.

Proteolytic enzyme activities were measured in skeletal muscle of Sprague-Dawley rats with streptozotocin-induced diabetes [tail vein injection of streptozotocin (100 mg/kg), under ether anesthesia]. Assay of rat muscle homogenates from diabetic rats revealed a significant increase in alkaline serine protease activity as compared to untreated control rats and diabetic rats given insulin. There were no significant changes in lysosomal cathepsin activities in diabetic muscle as compared to controls. Gel studies of myofibrils isolated from the three groups of rats, subjected to autolysis, revealed that the serine protease had copurified with the myofibrils. Treatment of rats with compound 48/80, which degranulates mast cells, abolished the alkaline protease activity. There was no serine protease activity associated with the myofibrils isolated from compound 48/80-treated rats. Results from this study indicate that serine proteases are not involved in muscle protein breakdown in diabetes and are of mast cell origin.

Lysosomes and proteolytic enzyme activities in cultured striated muscle cells.

Primary cell cultures prepared from chick embryonic skeletal muscle and the rat myogenic line L6 were examined morphologically and biochemically during several stages of development. The L6 cells were cultured to provide three morphologically distinct populations: prefusion, postfusion, and a subclone of cells that did not fuse even at high density. Ultrastructural studies revealed the characteristic morphology of healthy myoblasts. Acridine orange staining and cytochemical localization of acid phosphatase suggest the presence of presumptive lysosomal material. Enzymatic studies of lysosomal cathepsins B, D, H, and L revealed unusually high enzyme specific activities in these homogeneous myoblast populations. No activity was detected for the two nonlysosomal enzymes Ca2+-proteinase and serine proteinase. It is suggested that the lysosomal apparatus and its complement of enzymes play a significant role in the differentiation of muscle myotubes.

Properties of the lysosomal apparatus during differentiation of cultured striated muscle cells.

A technique for the preparation of relatively pure myoblasts from chick primary culture is described. Cultured rat myogenic cells (L6) were plated and grown to provide three morphologically distinct cell populations: perfusion, postfusion, and nonfusion. Homogenates of L6 cells demonstrated two major peaks of proteolytic activity at pH 3.0 and 5.5. The activity could be partially inhibited by leupeptin or pepstatin. Differential centrifugation indicated significant acid hydrolase activity in the "H" fraction of prefused cells, which shifted to the "L" fraction after fusion of the cells. Two populations of lysosomes were resolved in the myoblasts and myotubes after isopycnic centrifugation in Percoll. The equilibrium densities were 1.044 and 1.060-1.068. Cells were incubated with several protease inhibitors. Only chloroquine caused a large inhibition of protein degradation.