Partial structure and hormonal regulation of rabbit liver inhibitor-1; distribution of inhibitor-1 and inhibitor-2 in rabbit and rat tissues

Inhibitor-1 purified from rabbit liver could not be distinguished from the skeletal muscle protein by chromatographic, electrophoretic and immunological criteria. Amino acid sequences comprising 68% of rabbit liver inhibitor-1 were identical to the skeletal muscle protein indicating that they are products of a single gene. Total inhibitor-1 activity in heat-treated rabbit liver extracts was similar to that in skeletal muscle extracts, and the phosphorylation state of inhibitor-1 increased from 14% to 42% in rabbit liver in vivo after an intravenous injection of glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-1 recognised a single major protein of identical electrophoretic mobility (26 kDa) in each rabbit tissue examined (skeletal muscle, liver, brain, heart, kidney, uterus and adipose). The antibodies also recognised a single major (30 kDa) protein in the same rat tissues, except liver. The results show that while there are interspecies differences in apparent molecular mass, inhibitor-1 is likely to be the same gene product in each mammalian tissue. Inhibitor-1 was not detected in rat liver, either by activity measurements or immunoblotting, irrespective of the age, sex or strain of the animals. Immunoblotting also failed to detect inhibitor-1 in mouse liver, although it was present in guinea pig, porcine and sheep liver. The absence of inhibitor-1 in rat liver indicates that phosphorylation of this protein cannot underlie the increased phosphorylation of hydroxymethylglutaryl-CoA reductase observed after stimulation by glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-2 recognised a 31 kDa protein in each rabbit tissue, and a 33 kDa protein in all rat tissues including liver. The results suggest that inhibitor-2 is the same gene product in each mammalian tissue.     

Cloning and sequence analysis of a cDNA for 3-hydroxyisobutyrate dehydrogenase. Evidence for its evolutionary relationship to other pyridine nucleotide-dependent dehydrogenases

A 1.7-kilobase pair cDNA clone encoding 3-hydroxyisobutyrate dehydrogenase has been isolated by screening a rat liver lambda gt11 library with a 17-base oligonucleotide probe which corresponds to a portion of the N-terminal amino acid sequence of rabbit liver 3-hydroxyisobutyrate dehydrogenase. The cDNA contains an open reading frame of 1038 base pairs which includes an amino acid sequence that matches the N-terminal 35 amino acid sequence of rabbit 3-hydroxyisobutyrate dehydrogenase at 33 residues. The cDNA predicts a 300-amino acid mature protein with an amino acid composition and molecular weight very similar to that of rabbit liver 3-hydroxyisobutyrate dehydrogenase. Northern blot analysis of total RNA from several rat tissues shows an mRNA of approximately 2.0 kilobase pairs in each tissue. Relative mRNA levels were: kidney greater than liver = heart greater than muscle. The amino acid sequence of 3-hydroxyisobutyrate dehydrogenase shows similarity to several other pyridine nucleotide-dependent dehydrogenases. The resemblance to malate and lactate dehydrogenases suggests that the nucleotide-binding domain is located in the N-terminal region of the protein.     

Isolation and characterization of a cDNA clone encoding rat 5-lipoxygenase

A full-length cDNA clone encoding 5-lipoxygenase, a key enzyme in the formation of leukotrienes, was isolated from a rat basophilic leukemia cell lambda gt11 cDNA library. The 2.5-kilobase (kb) cDNA insert, whose identity was confirmed by hybrid-select translation and DNA sequence analysis, has a 2.0-kb open reading frame encoding a protein of Mr approximately 77,600 and includes 60 base pairs of 5'-untranslated region and 0.4 kb of 3'-untranslated region to the polyadenylation signal. The deduced amino acid sequence shows significant homology with published sequences for the rabbit reticulocyte lipoxygenase and soybean lipoxygenase-1; it also contains sequences similar to a consensus sequence found in several calcium-dependent membrane-binding proteins. The cDNA recognizes a 2.6-kb mRNA species which is detected in all tissues but is particularly abundant in RNA from lung.     

Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.     

The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression.

The inhibitor protein (PKI) of the cAMP-dependent protein kinase was first characterized from rabbit skeletal muscle. More recently a form of PKI was isolated and cloned from rat testis which shares relatively limited amino acid sequence with the rabbit skeletal muscle form. We have now isolated a cDNA from rat brain which encodes a protein corresponding to the rabbit skeletal muscle PKI. This establishes the presence of the "skeletal muscle" and "testis" proteins in the same species and therefore that they clearly represent distinct isoforms. We have also demonstrated that the isoform from testis, like the skeletal muscle isoform, is specific for the cAMP-dependent protein kinase and that it is able to inhibit this enzyme when expressed in cultured JEG-3 cells. Both forms contain the five specific amino acid recognition determinants which have been shown to be required for high affinity binding to the protein kinase catalytic site, although there is some noted lack of conservation of codons used for these residues. Overall, the two rat isoforms are only 41% identical at the amino acid level and 46% at the level of coding nucleotides. We propose that the rabbit skeletal muscle and rat testis forms be designated PKI alpha and PKI beta, respectively. Using Northern blot analysis, we have examined the tissue distribution of the two forms in the rat and their relative expression during development. In the adult rat, mRNA of the PKI alpha species is highest in muscle (both skeletal and cardiac) and brain (cortex and cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of the cDNA encoding rat skeletal muscle myosin light chain kinase. Sequence and tissue distribution

A cDNA clone encoding skeletal muscle myosin light chain kinase (MLCK) was isolated from a rat skeletal muscle library using oligonucleotide probes. The total length of the rat skeletal muscle MLCK cDNA was 2823 base pairs with an open reading frame of 1830 base pairs. The deduced sequence of the 610-amino acid protein exhibited 96% amino acid identity to rabbit skeletal muscle MLCK in the carboxyl-terminal portion of the molecule, which contains the catalytic and the calmodulin-binding domains, and 58% identity in the amino-terminal region. Analysis of total rat mRNA revealed a single mRNA species of 3.4 kilobases that was unique to skeletal muscle. Further analysis of skeletal muscle tissue using fast-twitch glycolytic, fast-twitch oxidative glycolytic, and slow-twitch oxidative fibers isolated from rat leg revealed that the mRNA level for MLCK varied among the three fiber types. The results of kinase assays performed on the fibers showed that MLCK activity levels paralleled the MLCK mRNA levels found in each of the three types of skeletal muscle fibers studied. Fast-twitch oxidative glycolytic (gastrocnemius red) and slow-twitch oxidative (soleus) exhibited 60 and 13%, respectively, of the enzymatic activity present in fast-twitch glycolytic (gastrocnemius white) fibers.     

Molecular cloning and sequence analysis of cDNA for the catalytic subunit 1 alpha of rat kidney type 1 protein phosphatase, and detection of the gene expression at high levels in hepatoma cells and regenerating livers as compared to rat livers.

A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.     

An alternative non-tyrosine protein kinase product of the c-src gene in chicken skeletal muscle.

While the c-src locus is expressed as a 4.0-kilobase (kb) mRNA coding for pp60c-src in various chicken tissues, including embryonic muscle, it is expressed as a novel 3.0-kb mRNA in adult skeletal muscle. We have analyzed the primary structure of this alternatively transcribed and spliced c-src mRNA. The sequence revealed three open reading frames, with the previously defined c-src exons 1 through 5 or 6 comprising the third, on the 3' untranslated region of this 3-kb mRNA. The exons coding for the tyrosine kinase domain of pp60c-src were excluded. On the 5' side, 2 kb of sequence upstream from the previously defined exon 1 of the c-src gene was included in this mRNA. The start site for the 3-kb mRNA probably lies downstream of that for the 4-kb mRNA. The first reading frame of the 3.0-kb mRNA, called sur (for src upstream region), encoded a 24-kilodalton (kDa) protein product rich in cysteine and proline residues. In vitro analysis indicated that the 24-kDa sur protein was membrane associated. Antibodies to sur protein detected in vivo a 24-kDa muscle-specific protein which was developmentally regulated and corresponded to the switch from the 4-kb to the 3-kb c-src mRNA. A striking kinetic pattern of appearance of sur protein and disappearance of pp60c-src suggests that the expression of these two proteins is inversely related.     

Cloning and characterization of cDNA sequences corresponding to myosin light chains 1, 2, and 3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin

A library of cDNA clones was constructed from adult rat skeletal muscle mRNA, from which a set of contractile protein clones was selected. These clones were identified by sequencing the cDNA inserts and comparing the derived amino acid sequences with published sequences of rabbit contractile proteins. In this manner, clones corresponding to myosin light chains 1, 2, and 3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin were identified. A high degree of amino acid sequence conservation was found upon comparison of the rat and rabbit proteins. Using the cDNA clone panel, we analyzed the expression of abundant rat muscle mRNAs. We show that abundant rat muscle mRNAs can be classified into four developmentally regulated groups, based upon their expression at different stages of myogenesis. One class of mRNAs is expressed during all stages of muscle development. Since these mRNAs are also present in nonmuscle tissues, we conclude that they code for housekeeping proteins. The second class of mRNAs is present in both embryonic and adult muscle, while a third class of mRNAs is expressed only in adult muscle. A small number of mRNAs, which are present at greater levels in undifferentiated myoblasts than in adult muscle, comprise a fourth class. These results suggest the existence of at least four modes of gene control during myogenesis.     

Cloning of a differentially expressed tropomyosin isoform from cultured rabbit aortic smooth muscle cells

The four known tropomyosin genes have highly conserved DNA and amino acid sequences, and at least 18 isoforms are generated by alternative RNA splicing in muscle and non-muscle cells. No rabbit tropomyosin nucleotide sequences are known, although protein sequences for alpha- and beta-tropomyosin expressed by rabbit skeletal muscle have been described. Subtractive hybridisation was used to select for genes differentially expressed in rabbit aortic smooth muscle cells (SMC), during the change in cell phenotype in primary culture that is characterised by a loss of cytoskeletal filaments and contractile proteins. This led to the cloning of a tropomyosin gene predominantly expressed in rabbit SMC during this change. The full-length cDNA clone, designated "rabbit TM-beta", contains an open reading frame of 284 amino acids, 5' untranslated region (UTR) of 117 base pairs and 3' UTR of 79 base pairs. It is closely related to the beta-gene isoforms in other species, with the highest homology in DNA and protein sequences to the human fibroblast isoform TM-1 (91.7% identity in 1035 bp and 93.3% identity in the entire 284 amino acid sequence of the protein). It differs from rabbit skeletal muscle beta-tropomyosin (81.7% homology at the protein level) mainly in two regions at amino acids 189-213 and 258-283 suggesting alternative splicing of exons 6a for 6b and 9d for 9a. Since this TM-beta gene was the only gene strongly enough expressed in SMC changing phenotype to be observed by the subtractive hybridisation screen, it likely plays a significant role in this process.     

Cloning of zebrafish BAD,a BH3-only proapoptotic protein,whose overexpression leads to apoptosis in COS-1 cells and zebrafish embryos

The BH3-only proapoptotic protein, BAD, was cloned from zebrafish embryos and its properties were characterized. Zebrafish BAD (zBAD) is a protein with 147 amino acids that contains a BH3 domain and a putative 14-3-3 binding site with the sequence of RPRSRS(84)AP, corresponding to S(136) in mouse BAD (mBAD). zBAD shares 34%, 28%, and 29% amino acid sequence identity to the human, mouse, and rat BAD, respectively. RT-PCR analysis revealed that the expression of zBAD gene is found in various parts of zebrafish tissues. The treatment with the z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zebrafish BAD fusion proteins (GFP-zBAD and HA-zBAD), indicating that zebrafish BAD fusion proteins may be cleaved by caspase(s). zBAD was shown to induce apoptosis when it was overexpressed in COS-1 cells. In addition, zBAD was also expressed in muscle cells under the muscle-specific promoter from zebrafish alpha-actin gene. Abnormality in the skeletal muscles and the loss of green fluorescence signal in the same region were observed. Taken together, our results indicate that zBAD could induce apoptosis in vitro and in vivo and may have biological implications in apoptosis during zebrafish development.     

cDNA cloning, functional expression, and mRNA tissue distribution of a third organellar Ca2+ pump

We describe the characterization of a rat kidney cDNA that encodes a novel Ca2+-transporting ATPase. The cDNA, termed RK 8-13, was isolated previously using an oligonucleotide hybridization probe corresponding to part of the ATP binding site of the sarcoplasmic reticulum Ca-ATPases (Gunteski-Hamblin, A.-M., Greeb, J., and Shull, G. E. (1988) J. Biol. Chem. 263, 15032-15040). The complete nucleotide sequence of the 4.5-kilobase cDNA has been determined, and the primary structure of the protein has been deduced. The enzyme consists of 999 amino acids, has an Mr of 109,223, and contains all of the conserved domains found in transport ATPases of the E1-E2 class. It exhibits 75-77% amino acid identity with the fast-twitch and slow-twitch/cardiac isoforms of the sarcoplasmic reticulum Ca-ATPase, and the hydropathy plots of the three enzymes are virtually identical. High levels of ATP-dependent Ca2+ uptake were demonstrated in microsomes of COS-1 cells that had been transfected with a construct consisting of the entire coding sequence of the cDNA in the expression vector p91023(B). Northern blot analyses of poly(A)+ RNA revealed that the mRNA for this protein is expressed in heart, skeletal muscle, uterus, brain, lung, liver, kidney, testes, small intestine, large intestine, and pancreas. These data show that the enzyme is a Ca2+-transporting ATPase and that its mRNA is expressed in a broad variety of both muscle and non-muscle tissues.     

Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: sequence similarity with the 70-kilodalton precursor to sulfatide/GM1 activator

Sulfated glycoprotein 1 (SGP-1) is one of the abundant proteins secreted by rat Sertoli cells. Pulse-chase labeling shows that SGP-1 is synthesized as a cotranslationally glycosylated 67-kilodalton (kDa) precursor which is posttranslationally modified to a 70-kDa form before secretion to the extracellular space. A plasmid cDNA library was constructed from immunopurified mRNA, and two overlapping clones coding for the entire protein coding sequence were isolated. The cDNAs represent 27 nucleotides of 5' noncoding sequence, 1554 nucleotides of coding sequence, and 594 nucleotides of 3' noncoding sequence. The derived SGP-1 sequence contains 554 amino acids and has a molecular weight of 61,123. Four potential N-glycosylation sites occur within the sequence. An internal region of SGP-1 shows 78% sequence identity with the 67 N-terminal amino acids described for human sulfatide/GM1 activator (SAP-1). Sequence comparisons suggest that SGP-1 is the precursor to sulfatide/GM1 activator; however, the secretion of the protein from Sertoli cells is distinct from the proteolytic processing and lysosomal compartmentalization which have been described for human fibroblasts. The presence of internal sequence similarity suggests that three additional binding sites may occur in SGP-1. Northern blots show similar levels of expression for the 2.6-kilobase SGP-1 mRNA in all tissues examined. The site of SGP-1 synthesis in testis was localized to Sertoli cells by immunofluorescence and in situ hybridization.     

Isolation and sequence analysis of a cDNA clone encoding a type-1 protein phosphatase catalytic subunit: homology with protein phosphatase 2A

A 1.5 kb clone containing the full-length coding sequence of a type-1 protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The protein sequence deduced from the cDNA contains 311 residues and has a molecular mass of 35.4 kDa. A single mRNA species at 1.6 kb was visualized by Northern blotting. The type-1 protein phosphatase was strikingly homologous to protein phosphatase 2A, 49% of the amino acids between residues 11 and 280 being identical. The first 10 and last 31 residues were dissimilar. Residues 1-101 of the type-1 protein phosphatase also showed 21% sequence identity with a region of mammalian alkaline phosphatases.